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Title:
VARIANTS OF GLUCOAMYLASE
Document Type and Number:
WIPO Patent Application WO/2012/001139
Kind Code:
A9
Abstract:
The present invention relates to combinatorial variants of a parent glucoamylase that have altered properties for reducing the synthesis of condensation products during hydrolysis of starch. Accordingly the variants of a parent glucoamylase are suitable such as for use within brewing and glucose syrup production. Also disclosed are DNA constructs encoding the variants and methods of producing the glucoamylase variants in host cells.

Inventors:
DEGN PETER EDVARD (DK)
BOTT RICHARD R (US)
VROEMEN CASPER WILLEM (NL)
SCHEFFERS MARTIJN SILVAN (NL)
AEHLE WOLFGANG (DE)
PETERSEN ELIN (DK)
Application Number:
PCT/EP2011/061082
Publication Date:
February 23, 2012
Filing Date:
June 30, 2011
Export Citation:
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Assignee:
DANISCO (DK)
DANISCO US INC (US)
DEGN PETER EDVARD (DK)
BOTT RICHARD R (US)
VROEMEN CASPER WILLEM (NL)
SCHEFFERS MARTIJN SILVAN (NL)
AEHLE WOLFGANG (DE)
PETERSEN ELIN (DK)
International Classes:
C12N9/34; C07K14/38; C12C5/00; C12C7/00; C12P19/14; C13K1/06
Attorney, Agent or Firm:
STAHR, Pia et al. (P.O. Box 45Kogle Allé 2, Hørsholm, DK)
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Claims:
CLAIMS

1. A glucoamylase variant comprising the following amino acid substitutions: a. 44R and 539R; or b. 44R, 611 and 539R, the positions corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase, wherein the glucoamylase variant has at least 80% sequence identity with SEQ ID NO: 1 or 2, or the parent glucoamylase.

2. The glucoamylase variant according to claim 1 comprising the following amino acid substitutions: a. D44R and A539R; or b. D44R, N61I and A539R, the positions corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase, wherein the glucoamylase variant has at least 80% sequence identity with SEQ ID NO: 1 or 2, or the parent glucoamylase.

3. The glucoamylase variant according to any one of claims 1-2 comprising the following amino acid substitutions: a. D44R, N61I and A539R, the positions corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase, wherein the glucoamylase variant has at least 80% sequence identity with SEQ ID NO: 1 or 2, or the parent glucoamylase.

4. The glucoamylase variant according to any one of claims 1-2 comprising the following amino acid substitutions: a. D44R and A539R, the positions corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase, wherein the glucoamylase variant has at least 80% sequence identity with SEQ ID NO: 1 or 2, or the parent glucoamylase.

5. The glucoamylase variant according to any one of claims 1-3, wherein the

glucoamylase variant has at least 85%, 90%, 95%, 98%, or 99.5% sequence identity with SEQ ID NO: 1 or 2.

6. The glucoamylase variant of claim 5, wherein the glucoamylase variant has at least 95% sequence identity with SEQ ID NO: 1 or 2.

7. The glucoamylase variant of claim 6, wherein the glucoamylase variant has at least 99.5% sequence identity with SEQ ID NO: 1 or 2.

8. The glucoamylase variant of any one of claims 1-7, wherein the parent glucoamylase comprises SEQ ID NO: 1 or 2.

9. The glucoamylase variant of claim 8, wherein the parent glucoamylase consists of SEQ ID NO: 1 or 2. 10. The glucoamylase variant according to any one of claims 1-9, wherein the

glucoamylase variant has a starch binding domain that has at least 96%, 97%, 98%, 99%, or 99.5% sequence identity with the starch binding domain of SEQ ID NO: 1, 2, 11, 385, 386, 387, 388, 389, or 390.

11. The glucoamylase variant according to any one of claims 1-10, wherein the glucoamylase variant has a catalytic domain that has at least 80%, 85%, 90%, 95%, or 99.5% sequence identity with the catalytic domain of SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9.

12. The glucoamylase variant according to any one of claims 1-11, wherein the parent glucoamylase is selected from a glucoamylase obtained from a Trichoderma spp., an Aspergillus spp., a Humicola spp., a PeniciUium spp., a Talaromyces spp., or a

Schizosaccharmyces spp.

13. The glucoamylase variant according to any one of claims 1-12, wherein the parent glucoamylase is obtained from a Trichoderma spp. or an Aspergillus spp.

14. The glucoamylase variant according to any one of claims 1-13, which glucoamylase exhibit an enhanced production of fermentable sugar(s) as compared to the parent glucoamylase.

15. The glucoamylase variant according to any one of claims 1- 14, which glucoamylase exhibit an enhanced production of fermentable sugars in the mashing step of the brewing process as compared to the parent glucoamylase.

16. The glucoamylase variant according to any one of claims 1-15, which glucoamylase exhibit an enhanced production of fermentable sugars in the fermentation step of the brewing process as compared to the parent glucoamylase.

17. The glucoamylase variant according to claim 16, wherein the fermentable sugar is glucose.

18. The glucoamylase variant according to any one of claims 1- 17, which glucoamylase exhibit a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase.

19. The glucoamylase variant according to any one of claims 1- 18, which glucoamylase exhibit a reduced starch hydrolysis activity which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase.

20. The glucoamylase variant according to any one of claims 1- 19, which glucoamylase exhibit an enhanced real degree of fermentation as compared to the parent glucoamylase.

21. The glucoamylase variant according to any one of claims 1-20, which glucoamylase forms a lower amount of condensation products than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under the same conditions.

22. The glucoamylase variant according to any one of claims 1-21, which glucoamylase forms an amount of condensation products which amount is essentially the same as, not more than 5%, not more than 8%, or not more than 10% higher than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under the same conditions.

23. The glucoamylase variant according to any one of claims 18-21, wherein the dosing of the glucoamylases are the same based on protein concentration.

24. The glucoamylase variant according to any one of claims 18-23, wherein the dosing of the glucoamylases are the same based on measurement of activity in activity assays.

25. The glucoamylase variant according to any one of claims 1-24, which glucoamylase has been purified.

26. A polynucleotide encoding a glucoamylase variant according to any of claims 1-25.

27. A vector comprising the polynucleotide according to claim 26, or capable of expressing a glucoamylase variant according to any of claims 1-25.

28. A host cell comprising a vector according to claim 27.

29. A host cell which has stably integrated into the chromosome a nucleic acid encoding the variant glucoamylase according to any of claims 1-25.

30. A cell capable of expressing a glucoamylase variant according to any one of claims claims 1-25.

31. The host cell according to any one of claims 28-29, or the cell according to claim 30, which is a bacterial, fungal or yeast cell.

32. The host cell according to claim 31, which is Trichoderma spp. such as Trichoderma reesei.

33. The host cell according to any one of claims 28-29 and 31-32, which is a protease deficient and/or xylanase deficient and/or glucanase deficient host cell.

34. A method of expressing a glucoamylase variant, the method comprising obtaining a host cell or a cell according to any one of claims 28-33 and expressing the glucoamylase variant from the cell or host cell, and optionally purifying the glucoamylase variant.

35. The method according to claim 34 comprising purifying the glucoamylase variant.

36. Use of a glucoamylase variant according to any one of claims 1-25 for the preparation of an enzymatic composition.

37. An enzymatic composition comprising at least one glucoamylase variant according to any one of claims 1-25.

38. The enzymatic composition according to claim 37 comprising at least one

glucoamylase variant according to any one of claims 1-25, wherein the composition is selected from among a starch hydrolyzing composition, a saccharifying composition, a detergent composition, an alcohol fermentation enzymatic composition, and an animal feed composition.

39. An enzymatic composition according to any one of claims 36-38 comprising at least one additional enzyme selected among amylase, protease, pullulanase, isoamylase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, phytase and a further glucoamylase.

40. The enzymatic composition according to any one of claims 36-39, wherein the composition comprises at least one additional enzyme selected among alpha-amylase and/or pullulanase. 41. The enzymatic composition according to any one of claims 36-40, wherein the composition comprises alpha-amylase and pullulanase.

42. The enzymatic composition according to any one of claims 36-41, which enzymatic composition comprises less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XL) of xylanase activity per GAU of a glucoamylase variant according to any one of claims 1-25.

43. The enzymatic composition according to any one of claims 36-42, which enzymatic composition comprises less than 400, less than 200, less than 50, less than 20, or less than 2 XU of xylanase activity per gram of the composition. 44. The enzymatic composition according to any one of claims 36-43, which enzymatic composition comprises between 0.1 - 20, 1-15, 2-10, or 3-10 SSU of alpha-amylase activity per GAU of a glucoamylase variant according to any one of claims 1-25.

45. The enzymatic composition according to any one of claims 36-44, which enzymatic composition comprises between 0.05-10, 0.1-10, 0.1-8, 0.1-5, 0.1 -3, 0.2-3, or 0.2-2 PL) of pullulanase activity per GAU of a glucoamylase variant according to any one of claims 1-25.

Description:
VARIANTS OF GLUCOAMYLASE

FIELD OF THE INVENTION

Disclosed are combinatorial variants of a parent glucoamylase that have altered properties and are suitable such as for use within brewing and glucose syrup production. Also disclosed are DNA constructs encoding the variants and methods of producing the glucoamylase variants in host cells.

BACKGROUND OF THE INVENTION

Glucoamylase enzymes (glucan 1, 4-a-glucohydrolases, EC 3.2.1.3) are starch hydrolyzing exo-acting carbohydrases, which catalyze the removal of successive glucose units from the non-reducing ends of starch or related oligo and polysaccharide molecules. Glucoamylases can hydrolyze both the linear and branched glucosidic linkages of starch (e.g. , amylose and amylopectin).

Glucoamylases are produced by numerous strains of bacteria, fungi, yeast and plants.

Particularly interesting, and commercially important, glucoamylases are fungal enzymes that are extracellularly produced, for example from strains of Aspergillus (Svensson et al.,

Carlsberg Res. Commun. 48: 529-544 (1983); Boel et al., EMBO J. 3 : 1097- 1102 (1984);

Hayashida et al., Agric. Biol. Chem. 53 : 923-929 ( 1989); U.S. Patent No. 5,024,941 ; U.S.

Patent No. 4,794,175 and WO 88/09795); Talaromyces (U.S. Patent No. 4,247,637; U.S.

Patent No. 6,255,084; and U.S. Patent No. 6,620,924); Rhizopus (Ashikari et al., Agric. Biol. Chem. 50: 957-964 ( 1986); Ashikari et al., App. Microbio. Biotech. 32: 129-133 ( 1989) and

U.S. Patent No. 4,863,864); Humicola (WO 05/052148 and U.S. Patent No. 4,618,579); and

Mucor (Houghton -La rsen et al., Appl. Microbiol. Biotechnol. 62 : 210-217 (2003)) . Many of the genes that code for these enzymes have been cloned and expressed in yeast, fungal and/or bacterial cells. Commercially, glucoamylases are very important enzymes and have been used in a wide variety of applications that require the hydrolysis of starch (e.g. , for producing glucose and other monosaccharides from starch). Glucoamylases are used to produce high fructose corn sweeteners, which comprise over 50% of the sweetener market in the United States. In general, glucoamylases may be, and commonly are, used with alpha-amylases in starch hydrolyzing processes to hydrolyze starch to dextrins and then glucose. The glucose may then be converted to fructose by other enzymes (e.g. , glucose isomerases); crystallized; or used in fermentations to produce numerous end products (e.g. , ethanol, citric acid, lactic acid, succinate, ascorbic acid intermediates, glutamic acid, glycerol and 1, 3-propanediol). Ethanol produced by using glucoamylases in the fermentation of starch and/or cellulose containing material may be used as a source of fuel or for alcoholic consumption.

At the high solids concentrations used commercially for high glucose corn syrup (HGCS) and high fructose corn syrup (HFCS) production, glucoamylase synthesizes di-, tri-, and tetra- saccharides from glucose by condensation reactions. This occurs because of the slow hydrolysis of alpha-( l-6)-D-glucosidic bonds in starch and the formation of various accumulating condensation products, mainly isomaltose, from D-glucose. Accordingly, the glucose yield in many conventional processes does not exceed 95% of theoretical yield. The amount of syrups produced worldwide by this process is very large and even very small increases in the glucose yield pr ton of starch are commercially important.

Glucoamylase is used in brewing mainly for production of low carb beer. In combination with other amylases (such as from the malt), glucoamylase gives a very extensive hydrolysis of starch, all the way down to glucose units. Glucose is readily converted to alcohol by yeast making it possible for the breweries to obtain a very high alcohol yield from fermentation and at the same time obtain a beer, which is very low in residual carbohydrate. The ferment is diluted down to the desired alcohol % with water, and the final beer is sold as "low carb".

Although glucoamylases have been used successfully in commercial applications for many years, a need still exists for new glucoamylases with altered properties, such as an improved specific activity, a reduced formation of condensation products such as isomaltose and increased thermostability.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

The glucoamylase variants and the use of glucoamylase variants for reducing the synthesis of condensation products during hydrolysis of starch are contemplated herein. These

glucoamylase variants contain amino acid substitutions within the catalytic domains and/or the starch binding domain. The variants display altered properties, such as an altered specific activity, a reduced formation of condensation products such as isomaltose and/or altered thermostability.

In one aspect, a glucoamylase variant is described herein comprising the following amino acid substitutions: a) 44R and 539R; or b) 44R, 611 and 539R, the positions corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase, wherein the glucoamylase variant has at least 80% sequence identity with SEQ ID NO: 1 or 2, or the parent glucoamylase. In a further aspect, the use is described of a glucoamylase variant for the preparation of an enzymatic composition. In a further aspect, the enzymatic composition comprises at least one additional enzyme selected among amylase, protease, pullulanase, isoamylase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, phytase and a further glucoamylase such as for example an pullulanase and a alpha-amylase.

In a further aspect, the use is described herein of a glucoamylase variant with a starch binding domain and a catalytic domain, said variant comprising two or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2 or equivalent parent glucoamylase in interconnecting loop 2',and/or in loop 1, and/or in helix 2, and/or in loop 11, and/or in helix 12 for reducing the synthesis of condensation products during hydrolysis of starch.

In a further aspect, the use is described of a glucoamylase variant comprising two or more amino acid substitutions relative to interconnecting loop 2' with the amino acid sequence from position 518 to position 543 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase for reducing the synthesis of condensation products during hydrolysis of starch. In a further aspect, the use is described of a glucoamylase variant comprising two or more amino acid substitutions relative to the amino acid sequence from position 518 to position 543 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 21 to position 51 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 52 to position 68 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 396 to position 420 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 421 to position 434 of SEQ ID NO : 2 or equivalent sequence of residues in a parent glucoamylase for reducing the synthesis of condensation products during hydrolysis of starch.

In a further aspect, the use is described of a glucoamylase variant wherein said two or more amino acid substitutions are relative to the interconnecting loop 2' with the amino acid sequence from position 518 to position 543 of SEQ ID NO: 2, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO: 2, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO: 2, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO: 2, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO: 2.

In a further aspect, the use of a glucoamylase variant which when in its crystal form has a crystal structure for which the atomic coordinates of the main chain atoms have a root-mean- square deviation from the atomic coordinates of the equivalent main chain atoms of TrGA (as defined in Table 20 in WO2009/067218) of less than 0.13 nm following alignment of equivalent main chain atoms, and which have a linker region, a starch binding domain and a catalytic domain, said variant comprising two or more amino acid substitutions relative to the amino acid sequence of the parent glucoamylase in interconnecting loop 2' of the starch binding domain, and/or in loop 1, and/or in helix 2, and/or in loop 11, and/or in helix 12 of the catalytic domain for reducing the synthesis of condensation products during hydrolysis of starch.

In one aspect, the glucoamylase variant comprises two or more amino acid substitutions, wherein an amino acid substitution is in position 539 and an amino acid substitution is in position 44, the positions corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase, and which sequence has at least 80% sequence identity to the parent glucoamylase, and wherein the amino acid substitution in position 44 is not 44C.

The present disclosure further relates to a polynucleotide encoding a glucoamylase variant as described herein. One aspect, is a plasmid comprising a nucleic acid. Another aspect, is a vector comprising a polynucleotide as described, or capable of expressing a glucoamylase variant as described. Another aspect, is a host cell comprising, e.g. transformed with, a plasmid or a vector as described. Another aspect, is a host cell, which has stably integrated into the chromosome a nucleic acid sequence encoding the variant glucoamylase. Another aspect is a cell capable of expressing a glucoamylase variant as described. Another aspect is a method of expressing a glucoamylase variant, the method comprising obtaining a host cell or a cell and expressing the glucoamylase variant from the cell or host cell, and optionally purifying the glucoamylase variant.

A further aspect of the disclosure is an enzymatic composition comprising at least one glucoamylase variant as described herein, and the use thereof.

A further aspect of the disclosure is a method for converting starch or partially hydrolyzed starch into a syrup containing glucose, which process includes saccharifying a liquid starch solution in the presence of at least one glucoamylase variant or an enzymatic composition as described herein. A further aspect of the disclosure is the use of a glucoamylase variant as described herein in a starch conversion process, such as in a continuous starch conversion process, in a process for producing oligosaccharides, maltodextrins or glucose syrups and in a process for producing high fructose corn syrup.

In a further aspect, the use of a glucoamylase variant as described herein in a alchohol fermentation process is provided.

A further aspect of the disclosure is a method for producing a wort for brewing comprising forming a mash from a grist, and contacting the mash with a glucoamylase variant as described or an enzymatic composition as described.

Yet a further aspect of the disclosure is a method for production of a beer which comprises: a) preparing a mash, b) filtering the mash to obtain a wort, and fermenting the wort to obtain a beer, wherein a glucoamylase variant as described is added to: step (a) and/or step (b) and/or step (c).

Yet a further aspect of the disclosure is the use of a glucoamylase variant as described to enhance the production of fermentable sugars in either the mashing step or the fermentation step of a brewing process.

Yet a further aspect of the disclosure is a beer, wherein the beer is produced by the steps of: a) preparing a mash, b) filtering the mash to obtain a wort, c) fermenting the wort to obtain a beer, and d) pasteurizing the beer, wherein a glucoamylase variant as described is added to: step (a) and/or step (b) and/or step (c). Accordingly, it is an object of the invention to not encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It is noted that in this disclosure and particularly in the claims and/or embodiments, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which :

FIG. 1A depicts a Trichoderma reesei glucoamylase (TrGA) having 632 amino acids (SEQ ID NO: 1). The signal peptide is underlined, the catalytic region (SEQ ID NO: 3) starting with amino acid residues SVDDFI (SEQ ID NO: 12) and having 453 amino acid residues is in bold; the linker region is in italics and the starch binding domain (SBD) is both italics and underlined. The mature protein of TrGA (SEQ ID NO : 2) includes the catalytic domain (SEQ ID NO: 3), linker region (SEQ ID NO: 10), and starch binding domain (SEQ ID NO: 11). With respect to the SBD numbering of the TrGA glucoamylase molecule, reference is made in the present disclosure to either a) positions 491 to 599 in SEQ ID NO:2 of the mature TrGA, and/or b) positions 1 to 109 in SEQ ID NO: 11, which represents the isolated SBD sequence of the mature TrGA. With respect to the catalytic domain numbering of the TrGA molecule, reference is made to SEQ ID NO: 2 and/or SEQ ID NO: 3. FIG. IB depicts the cDNA (SEQ ID NO:4) that codes for the TrGA. FIG. 1C depicts the precursor and mature protein TrGA domains.

FIG. 2 depicts the destination plasmid pDONR-TrGA which includes the cDNA (SEQ ID NO: 4) of the TrGA. FIG. 3 depicts the plasmid pTTT-Dest.

FIG. 4 depicts the final expression vector pTTT-TrGA.

FIGs. 5A and 5B depict an alignment comparison of the catalytic domains of parent glucoamylases from Aspergillus awamori (AaGA) (SEQ ID NO: 5); Aspergillus niger (AnGA) (SEQ ID NO: 6); Aspergillus oryzae (AoGA) (SEQ ID NO: 7); Trichoderma reesei (TrGA) (SEQ ID NO: 3); Humicola grisea (HgGA) (SEQ ID NO: 8); and Hypocrea vinosa (HvGA) (SEQ ID NO: 9). Identical amino acids are indicated by an asterisk (*). FIG. 5C depicts a

Talaromyces glucoamylase (TeGA) mature protein sequence (SEQ ID NO: 384). FIGs 5D and 5E depict an alignment comparing the Starch Binding Domain (SBD) of parent glucoamylases from Trichoderma reesei (SEQ ID NO: 11); Humicola grisea (HgGA) (SEQ ID NO: 385); Thermomyces lanuginosus (ThGA) (SEQ ID NO: 386); Talaromyces emersonii (TeGA) (SEQ ID NO: 387); Aspergillus niger (AnGA) (SEQ ID NO: 388); Aspergillus awamori (AaGA) (SEQ ID NO: 389); and Thielavia terrestris (TtGA) (SEQ ID NO: 390).

FIG. 6 depicts a comparison of the three dimensional structure of Trichoderma reesei glucoamylase (black) (SEQ ID NO: 2) and Aspergillus awamori glucoamylase (grey) (SEQ ID NO: 5) viewed from the side. The side is measured in reference to the active site and the active site entrance is at the "top" of the molecule.

FIG. 7 depicts a comparison of the three dimensional structures of Trichoderma reesei glucoamylase (black) (SEQ ID NO: 2) and Aspergillus awamori glucoamylase (grey) (SEQ ID NO: 5) viewed from the top. FIG. 8 depicts an alignment of the three dimensional structures of TrGA (SEQ ID NO: 2) and AnGA (SEQ ID NO: 6) viewed from the side showing binding sites 1 and 2.

FIG. 9 depicts a model of the binding of acarbose to the TrGA crystal structure.

Fig. 10 depicts a TLC plate with standards containing different concentrations of glucose, maltose and isomaltose and samples containing reaction products from glucose incubated with TrGA and AnGA. DETAILED DISCLOSURE OF THE INVENTION

Glucoamylases are commercially important enzymes in a wide variety of applications that require the hydrolysis of starch. The applicants have found that by introducing certain alterations in positions within specific regions of the amino acid sequence of a parent glucoamylase the rate of forming alpha-(l-6) bonds is reduced, and/or the formation of condensation products such as isomaltose is reduced. A reduction of the rate that

glucoamylase forms alpha-(l-6) bonds relative to the rate it cleaves alpha-(l-4) bonds has practical implications.

The present inventors have provided a number of variants of a parent glucoamylase, which variants in some embodiments show a reduced condensation and/or a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase. In some embodiments using a glucoamylase variant as described herein in a saccharification process produces a syrup with high glucose percentage. In some

embodiments using a glucoamylase variant as described herein results in an enhanced production of fermentable sugars in a mashing and/or fermentation step of a brewing step. In some embodiments using a glucoamylase variant as described herein results in an enhanced real degree of fermentation. These altered properties are obtained by mutating e.g.

substituting selected positions in a parent glucoamylase. This will be described in more detail below. Accordingly, in a further aspect, the use is described of a glucoamylase variant comprising two or more amino acid substitutions relative to interconnecting loop 2' with the amino acid sequence from position 518 to position 543 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO: 2 or equivalent sequence of residues in a parent

glucoamylase, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase for reducing the synthesis of condensation products during hydrolysis of starch.

In a further aspect, the use is described of a glucoamylase variant comprising two or more amino acid substitutions relative to the amino acid sequence from position 518 to position 543 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 21 to position 51 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 52 to position 68 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 396 to position 420 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 421 to position 434 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase for reducing the synthesis of condensation products during hydrolysis of starch.

Accordingly, in a further aspect, the use of a glucoamylase variant is described, which glucoamylase variant when in its crystal form has a crystal structure for which the atomic coordinates of the main chain atoms have a root-mean-square deviation from the atomic coordinates of the equivalent main chain atoms of TrGA (as defined in Table 20 in

WO2009/067218) of less than 0.13 nm following alignment of equivalent main chain atoms, and which have a linker region, a starch binding domain and a catalytic domain, said variant comprising two or more amino acid substitutions relative to the amino acid sequence of the parent glucoamylase in interconnecting loop 2' of the starch binding domain, and/or in loop 1, and/or in helix 2, and/or in loop 11, and/or in helix 12 of the catalytic domain for reducing the synthesis of condensation products during hydrolysis of starch. In a further aspect, the root-mean-square deviation from the atomic coordinates of the equivalent main chain atoms of TrGA (as defined in Table 20 in WO2009/067218) is less than 0.12 nm, such as less than 0.11 or such as less than 0.10.

In one aspect, the use is described herein of a glucoamylase variant with a starch binding domain and a catalytic domain, said variant comprising two or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2 or equivalent parent glucoamylase in interconnecting loop 2', and/or in loop 1, and/or in helix 2, and/or in loop 11, and/or in helix 12 for reducing the synthesis of condensation products during hydrolysis of starch.

In a further aspect, the use is described of a glucoamylase variant wherein said two or more amino acid substitutions are relative to the interconnecting loop 2' with the amino acid sequence from position 518 to position 543 of SEQ ID NO: 2 or equivalent sequence of residues in parent glucoamylase, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO: 2 or equivalent sequence of residues in parent glucoamylase, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO:2 or equivalent sequence of residues in parent glucoamylase, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO: 2 or equivalent sequence of residues in parent glucoamylase, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO:2 or equivalent sequence of residues in parent

glucoamylase. In a further aspect, the use is described of a glucoamylase variant wherein said two or more amino acid substitutions are relative to the interconnecting loop 2' with the amino acid sequence from position 518 to position 543 of SEQ ID NO:2, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO:2, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO:2, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO: 2, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO:2.

In a further aspect, the two or more amino acid substitutions are at least one such as one, two or three amino acid substitution in the interconnecting loop 2' and at least one such as one, two, three, four, five or six amino acid substitution in loop 1 and/or helix 2 and/or loop 11 and/or helix 12.

In a further aspect, the two or more amino acid substitutions are one, two, three or four amino acid substitutions in the interconnecting loop 2' and one, two, three or four amino acid substitutions in loop 1 and/or helix 2 and/or loop 11 and/or helix 12. In a further aspect, there are one, two, three or four amino acid substitutions in the interconnecting loop 2'. In a further aspect, there are one, two, three or four amino acid substitutions in loop 1. In a further aspect, there are one, two, three or four amino acid substitutions in helix 2. In a further aspect, there are one, two, three or four amino acid substitutions in loop 11. In a further aspect, there are one, two, three or four amino acid substitutions in helix 12. In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in loop 1.

In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in helix 2.

In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in loop 11.

In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in helix 12.

In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in loop 1 and at least one amino acid substitution in helix 2. In a further aspect, the glucoamylase variant has at least one amino acid substitution within position 520-543, 530-543, or 534-543 of interconnecting loop 2', the positions

corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase. In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 30-50, 35-48, or 40-46 of loop 1, the positions corresponding to the respective position in SEQ ID NO: 2 or equivalent positions in a parent glucoamylase.

In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 50-66, 55-64, or 58-63 of helix 2, the positions corresponding to the respective position in SEQ ID NO: 2 or equivalent positions in a parent glucoamylase.

In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 405-420, 410-420, or 415-420 of loop 11, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase.

In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 421-434, 425-434, or 428-434 of helix 12, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase.

In a further aspect, the glucoamylase variant has at least 80%, 85%, 90%, 95%, 98%, or 99.5% sequence identity to the parent glucoamylase, such as at least 80%, 85%, 90%, 95%, 98%, or 99.5% sequence identity to SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9. In one aspect, the glucoamylase variant has at least 80%, 85%, 90%, 95%, 98%, or 99.5% sequence identity to SEQ ID NO:2.

In a further aspect, the parent glucoamylase or the glucoamylase variant has a starch binding domain that has at least 96%, 97%, 98%, 99%, or 99.5% sequence identity with the starch binding domain of SEQ ID NO: 1, 2, 11, 385, 386, 387, 388, 389, or 390. In a further aspect, the parent glucoamylase or the glucoamylase variant has a catalytic domain that has at least 80%, 85%, 90%, 95%, or 99.5% sequence identity with the catalytic domain of SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9. In one aspect, the glucoamylase variant has an amino acid substitution in position 539 and one or more amino acid substitutions in a position selected from position 44, 61, 417 and 431, the positions corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase. In one aspect, the glucoamylase variant has an amino acid substitution in position 539 and a) an amino acid substitution in position 44 and/or b) amino acid substitutions in both positions 417 and 431, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the glucoamylase variant has an amino acid substitution in position 539 and an amino acid substitution in position 44, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the glucoamylase variant has an amino acid substitution in position 539 and amino acid substitutions in positions 417 and 431, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the glucoamylase variant has an amino acid substitution in position 539 and amino acid substitutions in positions 44 and 61, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the glucoamylase variant has an amino acid substitution in position 43, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the glucoamylase variant has an amino acid substitution in position 61, the position

corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 539 is 539R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 44 is 44R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 417 is 417R/V, the position corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 417 is 417R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 417 is 417V, the position corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase.. In one aspect, the amino acid substitution in position 431 is 431L, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 43 is 43R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 61 is 611, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the condensation product is isomaltose. In one aspect, the hydrolysis of starch is in a brewing process. In for example brewing, the formation of isomaltose is undesired because it can not be converted into alcohol during fermentation.

Beer is traditionally referred to as an alcoholic beverage derived from malt, such as malt derived from barley, and optionally adjuncts, such as cereal grains, and flavoured with hops.

Beer can be made from a variety of grains by essentially the same process. All grain starches are glucose homopolymers in which the glucose residues are linked by either alpha-1, 4- or alpha-l,6-bonds, with the former predominating.

The process of making fermented malt beverages is commonly referred to as brewing. The principal raw materials used in making these beverages are water, hops and malt. In addition, adjuncts such as common corn grits, refined corn grits, brewer's milled yeast, rice, sorghum, refined corn starch, barley, barley starch, dehusked barley, wheat, wheat starch, torrified cereal, cereal flakes, rye, oats, potato, tapioca, and syrups, such as corn syrup, sugar cane syrup, inverted sugar syrup, barley and/or wheat syrups, and the like may be used as a source of starch. The starch will eventually be converted into dextrins and fermentable sugars.

For a number of reasons, the malt, which is produced principally from selected varieties of barley, is believed to have the greatest effect on the overall character and quality of the beer. First, the malt is the primary flavouring agent in beer. Second, the malt provides the major portion of the fermentable sugar. Third, the malt provides the proteins, which will contribute to the body and foam character of the beer. Fourth, the malt provides the necessary enzymatic activity during mashing.

Hops also contribute significantly to beer quality, including flavouring. In particular, hops (or hops constituents) add desirable bittering substances to the beer. In addition, the hops act as protein precipitants, establish preservative agents and aid in foam formation and

stabilization.

The process for making beer is well known in the art, but briefly, it involves five steps: (a) mashing and/or adjunct cooking (b) wort separation and extraction (c) boiling and hopping of wort (d) cooling, fermentation and storage, and (e) maturation, processing and packaging. Typically, in the first step, milled or crushed malt is mixed with water and held for a period of time under controlled temperatures to permit the enzymes present in the malt to convert the starch present in the malt into fermentable sugars. In the second step, the mash is transferred to a "lauter tun" or mash filter where the liquid is separated from the grain residue. This sweet liquid is called "wort" and the left over grain residue is called "spent grain". The mash is typically subjected to an extraction, which involves adding water to the mash in order to recover the residual soluble extract from the spent grain.

In the third step, the wort is boiled vigorously. This sterilizes the wort and helps to develop the colour, flavour and odour and inactivates enzyme activities. Hops are added at some point during the boiling.

In the fourth step, the wort is cooled and transferred to a fermentor, which either contains the yeast or to which yeast is added. The yeast converts the sugars by fermentation into alcohol and carbon dioxide gas; at the end of fermentation the fermentor is chilled or the fermentor may be chilled to stop fermentation. The yeast flocculates and is removed.

In the last step, the beer is cooled and stored for a period of time, during which the beer clarifies and its flavour develops, and any material that might impair the appearance, flavour and shelf life of the beer settles out. Prior to packaging, the beer is carbonated and, optionally, filtered and pasteurized.

After fermentation, a beverage is obtained which usually contains from about 2% to about 10% alcohol by weight. The non-fermentable carbohydrates are not converted during fermentation and form the majority of the dissolved solids in the final beer. This residue remains because of the inability of malt amylases to hydrolyze the alpha- 1,6- linkages of the starch. The non-fermentable carbohydrates contribute about 50 calories per 12 ounces of beer.

Further information on conventional brewing processes, as well as definitions for terms used in the field of brewing technology to be applied for the present invention, may be found in "Technology Brewing and Malting" by Wolfgang Kunze of the Research and Teaching Institute of Brewing, Berlin (VLB), 2nd revised Edition 1999, ISBN 3-921690-39-0 or 3rd edition (2004) : ISBN 3-921690-49-8.

Recently, there has been a widespread popularization of brewed beverages called light beers, reduced calorie beers or low calorie beers, particularly in the U. S. market. As defined in the U. S., these beers have approximately 30% fewer calories than a manufacturer's "normal" beer. As used herein, the term "light beers, reduced calorie beers or low calorie beers", refers to the recent, widespread popularization of brewed beverages, particularly in the U. S. market. As defined in the U. S., these highly attenuated beers have approximately 30% fewer calories than a manufacturer's "normal beer". Further information on conventional brewing processes may be found in "Technology Brewing and Malting" by Wolfgang Kunze of the Research and Teaching Institute of Brewing, Berlin (VLB), 3rd completely updated edition, 2004, ISBN 3- 921690-49-8."

Disclosed herein is the use of a glucoamylase variant as described herein, wherein the production of fermentable sugar(s) is enhanced as compared to the parent glucoamylase, such as TrGA. Further disclosed herein is the use of a glucoamylase variant as described herein, wherein the production of fermentable sugars is enhanced in a mashing step of the brewing process as compared to the parent glucoamylase, such as TrGA. Disclosed herein is the use of a glucoamylase variant as described herein, wherein the production of fermentable sugars is enhanced in a fermentation step of a brewing process as compared to the parent glucoamylase, such as TrGA. Disclosed herein is the use of a glucoamylase variant as described herein, wherein the fermentable sugar is glucose.

A glucoamylase that can produce glucose with a significantly reduced amount of by-products would be of great commercial interest, e.g. in production of glucose syrup or in brewing. Further disclosed herein is the use of a glucoamylase variant as described herein, wherein the hydrolysis of starch is in a process for producing glucose syrup. In one aspect, the glucoamylase exhibit a reduced ratio between isomaltose synthesis (IS) and starch hydrolysis activity (SH) as compared to the parent glucoamylase, such as TrGA. In one aspect, the glucoamylase exhibit a reduced starch hydrolysis activity, which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase, such as TrGA. In one aspect, the glucoamylase exhibit an enhanced real degree of fermentation as compared to the parent glucoamylase such as TrGA. In one aspect, the glucoamylase forms a lower amount of condensation products than the amount of condensation products formed by the glucoamylase Aspergillus niger (AnGA) (SEQ ID NO: 6) under comparable conditions. In one aspect, the glucoamylase forms an amount of condensation products which amount is essentially the same as, not more than 5% higher, not more than 8% higher or not more than 10% higher than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under comparable conditions. In one aspect, dosing of the glucoamylases are the same based on protein concentration. In one aspect, dosing of the glucoamylases are the same based on measurement of activity in activity assays. Glucoamylase variants described herein contain amino acid substitutions within the catalytic domain and/or the starch binding domain. The variants may display altered properties such as improved thermostability, altered formation of condensation products such as isomaltose and/or an enhanced real degree of fermentation and/or a reduced ratio between isomaltose synthesis (IS) and starch hydrolysis activity (SH) and/or specific activity. The variants with reduced formation of condensation products such as isomaltose may significantly improve the ability to make desired products in the brewing industri, for example.

1. Definitions and Abbreviations

1.1. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton et al.. Dictionary Of Microbiology And Molecular Biology, 2nd ed., John Wiley and Sons, New York ( 1994), and Hale & Markham, The Harper Collins Dictionary Of Biology, Harper Perennial, N.Y. (1991) provide one of skill with the general meaning of many of the terms used herein. Certain terms are defined below for the sake of clarity and ease of reference.

As used herein, the term "glucoamylase (EC 3.2.1.3)" refers to an enzyme that catalyzes the release of D-glucose from the non-reducing ends of starch and related oligo- and

polysaccharides. The term "parent" or "parent sequence" refers to a sequence that is native or naturally occurring in a host cell. Parent glucoamylases include, but are not limited to, the

glucoamylase sequences set forth in SEQ ID NOs: 1, 2, 3, 5, 6, 7, 8, and 9, and

glucoamylases with at least 80% amino acid sequence identity to SEQ ID NO: 2.

As used herein, an "equivalent position" means a position that is common to two parent sequences that is based on an alignment of the amino acid sequence of the parent glucoamylase in question as well as alignment of the three-dimensional structure of the parent glucoamylase in question with the TrGA reference glucoamylase amino acid sequence (SEQ ID NO: 2) and three-dimensional structure. Thus either sequence alignment or structural alignment may be used to determine equivalence. The term "TrGA" refers to a parent Trichoderma reesei glucoamylase sequence having the mature protein sequence illustrated in SEQ ID NO: 2 that includes the catalytic domain having the sequence illustrated in SEQ ID NO: 3. The isolation, cloning and expression of the TrGA are described in WO 2006/060062 and U.S. Patent No. 7,413,887, both of which are incorporated herein by reference. In some embodiments, the parent sequence refers to a glucoamylase sequence that is the starting point for protein engineering. The numbering of the glucoamylase amino acids herein is based on the sequence alignment of a glucoamylase with TrGA (SEQ ID NO: 2 and/or 3).

The phrase "mature form of a protein or polypeptide" refers to the final functional form of the protein or polypeptide. A mature form of a glucoamylase may lack a signal peptide, for example. To exemplify, a mature form of the TrGA includes the catalytic domain, linker region and starch binding domain having the amino acid sequence of SEQ ID NO: 2. As used herein, the terms "glucoamylase variant" and "variant" are used in reference to glucoamylases that have some degree of amino acid sequence identity to a parent glucoamylase sequence. A variant is similar to a parent sequence, but has at least one substitution, deletion or insertion in their amino acid sequence that makes them different in sequence from a parent glucoamylase. In some cases, variants have been manipulated and/or engineered to include at least one substitution, deletion, or insertion in their amino acid sequence that makes them different in sequence from a parent. Additionally, a glucoamylase variant may retain the functional characteristics of the parent glucoamylase, e.g., maintaining a glucoamylase activity that is at least about 50%, about 60%, about 70%, about 80%, or about 90% of that of the parent glucoamylase. Can also have higher activity than 100% if that is what one has selected for.

"Variants" may have at least about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to a parent polypeptide sequence when optimally aligned for comparison. In some embodiments, the glucoamylase variant may have at least about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to the catalytic domain of a parent glucoamylase. In some embodiments, the glucoamylase variant may have at least at least about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to the starch binding domain of a parent

glucoamylase. The sequence identity can be measured over the entire length of the parent or the variant sequence. Sequence identity is determined using standard techniques known in the art (see e.g., Smith and Waterman, Adv. Appl. Math. 2 : 482 (1981); Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988); programs such as GAP, BESTHT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); and Devereux el al., Nucleic Acid Res. , 12: 387- 395 ( 1984)).

The "percent (%) nucleic acid sequence identity" or "percent (%) amino acid sequence identity" is defined as the percentage of nucleotide residues or amino acid residues in a candidate sequence that are identical with the nucleotide residues or amino acid residues of the starting sequence (e.g., SEQ ID NO 2). The sequence identity can be measured over the entire length of the starting sequence.

"Sequence identity" is determined herein by the method of sequence alignment. For the purpose of the present disclosure, the alignment method is BLAST described by Altschul et al., (Altschul et al., J. Mol. Biol. 215: 403-410 ( 1990); and Karlin et al, Proc. Natl. Acad. Sci. USA 90 : 5873-5787 (1993)). A particularly useful BLAST program is the WU-BLAST-2 program (see Altschul et al, Meth. Enzymol. 266: 460-480 ( 1996)) . WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. However, the values may be adjusted to increase sensitivity. A % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).

The term "optimal alignment" refers to the alignment giving the highest percent identity score.

As used herein the term "catalytic domain" refers to a structural region of a polypeptide, which contains the active site for substrate hydrolysis.

The term "linker" refers to a short amino acid sequence generally having between 3 and 40 amino acids residues that covalently bind an amino acid sequence comprising a starch binding domain with an amino acid sequence comprising a catalytic domain. The term "starch binding domain" refers to an amino acid sequence that binds preferentially to a starch substrate.

As used herein, the terms "mutant sequence" and "mutant gene" are used interchangeably and refer to a polynucleotide sequence that has an alteration in at least one codon occurring in a host cell's parent sequence. The expression product of the mutant sequence is a variant protein with an altered amino acid sequence relative to the parent. The expression product may have an altered functional capacity (e.g. , enhanced enzymatic activity) .

The term "property" or grammatical equivalents thereof in the context of a polypeptide, as used herein, refers to any characteristic or attribute of a polypeptide that can be selected or detected . These properties include, but are not limited to oxidative stability, substrate specificity, catalytic activity, thermal stability, pH activity profile, resistance to proteolytic degradation, K M , K C AT, K C AT/K m ratio, protein folding, ability to bind a substrate and ability to be secreted .

The term "property" or grammatical equivalent thereof in the context of a nucleic acid, as used herein, refers to any characteristic or attribute of a nucleic acid that can be selected or detected. These properties include, but are not limited to, a property affecting gene transcription (e.g. , promoter strength or promoter recognition), a property affecting RNA processing (e.g. , RNA splicing and RNA stability), a property affecting translation (e.g. , regulation, binding of mRNA to ribosomal proteins) . The terms "thermally stable" and "thermostable" refer to glucoamylase variants of the present disclosure that retain a specified amount of enzymatic activity after exposure to a temperature over a given period of time under conditions prevailing during the hydrolysis of starch substrates, for example, while exposed to altered temperatures.

The term "enhanced stability" in the context of a property such as thermostability refers to a higher retained starch hydrolytic activity over time as compared to another reference {i. e. , parent) glucoamylase.

The term "diminished stability" in the context of a property such as thermostability refers to a lower retained starch hydrolytic activity over time as compared to another reference glucoamylase. The term "specific activity" is defined as the activity per mg of glucoamylase protein . In some embodiments, the activity for glucoamylase is determined by the ethanol assay described herein and expressed as the amount of glucose that is produced from the starch substrate. In some embodiments, the protein concentration can be determined using the Caliper assay described herein.

The terms "active" and "biologically active" refer to a biological activity associated with a particular protein. It follows that the biological activity of a given protein refers to any biological activity typically attributed to that protein by those skilled in the art. For example, an enzymatic activity associated with a glucoamylase is hydrolytic and, thus an active glucoamylase has hydrolytic activity.

The terms "polynucleotide" and "nucleic acid", used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include, but are not limited to, a single-, double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases.

As used herein, the terms "DNA construct," "transforming DNA" and "expression vector" are used interchangeably to refer to DNA used to introduce sequences into a host cell or organism. The DNA may be generated in vitro by PCR or any other suitable technique(s) known to those in the art. The DNA construct, transforming DNA or recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector, DNA construct or transforming DNA includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In some embodiments, expression vectors have the ability to incorporate and express heterologous DNA fragments in a host cell.

As used herein, the term "vector" refers to a polynucleotide construct designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, cassettes, and the like.

As used herein in the context of introducing a nucleic acid sequence into a cell, the term "introduced" refers to any method suitable for transferring the nucleic acid sequence into the cell. Such methods for introduction include but are not limited to protoplast fusion, transfection, transformation, conjugation, and transduction.

As used herein, the terms "transformed" and "stably transformed" refers to a cell that has a non-native (heterologous) polynucleotide sequence integrated into its genome or as an episomal plasmid that is maintained for at least two generations. As used herein, the terms "selectable marker" and "selective marker" refer to a nucleic acid (e.g. , a gene) capable of expression in host cells that allows for ease of selection of those hosts containing the vector. Typically, selectable markers are genes that confer antimicrobial resistance or a metabolic advantage on the host cell to allow cells containing the exogenous DNA to be distinguished from cells that have not received any exogenous sequence during the transformation.

As used herein, the term "promoter" refers to a nucleic acid sequence that functions to direct transcription of a downstream gene. The promoter, together with other transcriptional and translational regulatory nucleic acid sequences (also termed "control sequences") is necessary to express a given gene. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader {i.e. , a signal peptide), is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. As used herein the term "gene" refers to a polynucleotide (e.g. , a DNA segment), that encodes a polypeptide and includes regions preceding and following the coding regions, as well as intervening sequences (introns) between individual coding segments (exons).

As used herein, "ortholog" and "orthologous genes" refer to genes in different species that have evolved from a common ancestral gene {i.e. , a homologous gene) by speciation.

Typically, orthologs retain the same function during the course of evolution. Identification of orthologs finds use in the reliable prediction of gene function in newly sequenced genomes.

As used herein, "paralog" and "paralogous genes" refer to genes that are related by duplication within a genome. While orthologs retain the same function through the course of evolution, paralogs evolve new functions, even though some functions are often related to the original one. Examples of paralogous genes include, but are not limited to genes encoding trypsin, chymotrypsin, elastase, and thrombin, which are all serine proteinases and occur together within the same species. As used herein, the term "hybridization" refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing, as known in the art.

A nucleic acid sequence is considered to be "selectively hybridizable" to a reference nucleic acid sequence if the two sequences specifically hybridize to one another under moderate to high stringency hybridization and wash conditions. Hybridization conditions are based on the melting temperature (T m ) of the nucleic acid binding complex or probe. For example, "maximum stringency" typically occurs at about T m - 5°C (5°C below the T m of the probe); "high stringency" at about 5-10°C below the T m ; "intermediate stringency" at about 10-20°C below the T m of the probe; and "low stringency" at about 20-25°C below the T m . Functionally, maximum stringency conditions may be used to identify sequences having strict identity or near-strict identity with the hybridization probe; while an intermediate or low stringency hybridization can be used to identify or detect polynucleotide sequence homologs.

Moderate and high stringency hybridization conditions are well known in the art. An example of high stringency conditions includes hybridization at about 42°C in 50% formamide, 5 χ SSC, 5 x Denhardt's solution, 0.5% SDS and 100 pg/ml denatured carrier DNA followed by washing two times in 2 χ SSC and 0.5% SDS at room temperature and two additional times in 0.1 SSC and 0.5% SDS at 42°C. An example of moderate stringent conditions include an overnight incubation at 37°C in a solution comprising 20% formamide, 5 χ SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 χ Denhardt's solution, 10% dextran sulfate and 20 mg/ml denaturated sheared salmon sperm DNA, followed by washing the filters in 1 χ SSC at about 37-50°C. Those of skill in the art know how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

As used herein, "recombinant" includes reference to a cell or vector, that has been modified by the introduction of a heterologous or homologous nucleic acid sequence or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention. In an embodiment of the disclosure, mutated DNA sequences are generated with site saturation mutagenesis in at least one codon. In another embodiment, site saturation mutagenesis is performed for two or more codons. In a further embodiment, mutant DNA sequences have more than about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98% identity with the parent sequence. In alternative embodiments, mutant DNA is generated in vivo using any known mutagenic procedure such as, for example, radiation, nitrosoguanidine, and the like. The desired DNA sequence is then isolated and used in the methods provided herein.

As used herein, "heterologous protein" refers to a protein or polypeptide that does not naturally occur in the host cell. An enzyme is "over-expressed" in a host cell if the enzyme is expressed in the cell at a higher level than the level at which it is expressed in a corresponding wild-type cell.

The terms "protein" and "polypeptide" are used interchangeability herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues are used. The 3-letter code for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

Variants of the disclosure are described by the following nomenclature: [original amino acid residue/position/substituted amino acid residue]. For example, the substitution of leucine for arginine at position 76 is represented as R76L. When more than one amino acid is substituted at a given position, the substitution is represented as 1) Q172C, Q172D or Q172R; 2) Q172C, D, or R, or 3) Q172C/D/R. When a position suitable for substitution is identified herein without a specific amino acid suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position. Where a variant glucoamylase contains a deletion in comparison with other glucoamylases the deletion is indicated with "*". For example, a deletion at position R76 is represented as R76*. A deletion of two or more consecutive amino acids is indicated for example as (76 - 78)*.

A "prosequence" is an amino acid sequence between the signal sequence and mature protein that is necessary for the secretion of the protein. Cleavage of the pro sequence will result in a mature active protein.

The term "signal sequence" or "signal peptide" refers to any sequence of nucleotides and/or amino acids that may participate in the secretion of the mature or precursor forms of the protein. This definition of signal sequence is a functional one, meant to include all those amino acid sequences encoded by the N-terminal portion of the protein gene, which participate in the effectuation of the secretion of protein. They are often, but not universally, bound to the N-terminal portion of a protein or to the N-terminal portion of a precursor protein. The signal sequence may be endogenous or exogenous. The signal sequence may be that normally associated with the protein (e.g. , glucoamylase), or may be from a gene encoding another secreted protein.

The term "precursor" form of a protein or peptide refers to a mature form of the protein having a prosequence operably linked to the amino or carbonyl terminus of the protein. The precursor may also have a "signal" sequence operably linked, to the amino terminus of the prosequence. The precursor may also have additional polynucleotides that are involved in post-translational activity (e.g. , polynucleotides cleaved therefrom to leave the mature form of a protein or peptide).

"Host strain" or "host cell" refers to a suitable host for an expression vector comprising DNA according to the present disclosure.

The terms "derived from" and "obtained from" refer to not only a glucoamylase produced or producible by a strain of the organism in question, but also a glucoamylase encoded by a DNA sequence isolated from such strain and produced in a host organism containing such DNA sequence. Additionally, the term refers to a glucoamylase that is encoded by a DNA sequence of synthetic and/or cDNA origin and that has the identifying characteristics of the glucoamylase in question.

A "derivative" within the scope of this definition generally retains the characteristic hydrolyzing activity observed in the wild-type, native or parent form to the extent that the derivative is useful for similar purposes as the wild-type, native or parent form. Functional derivatives of glucoamylases encompass naturally occurring, synthetically or recombinantly produced peptides or peptide fragments that have the general characteristics of the glucoamylases of the present disclosure.

The term "isolated" refers to a material that is removed from the natural environment if it is naturally occurring. A "purified" protein refers to a protein that is at least partially purified to homogeneity. In some embodiments, a purified protein is more than about 10% pure, about 20% pure, or about 30% pure, as determined by SDS-PAGE. Further aspects of the disclosure encompass the protein in a highly purified form (i.e., more than about 40% pure, about 60% pure, about 80% pure, about 90% pure, about 95% pure, about 97% pure, or about 99% pure), as determined by SDS-PAGE. As used herein, the term, "combinatorial mutagenesis" refers to methods in which libraries of variants of a starting sequence are generated. In these libraries, the variants contain one or several mutations chosen from a predefined set of mutations. In addition, the methods provide means to introduce random mutations that were not members of the predefined set of mutations. In some embodiments, the methods include those set forth in U.S. Patent No. 6,582,914, hereby incorporated by reference. In alternative embodiments, combinatorial mutagenesis methods encompass commercially available kits (e.g. , QuikChange® Multisite, Stratagene, San Diego, CA). As used herein, the term "library of mutants" refers to a population of cells that are identical in most of their genome but include different homologues of one or more genes. Such libraries can be used, for example, to identify genes or operons with improved traits.

As used herein the term "dry solids content (DS or ds)" refers to the total solids of a slurry in % on a dry weight basis. As used herein, the term "initial hit" refers to a variant that was identified by screening a combinatorial consensus mutagenesis library. In some embodiments, initial hits have improved performance characteristics, as compared to the starting gene.

As used herein, the term "improved hit" refers to a variant that was identified by screening an enhanced combinatorial consensus mutagenesis library. As used herein, the term "target property" refers to the property of the starting gene that is to be altered. It is not intended that the present disclosure be limited to any particular target property. However, in some embodiments, the target property is the stability of a gene product (e.g. , resistance to denaturation, proteolysis or other degradative factors), while in other embodiments, the level of production in a production host is altered. Indeed, it is contemplated that any property of a starting gene will find use in the present disclosure. Other definitions of terms may appear throughout the specification.

As used herein, the "process for making beer" may further be applied in the mashing of any grist.

As used herein, the term "grist" refers to any starch and/or sugar containing plant material derivable from any plant and plant part, including tubers (e.g. potatoes), roots (e.g. cassava [Manihot esculenta] roots), stems, leaves and seeds. The grist may comprise grain, such as grain from barley, wheat, rye, oat, corn/maize, rice, milo, millet and sorghum, and e.g. at least 10%, or at least 15%, or at least 25%, or at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w/w) of the grist of the wort is derived from grain. In some embodiments the grist may comprise the starch and/or sugar containing plant material obtained from cassava [Manihot esculenta] roots. The grist may comprise malted grain, such as barley malt. Often, at least 10%, or at least 15%, or at least 25%, or at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w/w) of the grist of the wort is derived from malted grain. The grist may comprise adjunct, such as up to 10%, or at least 10%, or at least 15%, or at least 25%, or at least 35%, or at least 50%, at least 75%, at least 90%, or even 100% (w/w) of the grist of the wort is adjunct. The term "adjunct" is understood as the part of the grist which is not barley malt. The adjunct may be any carbohydrate rich material. In term "adjunct" includes starch and/or sugar containing plant material as e.g. defined above under "grist".

The term "fermentation" means, in the context of brewing, the transformation of sugars in the wort, by enzymes in the brewing yeast, into ethanol and carbon dioxide with the formation of other fermentation by-products.

As used herein the term "malt" is understood as any malted cereal grain, such as barley.

As used herein, the term "malt beverage" includes such foam forming fermented malt beverages as full malted beer, ale, dry beer, near beer, light beer, low alcohol beer, low calorie beer, porter, bock beer, stout, malt liquor, non-alcoholic malt liquor and the like. The term "malt beverages" also includes non-foaming beer and alternative malt beverages such as fruit flavoured malt beverages, e. g. , citrus flavoured, such as lemon-, orange-, lime-, or berry-flavoured malt beverages, liquor flavoured malt beverages, e. g. , vodka-, rum-, or tequila-flavoured malt liquor, or coffee flavoured malt beverages, such as caffeine-flavoured malt liquor, and the like. The term "mash" is understood as aqueous starch slurry, e. g. comprising crushed barley malt, crushed barley, and/or other adjunct or a combination hereof, mixed with water later to be separated into wort + spent grains.

As used herein, the term "wort" refers to the unfermented liquor run-off following extracting the grist during mashing. As used herein, the term "spent grains" refers to the drained solids remaining when the grist has been extracted and the wort separated from the mash.

Included within the term "beer" is any fermented wort, produced by the brewing and fermentation of a starch-containing material, mainly derived from cereal grains, such as malted barley. Wheat, maize, and rice may also be used. As used herein, the term "extract recovery" in the wort is defined as the sum of soluble substances extracted from the grist (malt and adjuncts) expressed in percentage based on dry matter.

As used herein, the term "pasteurization" means heating (e.g. beer) at certain temperatures for certain time intervals. The purpose is normally killing of micro-organisms but

pasteurization can also cause inactivation of enzyme activity. Implementation of

pasteurisation in the brewing process is typically through the use of a flash pasteuriser or tunnel pasteuriser. As used herein, the term "pasteurisation units or PU" refers to a quantitative measure of pasteurisation. One pasteurisation unit ( 1 PU) for beer is defined as a heat retention of one minute at 60 degrees Celsius. One calculates that:

PU = t x 1.393 Λ (Τ - 60), where: t = time, in minutes, at the pasteurisation temperature in the pasteuriser

T = temperature, in degrees Celsius, in the pasteuriser

[ Λ (Τ -60) represents the exponent of (T-60)] Different minimum PU may be used depending on beer type, raw materials and microbial contamination, brewer and perceived effect on beer flavour. Typically, for beer

pasteurisation, 14 - 15 PU are required. Depending on the pasteurising equipment, pasteurisation temperatures are typically in the range of 64 - 72 degrees Celsius with a pasteurisation time calculated accordingly. Further information may be found in "Technology Brewing and Malting" by Wolfgang Kunze of the Research and Teaching Institute of Brewing, Berlin (VLB), 3rd completely updated edition, 2004, ISBN 3-921690-49-8..

As used herein, the term "non-alcoholic beer" or "low-alcohol beer" refers to a beer containing a maximum of 0.1% to 3.5% or 0.1% to 2.5% such as 0.1% to 0.5% alcohol by volume. Non-alcoholic beer is brewed by traditional methods, but during the finishing stages of the brewing process the alcohol is removed by vacuum evaporation, by taking advantage of the different boiling points of water and alcohol.

As used herein, the term "low-calorie beer" or "beer with a low carbohydrate content" is defined as a beer with a carbohydrate content of 1.5 g/100 g or less and with a real degree of fermentation of at least 80%. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods and materials are now described.

As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" includes a plurality of such candidate agents and reference to "the cell" includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior invention.

1.2. Abbreviations GA glucoamylase

GAU glucoamylase unit

wt % weight percent

°C degrees Centigrade

rpm revolutions per minute

H 2 0 water

dH 2 0 deionized water

dIH 2 0 deionized water, Milli-Q filtration

aa or AA amino acid

bp base pair

kb kilobase pair

kD kilodaltons

g or gm grams

g micrograms

mg milligrams μΙ and μί. microliters

ml and ml_ milliliters

mm millimeters

μΠΓΙ micrometer

M molar

mM millimolar

μΜ micromolar

U units

V volts

MW molecular weight

MWCO molecular weight cutoff

sec(s) or s(s) second/seconds

min(s) or m(s) minute/minutes

hr(s) or h(s) hour/hours

DO dissolved oxygen

ABS Absorbance

EtOH ethanol

PSS physiological salt solution

m/v mass/volume

MTP microtiter plate

N Normal

DPI monosaccharides

DP2 disaccharides

DP>3 oligosaccharides, sugars having a degree of polymerization greater than 3

ppm parts per million

SBD starch binding domain

CD catalytic domain

PCR polymerase chain reaction

WT wild-type

2. Parent Glucoamylases

In some embodiments, the present disclosure provides a glucoamylase variant. The glucoamylase variant is a variant of a parent glucoamylase, which may comprise both a catalytic domain and a starch binding domain. In some embodiments, the parent glucoamylase comprises a catalytic domain having an amino acid sequence as illustrated in SEQ ID NO: 1, 2, 3, 5, 6, 7, 8 or 9 or having an amino acid sequence displaying at least about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, or about 99.5% sequence identity with one or more of the amino acid sequences illustrated in SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9. In yet other embodiments, the parent glucoamylase comprises a catalytic domain encoded by a DNA sequence that hybridizes under medium, high, or stringent conditions with a DNA encoding the catalytic domain of a glucoamylase having one of the amino acid sequences of SEQ ID NO: 1, 2 or 3. In some embodiments, the parent glucoamylase comprises a starch binding domain having an amino acid sequence as illustrated in SEQ ID NO 1, 2, 11, 385, 386, 387, 388, 389, or 390, or having an amino acid sequence displaying at least about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, or about 99.5% sequence identity with one or more of the amino acid sequence illustrated in SEQ ID NO 1, 2, 11, 385, 386, 387, 388, 389, or 390. In yet other embodiments, the parent glucoamylase comprises a starch binding domain encoded by a DNA sequence that hybridizes under medium, high, or stringent conditions with a DNA encoding the starch binding domain of a glucoamylase having one of the amino acid sequences of SEQ ID NO: 1, 2, or 11.

Predicted structure and known sequences of glucoamylases are conserved among fungal species (Coutinho et al., 1994, Protein Eng. , 7 :393-400 and Coutinho et al., 1994, Protein Eng. , 7: 749-760). In some embodiments, the parent glucoamylase is a filamentous fungal glucoamylase. In some embodiments, the parent glucoamylase is obtained from a

Tricho erma strain (e.g., T. reesei, T. longibrachiatum, T. strictipilis, T. asperellum, T.

konilangbra and T. hazianum), an Aspergillus strain (e.g. A. niger, A. nidulans, A. kawachi, A. awamori and A. orzyae ), a Talaromyces strain (e.g. T. emersonii, T. thermophilus, and T. duponti ), a Hypocrea strain (e.g. H. gelatinosa , H. orientalis, H. vinosa, and H. citrina), a Fusarium strain (e.g., F. oxysporum , F. roseum, and F. venenatum), a Neurospora strain (e.g., N. crassa) and a Humicola strain (e.g., H. grisea, H. insolens and H. lanuginose), a Penicillium strain (e.g., P. notatum or P. chrysogenum), or a Saccharomycopsis strain (e.g. , S. fibuligera). In some embodiments, the parent glucoamylase may be a bacterial glucoamylase. For example, the polypeptide may be obtained from a gram-positive bacterial strain such as Bacillus (e.g. , B. alkalophilus, B. amyloliquefaciens, B. lentus, B. licheniformis, B.

stearothermophilus, B. subtilis and B. thuringiensis) or a Streptomyces strain (e.g., S.

lividans) . In some embodiments, the parent glucoamylase will comprise a catalytic domain having at least about 80%, about 85%, about 90%, about 93%, about 95%, about 97%, about 98%, or about 99% sequence identity with the catalytic domain of the TrGA amino acid sequence of SEQ ID NO: 3.

In other embodiments, the parent glucoamylase will comprise a catalytic domain having at least about 90%, about 93%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the catalytic domain of the Aspergillus parent glucoamylase of SEQ ID NO: 5 or SEQ ID NO: 6. In yet other embodiments, the parent glucoamylase will comprise a catalytic domain having at least about 90%, about 95%, about 97%, or about 99% sequence identity with the catalytic domain of the Humicola grisea (HgGA) parent glucoamylase of SEQ ID NO: 8.

In some embodiments, the parent glucoamylase will comprise a starch binding domain having at least about 80%, about 85%, about 90%, about 95%, about 97%, or about 98% sequence identity with the starch binding domain of the TrGA amino acid sequence of SEQ ID NO: 1, 2, or 11.

In other embodiments, the parent glucoamylase will comprise a starch binding domain having at least about 90%, about 95%, about 97%, or about 99% sequence identity with the catalytic domain of the Humicola grisea (HgGA) glucoamylase of SEQ ID NO: 385.

In other embodiments, the parent glucoamylase will comprise a starch binding domain having at least about 90%, about 95%, about 97%, or about 99% sequence identity with the catalytic domain of the Thielavia terrestris (TtGA) glucoamylase of SEQ ID NO: 390.

In other embodiments, the parent glucoamylase will comprise a starch binding domain having at least about 90%, about 95%, about 97%, or about 99% sequence identity with the catalytic domain of the Thermomyces lanuginosus (ThGA) glucoamylase of SEQ ID NO: 386.

In other embodiments, the parent glucoamylase will comprise a starch binding domain having at least about 90%, about 95%, about 97%, or about 99% sequence identity with the catalytic domain of the Talaromyces emersoniit (TeGA) glucoamylase of SEQ ID NO: 387. In yet other embodiments, the parent glucoamylase will comprise a starch binding domain having at least about 90%, about 93%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the starch binding domain of the Aspergillus parent glucoamylase of SEQ ID NO: 388 or 389.

In some embodiments, the parent glucoamylase will have at least about 80%, about 85%, about 88%, about 90%, about 93%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the TrGA amino acid sequence of SEQ ID NO: 1 or 2.

In further embodiments, a Trichoderma glucoamylase homologue will be obtained from a Trichoderma or Hypocrea strain. Some typical Trichoderma glucoamylase homologues are described in U.S. Patent No. 7,413,887 and reference is made specifically to amino acid sequences set forth in SEQ ID NOs: 17-22 and 43-47 of the reference. In some embodiments, the parent glucoamylase is TrGA comprising the amino acid sequence of SEQ ID NO: 2, or a Trichoderma glucoamylase homologue having at least about 80%, about 85%, about 88%, about 90%, about 93%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the TrGA sequence (SEQ ID NO: 2). A parent glucoamylase can be isolated and/or identified using standard recombinant DNA techniques. Any standard techniques can be used that are known to the skilled artisan. For example, probes and/or primers specific for conserved regions of the glucoamylase can be used to identify homologs in bacterial or fungal cells (the catalytic domain, the active site, etc.). Alternatively, degenerate PCR can be used to identify homologues in bacterial or fungal cells. In some cases, known sequences, such as in a database, can be analyzed for sequence and/or structural identity to one of the known glucoamylases, including SEQ ID NO:

2, or a known starch binding domains, including SEQ ID NO: 11. Functional assays can also be used to identify glucoamylase activity in a bacterial or fungal cell. Proteins having glucoamylase activity can be isolated and reverse sequenced to isolate the corresponding DNA sequence. Such methods are known to the skilled artisan.

3. Glucoamylase Structural Homology

The central dogma of molecular biology is that the sequence of DNA encoding a gene for a particular enzyme, determines the amino acid sequence of the protein, this sequence in turn determines the three-dimensional folding of the enzyme. This folding brings together disparate residues that create a catalytic center and substrate binding surface and this results in the high specificity and activity of the enzymes in question.

Glucoamylases consist of as many as three distinct structural domains, a catalytic domain of approximately 450 residues that is structurally conserved in all glucoamylases, generally followed by a linker region consisting of between 30 and 80 residues that are connected to a starch binding domain of approximately 100 residues. The structure of the Trichoderma reesei glucoamylase with all three regions intact was determined to 1.8 Angstrom resolution herein (see Table 20 in WO2009/067218 (Danisco US Inc., Genencor Division) page 94-216 incorporated herein by reference and Example 11 in WO2009/067218 (Danisco US Inc., Genencor Division) page 89-93 incorporated herein by reference ). Using the coordinates (see Table 20 in WO2009/067218 (Danisco US Inc., Genencor Division) page 94-216 incorporated herein by reference), the structure was aligned with the coordinates of the catalytic domain of the glucoamylase from Aspergillus awamori strain X100 that was determined previously (Aleshin, A.E., Hoffman, C, Firsov, L.M ., and Honzatko, R.B. Refined crystal structures of glucoamylase from Aspergillus awamori var. X100. J. Mol. Biol. 238: 575-591 ( 1994)). The Aspergillus awamori crystal structure only included the catalytic domain. As seen in FIGs. 6-7, the structure of the catalytic domains overlap very closely, and it is possible to identify equivalent residues based on this structural superposition. It is believed that all glucoamylases share the basic structure depicted in FIGs. 6-7.

The catalytic domain of TrGA thus has approximately 450 residues such as residues 1-453 of TrGA SEQ ID NO:2 and is a twelve helix double barrel domain. The helices and loops of the catalytic domain can be defined in terms of the residues of TrGA with SEQ ID NO:2 forming them : helix 1 residues 2-20,

loop 1 residues 21-51,

helix 2 residues 52-68,

loop 2 residues 69-71,

helix 3 residues 72-90,

loop 3 residues 91-125,

helix 4 residues 126-145,

loop 4 residues 146,

helix 5 residues 147-169,

helix 6 residues 186-206,

loop 6 residues 207-210,

helix 7 residues 211-227,

loop 7 residues 211-227,

helix 8 residues 250-275,

loop 8 residues 260-275,

helix 9 residues 276-292,

loop 9 residues 293-321,

helix 10 residues 322-342,

loop 10 residues 343-371,

helix 11 residues 372-395,

loop 11 residues 396-420,

helix 12 residues 421-434,

loop 12 residues 435-443,

helix 13 residues 444-447,

loop 13 residues 448-453

The linker domain has between 30 and 80 residues such as residues 454-490 of TrGA with SEQ ID NO: 2. The starch binding domain of TrGA has approximately 100 residues such as residues 496-596 of TrGA with SEQ ID NO: 2 consisting of the beta sandwich composed of two twisted three stranded sheets. The sheets, helices and loops of the starch binding domain can be defined in terms of the residues of TrGA with SEQ ID NO: 2 forming them : sheet 1' residues 496-504,

loop 1' residues 505-511,

sheet 2' residues 512-517,

interconnecting loop 2' residues 518-543,

sheet 3' residues 544-552,

loop 3' residues 553,

sheet 4' residues 554-565,

loop 4' residues 566-567,

sheet 5' residues 568-572,

inter-sheet segment residues 573-577,

sheet 5a' residues 578-582,

loop 5' residues 583-589,

sheet 6' residues 590-596,

It is possible to identify equivalent residues based on structural superposition in other glucoamylases as described in further detail below.

FIG. 6 is a comparison of the three dimensional structures of the Trichoderma reesei glucoamylase (black) of SEQ ID NO: 2 and of Aspergillus awamorii glucoamylase (grey) viewed from the side. In this view, the relationship between the catalytic domain and the linker region and the starch binding domain can be seen. FIG. 7 is a comparison of the three dimensional structures of the Trichoderma reesei glucoamylase (black) of SEQ ID NO: 2 and of Aspergillus awamorii glucoamylase (grey) viewed from the top. The glucoamylases shown here and indeed all known glucoamylases to date share this structural homology. The conservation of structure correlates with the conservation of activity and a conserved mechanism of action for all glucoamylases. Given this high homology, changes resulting from site specific variants of the Trichoderma glucoamylase resulting in altered functions would also have similar structural and therefore functional consequences in other glucoamylases. Therefore, the teachings of which variants result in desirable benefits can be applied to other glucoamylases.

A further crystal structure was produced using the coordinates in Table 20 in

WO2009/067218 (Danisco US Inc., Genencor Division) page 94-216 incorporated herein by reference for the Starch Binding Domain (SBD). The SBD for TrGA was aligned with the SBD for A. niger. As shown in FIG. 8, the structure of the A. niger and TrGA SBDs overlaps very closely. It is believed that while all starch binding domains share at least some of the basic structure depicted in FIG. 8, some SBDs are more structurally similar than others. For example, the TrGA SBD can be classified as within the carbohydrate binding module 20 family within the CAZY database (cazy.org) . The CAZY database describes the families of structurally-related catalytic and carbohydrate-binding modules (or functional domains) of enzymes that degrade, modify, or create glycosidic bonds. Given a high structural homology, site specific variants of the TrGA SBD resulting in altered function would also have similar structural and therefore functional consequences in other glucoamylases having SBDs with similar structure to that of the TrGA SBD, particularly those classified within the carbohydrate binding module 20 family. Thus, the teachings of which variants result in desirable benefits can be applied to other SBDs having structural similarity.

Thus, the amino acid position numbers discussed herein refer to those assigned to the mature Trichoderma reesei glucoamylase sequence presented in FIG. 1 (SEQ ID NO: 2). The present disclosure, however, is not limited to the variants of Trichoderma glucoamylase, but extends to glucoamylases containing amino acid residues at positions that are "equivalent" to the particular identified residues in Trichoderma reesei glucoamylase (SEQ ID NO: 2). In some embodiments of the present disclosure, the parent glucoamylase is a Talaromyces GA and the substitutions are made at the equivalent amino acid residue positions in Talaromyces glucoamylase (see e.g., SEQ ID NO: 12) as those described herein. In other embodiments, the parent glucoamylase comprises SEQ ID NOs: 5-9 (see FIGs. 5A and 5B). In further embodiments, the parent glucoamylase is a Penicillium glucoamylase, such as Penicillium chrysogenum (see e.g., SEQ ID NO: 13). "Structural identity" determines whether the amino acid residues are equivalent. Structural identity is a one-to-one topological equivalent when the two structures (three dimensional and amino acid structures) are aligned. A residue (amino acid) position of a glucoamylase is "equivalent" to a residue of T. reesei glucoamylase if it is either homologous {i.e. ,

corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in 7 " . reesei glucoamylase (having the same or similar functional capacity to combine, react, or interact chemically) .

In order to establish identity to the primary structure, the amino acid sequence of a glucoamylase can be directly compared to Trichoderma reesei glucoamylase primary sequence and particularly to a set of residues known to be invariant in glucoamylases for which sequence is known. For example, FIGs. 5A and 5B herein show the conserved residues between glucoamylases. FIGs. 5D and 5E show an alignment of starch binding domains from various glucoamylases. After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment {i.e. avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of Trichoderma reesei glucoamylase are defined. Alignment of conserved residues typically should conserve 100% of such residues. However, alignment of greater than about 75% or as little as about 50% of conserved residues is also adequate to define equivalent residues. Further, the structural identity can be used in combination with the sequence identity to identify equivalent residues.

For example, in FIGs. 5A and 5B, the catalytic domains of glucoamylases from six organisms are aligned to provide the maximum amount of homology between amino acid sequences. A comparison of these sequences shows that there are a number of conserved residues contained in each sequence as designated by an asterisk. These conserved residues, thus, may be used to define the corresponding equivalent amino acid residues of Trichoderma reesei glucoamylase in other glucoamylases such as glucoamylase from Aspergillus niger. Similarly, FIGs. 5D and 5E show the starch binding domains of glucoamylases from seven organisms aligned to identify equivalent residues.

Structural identity involves the identification of equivalent residues between the two structures. "Equivalent residues" can be defined by determining homology at the level of tertiary structure (structural identity) for an enzyme whose tertiary structure has been determined by X-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the Trichoderma reesei glucoamylase (N on N, CA on CA, C on C and O on O) are within 0.13 nm and optionally 0.1 nm after alignment. In one aspect, at least 2 or 3 of the four possible main chain atoms are within 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the glucoamylase in question to the

Trichoderma reesei glucoamylase. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available.

R factor =

Equivalent residues that are functionally analogous to a specific residue of Trichoderma reesei glucoamylase are defined as those amino acids of the enzyme that may adopt a conformation such that they either alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the Trichoderma reesei glucoamylase. Further, they are those residues of the enzyme (for which a tertiary structure has been obtained by X-ray crystallography) that occupy an analogous position to the extent that, although the main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of Trichoderma reesei glucoamylase. The coordinates of the three dimensional structure of Trichoderma reesei glucoamylase are set forth in Table 20 in WO2009/067218 (Danisco US Inc., Genencor Division) page 94-216 incorporated herein by reference and can be used as outlined above to determine equivalent residues on the level of tertiary structure.

Some of the residues identified for substitution are conserved residues whereas others are not. In the case of residues that are not conserved, the substitution of one or more amino acids is limited to substitutions that produce a variant that has an amino acid sequence that does not correspond to one found in nature. In the case of conserved residues, such substitutions should not result in a naturally-occurring sequence.

4. Glucoamylase Variants

The variants according to the disclosure include at least one substitution, deletion or insertion in the amino acid sequence of a parent glucoamylase that makes the variant different in sequence from a parent glucoamylase. In some embodiments, the variants of the disclosure will have at least about 20%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 97%, or about 100% of the glucoamylase activity as that of the TrGA (SEQ ID NO: 2), a parent glucoamylase that has at least 80% sequence identity to TrGA (SEQ ID NO: 2). In some embodiments, the variants according to the disclosure will comprise a substitution, deletion or insertion in at least one amino acid position of the parent TrGA (SEQ ID NO: 2), or in an equivalent position in the sequence of another parent glucoamylase having at least about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% sequence identity to the TrGA sequence (SEQ ID NO: 2). In other embodiments, the variant according to the disclosure will comprise a substitution, deletion or insertion in at least one amino acid position of a fragment of the parent TrGA, wherein the fragment comprises the catalytic domain of the TrGA sequence (SEQ ID NO: 3) or in an equivalent position in a fragment comprising the catalytic domain of a parent glucoamylase having at least about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% sequence identity to the catalytic-domain-containing fragment of the SEQ ID NO: 3, 5, 6, 7, 8, or 9. In some embodiments, the fragment will comprise at least about 400, about 425, about 450, or about 500 amino acid residues of TrGA catalytic domain (SEQ ID NO: 3).

In other embodiments, the variant according to the disclosure will comprise a substitution, deletion or insertion in at least one amino acid position of a fragment of the parent TrGA, wherein the fragment comprises the starch binding domain of the TrGA sequence (SEQ ID NO: 11) or in an equivalent position in a fragment comprising the starch binding domain of a parent glucoamylase having at least about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% sequence identity to the starch-binding-domain-containing fragment of SEQ ID NO: 11, 385, 386, 387, 388, 389, and 390. In some embodiments, the fragment will comprise at least about 40, about 50, about 60, about 70, about 80, about 90, about 100, or about 109 amino acid residues of TrGA starch binding domain (SEQ ID NO: 11).

In some embodiments, when the parent glucoamylase includes a catalytic domain, a linker region, and a starch binding domain, the variant will comprise a substitution, deletion or insertion in at least one amino acid position of a fragment comprising part of the linker region. In some embodiments, the variant will comprise a substitution deletion, or insertion in the amino acid sequence of a fragment of the TrGA sequence (SEQ ID NO: 2).

Structural identity with reference to an amino acid substitution means that the substitution occurs at the equivalent amino acid position in the homologous glucoamylase or parent glucoamylase. The term equivalent position means a position that is common to two parent sequences that is based on an alignment of the amino acid sequence of the parent glucoamylase in question as well as alignment of the three-dimensional structure of the parent glucoamylase in question with the TrGA reference glucoamylase amino acid sequence and three-dimensional sequence. For example, with reference to FIG. 5A, position 24 in TrGA (SEQ ID NO: 2 or 3) is D24 and the equivalent position for Aspergillus niger (SEQ ID NO: 6) is position D25, and the equivalent position for Aspergillus oryzea (SEQ ID NO: 7) is position D26. See FIGs. 6 and 7 for an exemplary alignment of the three-dimensional sequence.

Accordingly, in one aspect, a glucoamylase variant is described, which glucoamylase variant when in its crystal form has a crystal structure for which the atomic coordinates of the main chain atoms have a root-mean-square deviation from the atomic coordinates of the equivalent main chain atoms of TrGA (as defined in Table 20 in WO2009/067218) of less than 0.13 nm following alignment of equivalent main chain atoms, and which have a linker region, a starch binding domain and a catalytic domain, said variant comprising two or more amino acid substitutions relative to the amino acid sequence of the parent glucoamylase in interconnecting loop 2' of the starch binding domain, and/or in loop 1, and/or in helix 2, and/or in loop 11, and/or in helix 12 of the catalytic domain. In a further aspect, the root- mean-square deviation from the atomic coordinates of the equivalent main chain atoms of TrGA (as defined in Table 20 in WO2009/067218) is less than 0.12 nm, such as less than 0.11 or such as less than 0.10.

In one aspect, a glucoamylase variant is described, which glucoamylase variant comprises a starch binding domain and a catalytic domain, said variant comprising two or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO:2 or equivalent parent glucoamylase in interconnecting loop 2', and/or in loop 1, and/or in helix 2, and/or in loop 11, and/or in helix 12 for reducing the synthesis of condensation products during hydrolysis of starch.

In a further aspect, a glucoamylase variant is described, which glucoamylase variant comprises two or more amino acid substitutions relative to interconnecting loop 2' with the amino acid sequence from position 518 to position 543 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase.

In a further aspect, a glucoamylase variant is described, which glucoamylase variant comprises two or more amino acid substitutions relative to the amino acid sequence from position 518 to position 543 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 21 to position 51 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 52 to position 68 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 396 to position 420 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 421 to position 434 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase.

In one aspect, the two or more amino acid substitutions are relative to the interconnecting loop 2' with the amino acid sequence from position 518 to position 543 e.g. in one or more of positions 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542 and/or 543 of SEQ ID NO: 2, and/or loop 1 with the amino acid sequence from position 21 to position 51 e.g. in one or more of positions 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 and/or 51 of SEQ ID NO:2, and/or helix 2 with the amino acid sequence from position 52 to position 68 e.g. in one or more of positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and/or 68 of SEQ ID NO: 2, and/or loop 11 with the amino acid sequence from position 396 to position 420 e.g. in one or more of positions 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419 and/or 420 of SEQ ID NO: 2, and/or helix 12 with the amino acid sequence from position 421 to position 434 e.g. in one or more of positions 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433 and/or 534 of SEQ ID NO:2.

In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in the interconnecting loop 2' and at least one amino acid substitution in loop 1 and/or helix 2 and/or loop 11 and/or helix 12. In a further aspect, the two or more amino acid substitutions are 1, 2, 3 or 4 amino acid substitutions in the interconnecting loop 2' and 1, 2, 3 or 4 amino acid substitutions in loop 1 and/or helix 2 and/or loop 11 and/or helix 12.

In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in loop 1. In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in helix 2. In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in loop 11. In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in helix 12. In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in loop 1 and at least one amino acid substitution in helix 2. In a further aspect, the glucoamylase variant has at least one amino acid substitution within position 520-543, 530-543, or 534-543 of interconnecting loop 2', the positions corresponding to the respective position in SEQ ID NO: 2 or equivalent positions in a parent glucoamylase. In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 30-50, 35-48, or 40-46 of loop 1, the positions corresponding to the respective position in SEQ ID NO: 2 or equivalent positions in a parent glucoamylase. In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 50-66, 55-64, or 58-63 of helix 2, the positions corresponding to the respective position in SEQ ID NO: 2 or equivalent positions in a parent glucoamylase. In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 405-420, 410-420, or 415-420 of loop 11, the positions corresponding to the respective position in SEQ ID NO: 2 or equivalent positions in a parent glucoamylase. In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 421-434, 425-434, or 428-434 of helix 12, the positions corresponding to the respective position in SEQ ID NO: 2 or equivalent positions in a parent glucoamylase.

In one aspect, the glucoamylase variant comprises two or more amino acid substitutions, wherein an amino acid substitution is in position 539 and an amino acid substitution is in position 44, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase, and which sequence has at least 80% sequence identity to the parent glucoamylase, and wherein the amino acid substitution in position 44 is not 44C.

In a further aspect, the glucoamylase variant comprises two or more amino acid

substitutions, wherein an amino acid substitution is in position 539 and an amino acid substitution is 44R, the positions corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase. In a further aspect, the glucoamylase variant comprises an amino acid substitution in position 61, the position corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase. In a further aspect, the amino acid substitution in position 539 is 539R, the position corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase. In a further aspect, the amino acid substitution in position 44 is 44R, the position

corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase. In a further aspect, the amino acid substitution in position 61 is 611, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.

In a further aspect, the glucoamylase variant comprises the following amino acid

substitutions: a) D44R and A539R; or b) D44R, N61I and A539R, the positions corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase. In a further aspect, the glucoamylase variant consist of SEQ ID NO: 2 and has the following amino acid substitutions: a) D44R and A539R; or b) D44R, N61I and A539R, the positions corresponding to the respective position in SEQ ID NO: 2.

In a further aspect, the glucoamylase variant has a starch binding domain that has at least 96%, 97%, 98%, 99%, or 99.5% sequence identity with the starch binding domain of SEQ ID NO: 1, 2, 11, 385, 386, 387, 388, 389, or 390. In a further aspect, the glucoamylase variant has a catalytic domain that has at least 80%, 85%, 90%, 95%, or 99.5% sequence identity with the catalytic domain of SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9.

In a further aspect, the parent glucoamylase is a fungal glucoamylase.

In a further aspect, the parent glucoamylase is selected from a glucoamylase obtained from a Trichoderma spp. , an Aspergillus spp. , a Humicola spp. , a Penicillium spp. , a Talaromycese spp. , or a Schizosaccharmyces spp. In a further aspect, the parent glucoamylase is obtained from a Trichoderma spp. or an Aspergillus spp.

In a further aspect, the glucoamylase has been purified. The glucoamylases of the present disclosure may be recovered or purified from culture media by a variety of procedures known in the art including centrifugation, filtration, extraction, precipitation and the like. In some embodiments, the glucoamylase variant will include at least two substitutions in the amino acid sequence of a parent. In further embodiments, the variant may have more than two substitutions. For example, the variant may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 amino acid substitutions, deletions, or insertions as compared to a corresponding parent glucoamylase. In some embodiments, a glucoamylase variant comprises a substitution, deletion or insertion, and typically a substitution in at least one amino acid position in a position corresponding to the regions of non-conserved amino acids as illustrated in FIGs. 5A, 5B, 5D, and 5E (e.g. , amino acid positions corresponding to those positions that are not designated by "*" in FIGs. 5A, 5B, 5D, and 5E). While the variants may have substitutions in any position of the mature protein sequence (SEQ ID NO: 2), in some embodiments, a glucoamylase variant comprises two or more substitutions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2 : 23, 42, 43, 44, 59, 60, 61, 65, 67, 68, 410, 417, 418, 430, 431, 433, 518, 519, 520, 527, 531, 535, 536, 537 or 539, or in an equivalent position in a parent glucoamylase. In a further aspect, the glucoamylase variant comprises one or more further substitutions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2: 10, 14, 15, 72, 73, 97, 98, 99, 102, 110, 113, 114, 133, 140, 144, 145, 147, 152, 153, 164, 182, 204, 205, 214, 216, 219, 228, 229, 230, 231, 236, 239, 241, 242, 263, 264, 265, 268, 269, 276, 284, 291, 294, 300, 301, 303, 311, 338, 342, 344, 346, 349, 359, 361, 364, 375, 379, 382, 390, 391, 393, 394, 436, 442, 444, 448, 451, 493, 494, 495, 502, 503, 508, 511, 563, or 577, or in an equivalent position in a parent glucoamylase. In some embodiments, the parent

glucoamylase will have at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO: 2. In other embodiments, the parent glucoamylase will be a Trichoderma

glucoamylase homologue. In some embodiments, the variant will have altered properties. In some embodiments, the parent glucoamylase will have structural identity with the

glucoamylase of SEQ ID NO: 2.

In some embodiments, the glucoamylase variant comprises two or more substitutions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2 : P23, T42, 143, D44, P45, D46, F59, K60, N61, T67, E68, R408, S410, S415, L417, H418, T430, A431, R433, N518, A519, A520, T527, V531, A535, V536, N537, and A539 or an equivalent position in parent glucoamylase (e.g. , a Trichoderma glucoamylase homologue). In a further aspect, the glucoamylase variant comprises one or more substitutions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2: T10, L14, N15, A72, G73, S97, L98, A99, S102, K108, E110, L113, K114, R122, Q124, R125, 1133, K140, N 144, N 145, Y147, S152, N 153, N 164, F175, N 182, A204, T205, S214, V216, Q219, W228, V229, S230, S231, D236, 1239, N240, T241, N242, G244, N263, L264, G265, A268, G269, D276, V284, S291, G294, P300, A301, A303, Y310, A311, D313, Y316, V338, T342, S344, T346, A349, V359, G361, A364, T375, N379, S382, S390, E391, A393, K394, 1436, A442, N443, S444, T448, S451, T493, P494, T495, H502, E503, Q508, Q511, N563, and N577 or in an equivalent position in a parent glucoamylase. In some embodiments, the variant will have altered properties as compared to the parent glucoamylase.

In some embodiments, the glucoamylase variant may differ from the parent glucoamylase only at the specified positions. In further embodiments, the variant of a glucoamylase parent comprises at least two of the following substitutions in the following positions in an amino acid sequence set forth in SEQ ID NO: 2: T42V, I43Q/R, D44R/C, N61I, T67M, E68C/M, L417K/R/V, T430A/K, A431I/L Q, R433C/E/G/L/N/S/V/Y, A519I/K/R/Y, A520C/L/P, V531L, A535K/N/P/R, V536M, or

A539E/R/S, or a substitution in an equivalent position in a parent glucoamylase. In a further aspect, the glucoamylase variant comprises one or more substitutions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2 : TIOS, A72Y, G73F/W, S97N, S102A/M/R, K114M/Q, I133T/V, N 145I, N153A/D/E/ /S/V, T205Q, Q219S, W228A/F/H/M/V, V229I/L, S230C/F/G/L/N/Q/R, S231L/V, D236R, I239V/Y, N263P, L264D/K, A268C/D/G/K, S291A/F/H/M/T, g294c, A301P/R, V338I/N/Q, T342V, S344M/P/Q/R/V,

G361D/E/F/I/L/M/P/S/W/Y, A364D/E/F/G/K/L/M/R/S/T/V/W, T375N, K394S, I436H, T451K, T495K/M/S, E503A/C/V, Q508R, Q511H, N563C/E/I/K/K/Q/T/V, or N577K/P/R, or in an equivalent position in a parent glucoamylase.

In further embodiments, the glucoamylase variant comprises one of the following sets of substitutions, at the relevant positions of SEQ ID NO: 2, or at equivalent positions in a parent glucoamylase:

N61I/L417V/A431L/A539R;

I43Q/N61I/L417V/A431L/A539R;

N611/ L417 V/A431 L/A535 R/A539 R

I43Q/L417V/A431L7A535R/A539R;

I43Q/N61I/L417V/A431L/A535R/A539R;

I43Q/N61I/L417V/T430A/A431L/A535R/A539R;

I43Q/L417V/T430A/A431L7Q511H/A535R/A539R/N563I;

N61I/L417V/T430A/A431I./Q511H/A535R/A539R N563I;

I43Q/N61I/L417V/T430A/A431I7Q511H/A535R/A539R/N563I;

I43R/N61I/L417V/A431L/A539R;

I43R/N61I/L417V/T430A/A431L/A535R/A539R;

G73F/L417R/E503V/A539R N563K;

I43R/G73F/L417R/E503V/A539R N563K; and

I43R/G73F/E503V/Q511H/N563K.

In further embodiments, the glucoamylase variant comprises one of the following sets of substitutions, at positions of SEQ ID NO: 2 or equivalent positions in a parent glucoamylase: L417V/A431L/A539R;

I43Q/L417V/A431L/A539R;

L417V/A431L7A535R/A539R

I43R/L417V/A431L/A539R;

L417R/A431L/A539R; or L417G/A431L7A539R;

wherein the glucoamylase variant does not have any further substitutions relative to the parent glucoamylase, and wherein the parent glucoamylase has a catalytic domain that has at least 80% sequence identity with SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9. Thus the parent glucoamylase may be any of those described elsewhere.

The parent glucoamylase may comprise a starch binding domain that has at least 95% sequence identity with SEQ ID NO: 1, 2, 11, 385, 386, 387, 388, 389, or 390. The parent glucoamylase may have at least 80% sequence identity with SEQ ID NO: 1 or 2; for example it may comprise SEQ ID NO: 1 or 2. Optionally the parent glucoamylase may consist of SEQ ID NO: 1 or 2.

Glucoamylase variants of the disclosure may also include chimeric or hybrid glucoamylases with, for example a starch binding domain (SBD) from one glucoamylase and a catalytic domain and linker from another. For example, a hybrid glucoamylase can be made by swapping the SBD from AnGA (SEQ ID NO: 6) with the SBD from TrGA (SEQ ID NO: 2), making a hybrid with the AnGA SBD and the TrGA catalytic domain and linker. Alternatively, the SBD and linker from AnGA can be swapped for the SBD and linker of TrGA.

In some aspects, the variant glucoamylase exhibits altered thermostability as compared to the parent glucoamylase. In some aspects, the altered thermostability may be increased thermostability as compared to the parent glucoamylase. In some embodiments, the altered property is altered specific activity compared to the parent glucoamylase. In some embodiments, the altered specific activity may be increased specific activity compared to the parent glucoamylase. In some embodiments, the altered property is increased

thermostability at lower temperatures as compared to the parent glucoamylase. In some embodiments, the altered property is both increased specific activity and increased thermostability as compared to the parent glucoamylase.

In one embodiment, some variants may include the substitutions at positions:

D44R N61I/A539R;

D44R/A539R;

I43Q/D44C/L417V/E503A/Q511H/A539R;

I43Q/L417V/E503A/Q511 H/A539R;

I43Q/D44C/N61I/L417V/E503A/Q511H/A539R;

I43Q/N61I/L417V/E503A/Q511H/A539R;

I43R/L417V/E503A/Q511H/A539R;

I43R/N61I/L417V/E503A/Q511H/A539R;

I43R/L417R/E503A/A539R;

I43R/N61I/L417R/E503A/Q511H/A539R;

G73F/T430A/Q511H;

I43R G73F/T430A; G73F/T430A/E503V/Q511H;

D44C/G73F/N563K;

D44C/G73F/E503V/Q511H;

D44C/G73F/N563K;

D44C/G73F/L417R/N563K;

D44C/G73F/N563K;

I43R/T430A;

I43Q/T430A;

I43Q/T430A/Q511H;

D44C/L417R/N563K;

L417V/T430A/A431L/Q511H/A535R/A539R/N563I;

L417V/T430A/A431Q/Q511H/A535R A539R/N563I;

L417V/T430A/Q511H/A535R/N563I;

L417V/T430A/Q511H/A539R/N563I;

G294C/L417R/A431L;

G294C/L417V/A431Q;

G294C/L417V/A431 L/Q511 H ;

G294C/L417R/A431Q/Q511H ;

L417R/A431L/Q511H;

L417V/A431Q/Q511H;

I43Q/T430A/Q511H/N61I;

I43Q/T430A/Q511 H/L417V;

I43Q/T430A/Q511 H/A431L;

I43Q/T430A/Q511 H/E503A;

I43Q/T430A/Q511 H/A539R;

I43Q/T430A/Q511H/N61I/A539R;

I43Q/T430A/Q511H/L417V/A539R;

I43Q/T430A/Q511 H/A431 t_/A539R;

I43Q/T430A/Q511 H/A431 L/E503 A;

I43Q/T430A/Q511 H/N61I/A539R/A431 L;

I43Q/T430A/Q511 H/L417V/A539R/A431 L;

I43Q/Q511H/N61I;

I43Q/Q511H/L417V;

I43Q/Q511H/A431L;

I43Q/Q511H/A539R;

I43Q/Q511H/A539R/N61I;

I43Q/Q511H/A539R/E503A;

I43Q/Q511H/A539R/T430M;

I43Q/Q511H/A539R/T430M/N61I;

I43Q/Q511H/A539R/T430I /N61I/L417V;

I43R/T430A/E503V/A535R/N563K;

D44R/E503A/Q511H/N563I;

E503A/N563I;

I43R/T430A/E503A/Q511H/N563K;

D44R/T430A/Q511H/A535R;

L417V/A431I7A539R;

L417V/A431L/A539R/I43Q;

L417V/A431L7A539R/N61I;

L417V/A431L7A539R/A535R;

L417V/A431L7A539R/I43Q/N61I;

L417V/A431I7A539R/N61I/A535R;

L417V/A431I7A539R/A535R/I43Q;

L417V/A431I7A539R/I43Q/N61I/A535R;

L417V/A431I7A539R/I43Q/N61I/A535R/T430A;

L417V/T430A/A431L/Q511H/A535R/A539R/N563I/I43Q;

L417V/T430A/A431L7Q511H/A535R/A539R/N563I/N61I;

L417V/T430A/A431LyQ511H/A535R A539R/N563I/I43Q/N61I;

L417V/A431LJA539R/I43R;

L417V/A431LJA539R/I43R/N61I; L417V/A431L7A539R/I43R/N61I/A535R/T430A;

L417R/A431I7A539R;

L417G/A431L/A539R;

G73F/E503V/N563K/L417R/A539R;

G73F/E503V/N563K/I43R/L417R A539R; and

G73F/E503V/N563K/I43R/Q511H of SEQ ID NO: 2, or equivalent positions in parent glucoamylases and particularly Trichoderma glucoamylase homologues.

In a further embodiment, some variants may include the substitutions at positions: D44R/N61I/A539R;

D44R/A539R;

I43OJD44C/L417V/E503A/Q511H/A539R;

I43Q/L417V/E503A/Q511H/A539R;

I43Q/D44C/N61I/L417V/E503A/Q511H/A539R;

I43Q/N61I/L417V/E503A/Q511H/A539R;

I43R/L417V/E503A/Q511H/A539R;

I43R/N61I/L417V/E503A/Q511H/A539R;

I43R/L417R E503A/A539R;

I43R/N61I/L417R/E503A/Q511H/A539R;

L417V/T430A/A431L/Q511H/A535R/A539R/N563I;

L417V/T430A/A431Q/Q511H/A535R/A539R/N563I;

L417V/T430A/Q511H/A539R/N563I;

I43OJT430A/Q511H/A539R;

I43QJT430A/Q511H/N61I/A539R;

I43OJT430A/Q511H/L417V/A539R;

I43OJT430A/Q511H/A431L/A539R;

I43OJT430A/Q511H/N61I/A539R A431L;

I43OJT430A/Q511H/L417V/A539R A431L;

I43Q/Q511H/A539R;

I43Q/Q511H/A539R/N61I;

I43OJQ511H/A539R/E503A;

I43OJQ511H/A539R/T430M;

I43OJQ511H/A539R/T430M/N61I;

I43QJQ511H/A539R/T430 /N61I/L417V;

L417V/A431L/A539R;

L417V/A431I7A539R/I43Q;

L417V/A431L/A539R/N61I;

L417V/A431L/A539R/A535R;

L417V/A431L7A539R/I43Q/N61I;

L417V/A431L/A539R/N61I/A535R;

L417V/A431L7A539R/A535R/I43Q;

L417V/A431iyA539R/I430JN61I/A535R;

L417V/A4311JA539R/I43QJN61I/A535R/T430A;

L417V/T430A/A431L/Q511H/A535R/A539R/N563I/I43Q;

L417V/T430A/A431LVQ511H/A535R/A539R N563I/N61I;

L417V/T430A/A431L- Q511H/A535R/A539R N563I/I43OJN61I;

L417V/A431L/A539R/I43R;

L417V/A431I7A539R/I43R/N61I;

L417V/A431iyA539R/I43R/N61I/A535R/T430A;

L417R/A431L/A539R;

L417G/A431L/A539R;

G73F/E503V/N563K/L417R A539R; and G73F/E503V/N563K/I43R/L417R/A539R

of SEQ ID NO: 2, or equivalent positions in parent glucoamylases and particularly Trichoderma glucoamylase homologues. In a further embodiment, some variants may include the substitutions at positions:

D44R/N61I/A539R;

D44R/A539R;

I43Q/D44C/L417V/E503A/Q511H/A539R;

I43OJL417V/E503A/Q511H/A539R;

I43Q/D44C/N61I/L417V/E503A/Q511H/A539R;

I43Q/N61I/L417V/E503A/Q511H/A539R;

I43R/L417V/E503A/Q511H/A539R;

I43R/N61I/L417V/E503A/Q511 H/A539R;

I43R/L417R/E503A/A539R;

I43R/N61I/L417R/E503A/Q511H/A539R;

L417V/T430A/A431L/Q511H/A535R/A539R/N563I;

L417V/T430A/A431Q/Q511H/A535R/A539R/N563I;

L417V/T430A/Q511H/A539R/ 563I;

I43OJT430A/Q511H/A539R;

I43Q/T430A/Q511H/N61I/A539R;

I43OJT430A/Q511H/L417V/A539R;

I43OJT430A/Q511H/A431I7A539R;

I43Q/T430A/Q511H/N61I/A539R/A431L;

I43Q/T430A/Q511H/L417V/A539R/A431L;

I430JQ511H/A539R;

I43Q/Q511H/A539R/N61I;

I43QJQ511H/A539R/E503A;

I43Q/Q511H/A539R/T430M;

I43OJQ511H/A539R/T430M/N61I;

I43OJQ511H/A539R/T430M/N61I/L417V;

L417V/A431I7A539R;

L417V/A431L7A539R/I43Q;

L417V/A431L/A539R/N61I;

L417V/A431L/A539R/A535R;

L417V/A431L/A539R/I43Q/N61I;

L417V/A431L/A539R/N61I/A535R;

L417V/A431L7A539R/A535R/I43Q;

L417V/A431L7A539R/I43Q N61I/A535R;

L417V/A431L/A539R/I43OJN61I/A535R/T430A;

L417V/T430A/A431I7Q511H/A535R/A539R/N563I/I43Q;

L417V/T430A/A431I7Q511H/A535R/A539R/N563I/N61I;

L417V/T430A/A431I7Q511H/A535R/A539R/N563I/I43OJN61I;

L417V/A431L7A539R/I43R;

L417V/A431L7A539R/I43R/N61I;

L417V/A431L7A539R/I43R/N61I/A535R/T430A;

L417R/A431I7A539R;

L417G/A431L7A539R;

G73F/E503V/N563K/L417R A539R; and

G73F/E503V/N563K/I43R L417R/A539R

of SEQ ID NO: 2, or equivalent positions in parent glucoamylases and particularly Trichoderma glucoamylase homologues. In a further embodiment, some variants may include the substitutions at positions:

D44R/N61I/A539R;

D44R A539R;

L417V/A431LJA539R;

L417V/A431U/A539R/I43Q;

L417 V/A431 LV A539 R/ N611 ;

of SEQ ID NO: 2, or equivalent positions in parent glucoamylases and particularly

Trichoderma glucoamylase homologues.

In a further embodiment, some variants may include the substitutions at positions:

D44R/N61I/A539R;

D44R/A539R;

of SEQ ID NO: 2, or equivalent positions in parent glucoamylases and particularly

Trichoderma glucoamylase homologues.

In a further embodiment, some variants has the following substitutions: D44R/N61I/A539R or D44R/A539R of SEQ ID NO: 2.

In a further embodiment, the variant comprises SEQ ID NO: 1098. In yet a further

embodiment, the variant consists of SEQ ID NO: 1098. In a further embodiment, the variant comprises SEQ ID NO: 1099. In yet a further embodiment, the variant consists of SEQ ID NO: 1099.

A number of parent glucoamylases have been aligned with the amino acid sequence of TrGA. Figure 5 includes the catalytic domain of the following parent glucoamylases Aspergillus awamori (AaGA) (SEQ ID NO: 5); Aspergillus nlger (AnGA) (SEQ ID NO: 6); Aspergillus orzyae (AoGA) (SEQ IDNO: 7); Humicola grisea (HgGA) (SEQ ID NO: 8); and Hypocrea vinosa (HvGA) (SEQ ID NO: 9). The % identity of the catalytic domains is represented in Table 1 below.

Table 1: Sequence homology between various fungal glucoamylases

In some embodiments, for example, the variant glucoamylase will be derived from a parent glucoamylase that is an Aspergillus glucoamylase, a Humicola glucoamylase, or a Hypocrea glucoamylase.

5. Characterization of Variant Glucoamylases The present disclosure also provides glucoamylase variants having at least one altered property (e.g., improved property) as compared to a parent glucoamylase and particularly to the TrGA. In some embodiments, at least one altered property (e.g., improved property) is selected from the group consisting of IS/SH-ratio, starch hydrolysis activity, real degree of fermentation, reduced formation of condensation products, acid stability, thermal stability and specific activity. Typically, the altered property is reduced IS/SH-ratio, enhanced real degree of fermentation, reduced formation of condensation products, increased thermal stability and/or increased specific activity. The increased thermal stability typically is at higher temperatures. In one embodiment, the increased pH stability is at high pH. In a further embodiment, the increased pH stability is at low pH. The glucoamylase variants of the disclosure may also provide higher rates of starch hydrolysis at low substrate concentrations as compared to the parent glucoamylase. The variant may have a higher V max or lower K m than a parent glucoamylase when tested under the same conditions. For example the variant glucoamylase may have a higher V max at a temperature range of about 25°C to about 70°C (e.g. , about 25°C to about 35°C; about 30°C to about 35°C; about 40°C to about 50°C; at about 50°C to about 55°C, or about 55°C to about 62°C). The Michaelis-Menten constant, K m and V max values can be easily determined using standard known procedures. In another aspect, the glucoamylase may also exhibit a reduced starch hydrolysis activity which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase such as TrGA. 5.1. Variant Glucoamylases with Altered Thermostability

In some aspects, the disclosure relates to a variant glucoamylase having altered thermal stability as compared to a parent (wild-type). Altered thermostability can be at increased temperatures or at decreased temperatures. Thermostability is measured as the % residual activity after incubation for 1 hour at 64°C in NaAc buffer pH 4.5. Under these conditions, TrGA has a residual activity of between about 15% and 44% due to day-to-day variation as compared to the initial activity before incubation. Thus, in some embodiments, variants with increased thermostability have a residual activity that is between at least about 1% and at least about 50% more than that of the parent (after incubation for 1 hour at 64°C in NaAc buffer pH 4.5), including about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, and about 50% as compared to the initial activity before incubation. For example, when the parent residual activity is 15%, a variant with increased thermal stability may have a residual activity of between about 16% and about 75%. In some embodiments, the glucoamylase variant will have improved thermostability such as retaining at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% enzymatic activity after exposure to altered temperatures over a given time period, for example, at least about 60 minutes, about 120 minutes, about 180 minutes, about 240 minutes, or about 300 minutes. In some embodiments, the variant has increased thermal stability compared to the parent glucoamylase at selected temperatures in the range of about 40°C to about 80°C, also in the range of about 50°C to about 75°C, and in the range of about 60°C to about 70°C, and at a pH range of about 4.0 to about 6.0. In some embodiments, the thermostability is determined as described in the Assays and Methods. That method may be adapted as appropriate to measure thermostability at other

temperatures. Alternatively the thermostability may be determined at 64°C as described there. In some embodiments, the variant has increased thermal stability at lower temperature compared to the parent glucoamylase at selected temperature in the range of about 20°C to about 50°C, including about 35°C to about 45°C and about 30°C to about 40°C. In some embodiments, variants having an improvement in thermostability include one or more deletions, substitutions or insertions and particularly substitutions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2: 10, 42, 43, 44, 59, 61, 68, 72, 73, 97, 98, 99, 102, 114, 133, 140, 144, 152, 153, 182, 204, 205, 214, 216, 228, 229, 230, 231, 236, 241, 242, 263, 264, 265, 268, 269, 276, 284, 291, 294 300, 301, 303, 311, 338, 342, 344, 346, 349, 359, 361, 364, 375, 379, 382, 390, 391, 393, 394, 410, 417, 430, 431, 433, 436, 442, 444, 448, 451, 493, 495, 503, 508, 511, 518, 519, 520, 527, 531, 535, 536, 537, 539, 563, or 577, or an equivalent position in a parent glucoamylase. In some embodiments, the parent glucoamylase will be a Trichoderma glucoamylase homologue and in further embodiments, the parent glucoamylase will have at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 98% sequence identity to SEQ ID NO: 2. In some embodiments, the parent glucoamylase will also have structural identity to SEQ ID NO: 2. In some embodiments, the variant having increased thermostability has a substitution in at least one of the positions: T10S, T42V, I43Q, I43R, D44C, D44R, E68C, E68M, G73F, G73W, K114M, K114Q, I133V, N153A, N153E, N153M, N153S, N153V, W228V, V229I, V229L, S230Q, S231V, D236R, L264D, L264K, A268D, S291A, S291F, S291H, S291M, S291T, G294C, A301P, A301R, V338I, V338N, V338Q, S344M, S344P, S344Q, S344R, S344V, G361D, G361E, G361F, G361I, G361L, G361M, G361P, G361S, G361W, G361Y, A364D, A364E, A364F, A364G, A364K, A364L, A364M, A364R, A364S, A364T, A364V,

A364W, T375N, L417K, L417R, R433C, R433E, R433G, R433L, R433N, R433S, R433V, I436H, T495K, T495S, E503A, E503C, E503V, Q508R, Q511H, A519 , A519R, A519Y, V531L, A535K, A535N, A535P, A535R, A539E, A539R, A539S, N563C, N563E, N563I, N563K, N563L, N563Q, N563T, N563V, N577K, N577P, or N577R of SEQ ID NO: 2. 5.2. Variant Glucoamylases with Altered Specific Activity

As used herein, specific activity is the activity of the glucoamylase per mg of protein. Activity was determined using the ethanol assay. The screening identified variants having a

Performance Index (PI) > 1.0 compared to the parent TrGA PI. The PI is calculated from the specific activities (activity/mg enzyme) of the wild-type (WT) and the variant enzymes. It is the quotient "Variant-specific activity/WT-specific activity" and can be a measure of the increase in specific activity of the variant. A PI of about 2 should be about 2 fold better than WT. In some aspects, the disclosure relates to a variant glucoamylase having altered specific activity as compared to a parent or wild-type glucoamylase. In some embodiments, the altered specific activity is increased specific activity. Increased specific activity can be defined as an increased performance index of greater than or equal to about 1, including greater than or equal to about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, and about 2. In some embodiments, the increased specific activity is from about 1.0 to about 5.0, including about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2., about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, and about 4.9. In some embodiments, the variant has an at least about 1.0 fold higher specific activity than the parent glucoamylase, including at least about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, about 2.0 fold, about 2.2 fold, about 2.5 fold, about 2.7 fold, about 2.9 fold, about 3.0 fold, about 4.0 fold, and about 5.0 fold.

In some embodiments, variants having an improvement in specific activity include one or more deletions, substitutions or insertions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2: 10, 14, 15, 23, 59, 60, 61, 65, 67, 68, 72, 73, 97, 98, 99, 102, 110, 113, 133, 140, 144, 145, 147, 152, 153, 164, 182, 204, 205, 214, 216, 219, 228, 229, 230, 231, 236, 239, 241, 242, 263, 264, 265, 268, 269, 276, 284, 291, 300, 301, 303, 311, 338, 342, 344, 346, 349, 359, 361, 364, 375, 379, 382, 390, 391, 393, 394, 410, 417, 418, 430, 431, 433, 442, 444, 448, 451, 493, 494, 495, 502, 503, 508, 511, 518, 519, 520, 531, 535, 536, 539, or 563, or an equivalent position in a parent glucoamylase. In some embodiments, the parent glucoamylase will comprise a sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments, the parent glucoamylase will also have structural identity to SEQ ID NO: 2. In some embodiments, variants of the disclosure having improved specific activity include a substitution in the following positions in the amino acid sequence set forth in SEQ ID NO: 2 : I43Q, I43R, D44C, D44R, N061I, T067M, A072Y, S097N, S102A, S102M, S102R, I133T, N 145I, N153D, T205Q, Q219S, W228A, W228F, W228H, W228M, S230C, S230F, S230G, S230L, S230N, S230Q, S230R, S231L, I239V, I239Y, N263P, A268C, A268G, A268K, S291A, G294C, T342V, K394S, L417R, L417V, T430K, A431I, A431L, A431Q, R433Y, T451K, T495M, A519I, A520C, A520L, A520P, A535R, V536M, A539R, N563K, or N563I, or an equivalent position in a parent glucoamylase. In some embodiments, the specific activity of the parent as compared to the variant is determined as described in the Assays and Methods.

5.3. Variant G I ucoa myiases with Both Altered Thermostability and Altered

Specific Activity In some aspects, the disclosure relates to a variant glucoamylase having both altered thermostability and altered specific activity as compared to a parent (e.g. , wild-type). In some embodiments, the altered specific activity is an increased specific activity. In some embodiments, the altered thermostability is an increased thermostability at high

temperatures (e.g. , at temperatures above 80°C) as compared to the parent glucoamylase. In some embodiments, variants with an increased thermostability and increased specific activity include one or more deletions, substitutions or insertions and substitutions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2 : 10, 15, 43, 44, 59, 61, 68, 72, 73, 97, 99, 102, 140, 153, 182, 204, 205, 214, 228, 229, 230, 231, 236, 241, 242, 264, 265, 268, 276, 284, 291, 294, 300, 301, 303, 311, 338, 344, 346, 349, 359, 361, 364, 375, 379, 382, 391, 393, 394, 410, 430, 433, 444, 448, 451, 495, 503, 511, 520, 531, 535, 536, 539, or 563, or an equivalent position in a parent glucoamylase. In some embodiments, the parent glucoamylase will be a Trichoderma glucoamylase homologue and in further embodiments, the parent glucoamylase will have at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 98% sequence identity to SEQ ID NO: 2. In some embodiments, the parent glucoamylase will also have structural identity to SEQ ID NO: 2. In some embodiments, the variant having increased thermostability and specific activity has a substitution in at least one of the positions: I43Q/ , D44C/R,

W228F/H/M, S230C/F/G/N/Q/R, S231L, A268C/D/G/K, S291A, G294C, R433Y, S451 , E503C, Q511H, A520C/L/P, or A535N/P/R of SEQ ID NO : 2.

5.4. Variant Glucoamylases with production of fermentable sugar(s) In a further aspect, the glucoamylase exhibit an enhanced production of fermentable sugar(s) as compared to the parent glucoamylase such as TrGA. In a further aspect, the glucoamylase exhibit an enhanced production of fermentable sugars in the mashing step of the brewing process as compared to the parent glucoamylase such as TrGA. In a further aspect, the glucoamylase exhibit an enhanced production of fermentable sugars in the fermentation step of the brewing process as compared to the parent glucoamylase such as TrGA. In a further aspect, the the fermentable sugar is glucose. A skilled person within the field can determine the production of fermentable sugar(s) by e.g. HPLC techniques.

5.5 Variant Glucoamylases with a altered ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) In a further aspect, the glucoamylase exhibit a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase such as TrGA. In a further aspect, the glucoamylase exhibit a starch hydrolysis activity which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase such as TrGA. In one aspect, a screening method for identification of a glucoamylase variant having a reduced synthesis of condensation products during hydrolysis of starch and the glucoamylse variants obtained by the method is provided, the method comprising the steps of measuring the isomaltose synthesis and starch hydrolysis activity of glucoamylase variants and selecting the variants having a reduced starch hydrolysis activity which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase and having a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase.

In some embodiments the glucoamylase variants are selecting for having a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase.

In some embodiments the glucoamylase variants are selecting for having the same or increased starch hydrolysis activity and reduced isomaltose synthesis, which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase and thereby having a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase.

In a further aspect, the glucoamylase exhibit an enhanced real degree of fermentation as compared to the parent glucoamylase such as TrGA.

5.6 Variant Glucoamylases with an altered formation of condensation products

In one aspect, the glucoamylase forms a lower amount of condensation products than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under the same conditions. In a further aspect, the glucoamylase forms an amount of condensation products which amount is essentially the same as, not more than 5%, not more than 8% or not more than 10% higher than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under the same conditions. In a further aspect, the dosing of the glucoamylases are the same based on protein concentration. In a further aspect, the dosing of the glucoamylases are the same based on measurement of activity in activity assays such as a GAU activity assay as described herein or a starch hydrolysation-activity assay also as described herein.

6. Polynucleotides Encoding Glucoamylases

The present disclosure also relates to isolated polynucleotides encoding the variant glucoamylase. The polynucleotides may be prepared by established techniques known in the art. The polynucleotides may be prepared synthetically, such as by an automatic DNA synthesizer. The DNA sequence may be of mixed genomic (or cDNA) and synthetic origin prepared by ligating fragments together. The polynucleotides may also be prepared by polymerase chain reaction (PCR) using specific primers. In general, reference is made to Minshull J. et al., Methods 32(4) :416-427 (2004). DNA may also be synthesized by a number of commercial companies such as Geneart AG, Regensburg, Germany.

The present disclosure also provides isolated polynucleotides comprising a nucleotide sequence (i) having at least about 50% identity to SEQ ID NO: 4, including at least about 60%, about 70%, about 80%, about 90%, about 95%, and about 99%, or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence set forth in SEQ ID NO: 4, under conditions of intermediate to high stringency, or (iii) being complementary to a nucleotide sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 4. Probes useful according to the disclosure may include at least about 50, about 100, about 150, about 200, about 250, about 300 or more contiguous nucleotides of SEQ ID NO: 4. In some embodiments, the encoded polypeptide also has structural identity to SEQ ID NO: 2.

The present disclosure further provides isolated polynucleotides that encode variant glucoamylases that comprise an amino acid sequence comprising at least about 50%, about 60%, about 70%, about 80%, about 90%, about 93%, about 95%, about 97%, about 98%, or about 99% amino acid sequence identity to SEQ ID NO: 2. Additionally, the present disclosure provides expression vectors comprising any of the polynucleotides provided above. The present disclosure also provides fragments {i.e. , portions) of the DNA encoding the variant glucoamylases provided herein. These fragments find use in obtaining partial length DNA fragments capable of being used to isolate or identify polynucleotides encoding mature glucoamylase enzymes described herein from filamentous fungal cells {e.g. , Trichoderma , Aspergillus, Fusarium, Penicillium, and Humicola), or a segment thereof having glucoamylase activity. In some embodiments, fragments of the DNA may comprise at least about 50, about 100, about 150, about 200, about 250, about 300 or more contiguous nucleotides. In some embodiments, portions of the DNA provided in SEQ ID NO: 4 may be used to obtain parent glucoamylases and particularly Trichoderma glucoamylase homologues from other species, such as filamentous fungi that encode a glucoamylase.

7. Production of Glucoamylases 7.1. DNA Constructs and Vectors

According to one embodiment of the disclosure, a DNA construct comprising a polynucleotide as described above encoding a variant glucoamylase encompassed by the disclosure and operably linked to a promoter sequence is assembled to transfer into a host cell. In one aspect, a polynucleotide encoding a glucoamylase variant as disclosed herein is provided. The DNA construct may be introduced into a host cell using a vector. In one aspect, a vector comprising the polynucleotide, or capable of expressing a glucoamylase variant as disclosed herein is provided. The vector may be any vector that when introduced into a host cell is stably introduced. In some embodiments, the vector is integrated into the host cell genome and is replicated. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like. In some embodiments, the vector is an expression vector that comprises regulatory sequences operably linked to the glucoamylase coding sequence. Examples of suitable expression and/or integration vectors are provided in Sambrook et al. ( 1989) supra, and Ausubel (1987) supra, and van den Hondel et al. (1991) in Bennett and Lasure (Eds.) More Gene Manipulations In Fungi, Academic Press pp. 396-428 and U.S.

Patent No. 5,874,276. Reference is also made to the Fungal Genetics Stock Center Catalogue of Strains (FGSC, http://www.fgsc.net) for a list of vectors. Particularly useful vectors include vectors obtained from for example Invitrogen and Promega.

Suitable plasmids for use in bacterial cells include pBR322 and pUC19 permitting replication in E. coli and pE194 for example permitting replication in Bacillus. Other specific vectors suitable for use in E. coli host cells include vectors such as pFB6, pBR322, pUC18, pUClOO, pDONR™201, 10 pDONR™221, pENTR™, pGEM ® 3Z and pGEM ® 4Z.

Specific vectors suitable for use in fungal cells include pRAX, a general purpose expression vector useful in Aspergillus, pRAX with a glaA promoter, and in Hypocrea/Trichoderma includes pTrex3g with a cbhl promoter.

In some embodiments, the promoter that shows transcriptional activity in a bacterial or a fungal host cell may be derived from genes encoding proteins either homologous or heterologous to the host cell. The promoter may be a mutant, a truncated and/or a hybrid promoter. The above-mentioned promoters are known in the art. Examples of suitable promoters useful in fungal cells and particularly filamentous fungal cells such as Trichoderma or Aspergillus cells include such exemplary promoters as the T. reesei promoters cbhl, cbhl, eg/1, eg/2, eg5, xlnl and xln2. Other examples of useful promoters include promoters from A. awamori and A. niger glucoamylase genes (glaA) (see Nunberg et al., Mol. Cell Biol. 4: 2306-2315 (1984) and Boel et al., EMBO J. 3 : 1581- 1585 (1984)), A. oryzae TAKA amylase promoter, the TPI (triose phosphate isomerase) promoter from S. cerevisiae, the promoter from Aspergillus nidulans acetamidase genes and Rhizomucor miehei lipase genes. Examples of suitable promoters useful in bacterial cells include those obtained from the E. coli lac operon; Bacillus licheniformis alpha-amylase gene (amyL), B. stearothermophilus amylase gene (amyS) ; Bacillus subtilis xylA and xylB genes, the beta-lactamase gene, and the tac promoter. In some embodiments, the promoter is one that is native to the host cell. For example, when T. reesei is the host, the promoter is a native T. reesei promoter. In other embodiments, the promoter is one that is heterologous to the fungal host cell. In some embodiments, the promoter will be the promoter of a parent glucoamylase (e.g. , the TrGA promoter).

In some embodiments, the DNA construct includes nucleic acids coding for a signal sequence, that is, an amino acid sequence linked to the amino terminus of the polypeptide that directs the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of the nucleic acid sequence may naturally include a signal peptide coding region that is naturally linked in translation reading frame with the segment of the glucoamylase coding sequence that encodes the secreted glucoamylase or the 5' end of the coding sequence of the nucleic acid sequence may include a signal peptide that is foreign to the coding sequence. In some embodiments, the DNA construct includes a signal sequence that is naturally associated with a parent glucoamylase gene from which a variant glucoamylase has been obtained. In some embodiments, the signal sequence will be the sequence depicted in SEQ ID NO: 1 or a sequence having at least about 90%, about 94, or about 98% sequence identity thereto. Effective signal sequences may include the signal sequences obtained from other filamentous fungal enzymes, such as from Trichoderma (7. reesei glucoamylase, cellobiohydrolase I, cellobiohydrolase II, endoglucanase I, endoglucanase II, endoglucanase II, or a secreted proteinase, such as an aspartic proteinase), Humicola (H. insolens cellobiohydrolase or endoglucanase, or H. grisea glucoamylase), or Aspergillus [A. niger glucoamylase and A. oryzae TAKA amylase). In additional embodiments, a DNA construct or vector comprising a signal sequence and a promoter sequence to be introduced into a host cell are derived from the same source. In some embodiments, the native glucoamylase signal sequence of a Trichoderma glucoamylase homologue, such as a signal sequence from a Hypocrea strain may be used.

In some embodiments, the expression vector also includes a termination sequence. Any termination sequence functional in the host cell may be used in the present disclosure. In some embodiments, the termination sequence and the promoter sequence are derived from the same source. In another embodiment, the termination sequence is homologous to the host cell. Useful termination sequences include termination sequences obtained from the genes of Trichoderma reesei cbll; A. niger or A. awamori glucoamylase (Nunberg et al.

( 1984) supra, and Boel et al., (1984) supra), Aspergillus nidulans anthranilate synthase,

Aspergillus oryzae TAKA amylase, or A. nidulans trpC (Punt et al., Gene 56 : 117-124 ( 1987)).

In some embodiments, an expression vector includes a selectable marker. Examples of selectable markers include ones that confer antimicrobial resistance (e.g. , hygromycin and phleomycin). Nutritional selective markers also find use in the present disclosure including those markers known in the art as amdS (acetamidase), argB (ornithine

carbamoyltransferase) and pyrG (orotidine-5'phosphate decarboxylase). Markers useful in vector systems for transformation of Trichoderma are known in the art (see, e.g. , Finkelstein, Chapter 6 in Biotechnology Of Filamentous Fungi, Finkelstein et al. ( 1992) Eds. Butterworth- Heinemann, Boston, MA; Kinghorn et al. ( 1992) Applied Molecular Genetics Of Filamentous Fungi, Blackie Academic and Professional, Chapman and Hall, London; Berges and Barreau, Curr. Genet. 19 : 359-365 ( 1991); and van Hartingsveldt et al., Mol. Gen. Genet. 206: 71-75 (1987)). In some embodiments, the selective marker is the amdS gene, which encodes the enzyme acetamidase, allowing transformed cells to grow on acetamide as a nitrogen source. The use of A. nidulans amdS gene as a selective marker is described in Kelley et al., EMBO J. 4:475-479 ( 1985) and Penttila et al., Gene 61 : 155-164 (1987). Methods used to ligate the DNA construct comprising a nucleic acid sequence encoding a variant glucoamylase, a promoter, a termination and other sequences and to insert them into a suitable vector are well known in the art. Linking is generally accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide linkers are used in accordance with conventional practice (see Sambrook et al. ( 1989) supra, and Bennett and Lasure, More Gene Manipulations In Fungi, Academic Press, San Diego (1991) pp 70-76.). Additionally, vectors can be constructed using known recombination techniques (e.g. , Invitrogen Life Technologies, Gateway Technology).

7.2. Host Cells and Transformation of Host Cells

1. The present disclosure also relates to host cells comprising a polynucleotide encoding a variant glucoamylase of the disclosure. In some embodiments, the host cells are chosen from bacterial, fungal, plant and yeast cells. The term host cell includes both the cells, progeny of the cells and protoplasts created from the cells that are used to produce a variant glucoamylase according to the disclosure. In one aspect, a host cell comprising, preferably transformed with a vector is disclosed. In a further aspect, a cell capable of expressing a glucoamylase variant is provided. In a further aspect, the host cell is a protease deficient and/or xylanase deficient and/or glucanase deficient host cell. A protease deficient and/or xylanase deficient and/or native glucanase deficient host cell may be obtained by deleting or silencing the genes coding for the mentioned enzymes. As a consequence the host cell containing the GA-variant is not expressing the mentioned enzymes In some embodiments, the host cells are fungal cells and optionally filamentous fungal host cells. The term "filamentous fungi" refers to all filamentous forms of the subdivision

Eumycotina (see, Alexopoulos, C. J. ( 1962), Introductory Mycology, Wiley, New York). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi of the present disclosure are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism is obligatory aerobic. In the present disclosure, the filamentous fungal parent cell may be a cell of a species of, but not limited to, Trichoderma (e.g. , Trichoderma reesei, the asexual morph of Hypocrea jecorina, previously classified as T. longibrachiatum , Trichoderma viride, Trichoderma koningii, Trichoderma harzianum) (Sheir-Neirs et al., Appl. Microbiol. Biotechnol. 20:46-53 (1984); ATCC No. 56765 and ATCC No. 26921), Penicilliurn sp., Humicola sp. (e.g. , H.

insolens, H. lanuginosa and H. grisea), Chrysosporium sp. (e.g., C. lucknowense) ,

Gliocladium sp. , Aspergillus sp. (e.g., A. oryzae, A. niger, A sojae, A. japonicus, A. nidulans, and A. awamori) (Ward et al., Appl. Microbiol. Biotechnol. 39:738-743 (1993) and

Goedegebuur et al., Curr. Genet 41 :89 -98 (2002)), Fusarium sp.,(e.g., F. roseum, F.

graminum, F. cerealis, F. oxysporum, and F. venenatum), Neurospora sp., (N. crassa), Hypocrea sp., Mucor sp. (M. miehei), Rhizopus sp., and Emericella sp. (see also, Innis et al., Science 228:21 -26 (1985)). The term "Trichoderma" or "Trichoderma sp. " or "Trichoderma spp." refer to any fungal genus previously or currently classified as Trichoderma. In some embodiments, the host cells will be gram-positive bacterial cells. Non-limiting examples include strains of Streptomyces (e.g., S. lividans, S. coelicolor, and S. griseus) and Bacillus. As used herein, "the genus Bacillus" includes all species within the genus "Bacillus," as known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named "Geobacillus tearothermophilus." In some embodiments, the host cell is a gram-negative bacterial strain, such as E. coli or Pseudomonas sp. In other embodiments, the host cells may be yeast cells such as

Saccharomyces sp., Schizosaccharomyces sp., Pichia sp. , or Candida sp. In other

embodiments, the host cell will be a genetically engineered host cell wherein native genes have been inactivated, for example by deletion in bacterial or fungal cells. Where it is desired to obtain a fungal host cell having one or more inactivated genes known methods may be used (e.g., methods disclosed in U.S. Patent No. 5,246,853, U.S. Patent No. 5,475,101, and WO 92/06209). Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation or by any other means that renders a gene nonfunctional for its intended purpose (such that the gene is prevented from expression of a functional protein). In some embodiments, when the host cell is a Trichoderma cell and particularly a T. reesei host cell, the cbhl, cbhl, eg/1 and eg/2 genes will be inactivated and/or deleted. Exemplary Trichoderma reesei host cells having quad-deleted proteins are set forth and described in U.S. Patent No. 5,847,276 and WO 05/001036. In other embodiments, the host cell is a protease deficient or protease minus strain. Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, (e.g., lipofection-mediated and DEAE-Dextrin mediated transfection); incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion. General transformation techniques are known in the art (see, e.g. , Ausubel et al. ( 1987) supra, chapter 9; and Sambrook et al. (1989) supra, and Campbell et al., Curr. Genet. 16: 53-56 (1989)).

Transformation methods for Bacillus are disclosed in numerous references including

Anagnostopoulos C. and J. Spizizen, J. Bacteriol. 81 : 741-746 ( 1961) and WO 02/14490.

Transformation methods for Aspergillus are described in Yelton et al., Proc. Natl. Acad. Sci. USA 81 : 1470-1474 ( 1984); Berka et al., ( 1991) in Applications of Enzyme Biotechnology, Eds. Kelly and Baldwin, Plenum Press (NY); Cao et al., Protein Sci. 9 :991-1001 (2000); Campbell et al., Curr. Genet. 16 : 53-56 (1989), and EP 238 023. The expression of heterologous protein in Trichoderma is described in U.S. Patent No. 6,022,725; U.S. Patent No. 6,268,328; Harkki et al. Enzyme Microb. Technol. 13 : 227-233 ( 1991); Harkki et al., BioTechnol. 7: 596-603 ( 1989); EP 244,234; EP 215,594; and Nevalainen et al., "The Molecular Biology of Trichoderma and its Application to the Expression of Both Homologous and Heterologous Genes", in Molecular Industrial Mycology, Eds. Leong and Berka, Marcel Dekker Inc. , NY (1992) pp. 129- 148). Reference is also made to W096/00787 and Bajar et al., Proc. Natl. Acad. Sci. USA 88: 8202-8212 ( 1991) for transformation of Fusarium strains.

In one specific embodiment, the preparation of Trichoderma sp. for transformation involves the preparation of protoplasts from fungal mycelia (see, Campbell et al., Curr. Genet. 16: 53- 56 (1989); Pentilla et al., Gene 61 : 155-164 ( 1987)). Agrobacterium tumefaciens-med ated transformation of filamentous fungi is known (see de Groot et al., Nat. Biotechnol. 16:839- 842 (1998)). Reference is also made to U.S. Patent No. 6,022,725 and U.S. Patent No. 6,268,328 for transformation procedures used with filamentous fungal hosts. In some embodiments, genetically stable transformants are constructed with vector systems whereby the nucleic acid encoding the variant glucoamylase is stably integrated into a host strain chromosome. Transformants are then purified by known techniques.

In some further embodiments, the host cells are plant cells, such as cells from a monocot plant (e.g. , corn, wheat, and sorghum) or cells from a dicot plant (e.g. , soybean) . Methods for making DNA constructs useful in transformation of plants and methods for plant transformation are known. Some of these methods include Agrobacterium tumefaciens mediated gene transfer; microprojectile bombardment, PEG mediated transformation of protoplasts, electroporation and the like. Reference is made to U.S. Patent No. 6,803,499, U.S. Patent No. 6,777,589; Fromm et al., BioTechnol. 8:833-839 (1990); Potrykus et al., Mol. Gen. Genet. 199 : 169-177 (1985).

7.3. Production of Glucoamylases

The present disclosure further relates to methods of producing the variant glucoamylases, which comprises transforming a host cell with an expression vector comprising a

polynucleotide encoding a variant glucoamylase according to the disclosure, culturing the host cell under conditions suitable for expression and production of the variant glucoamylase and optionally recovering the variant glucoamylase. In one aspect, a method of expressing a variant glucoamylase according to the disclosure, the method comprising obtaining a host cell or a cell as disclosed herein and expressing the glucoamylase variant from the cell or host cell, and optionally purifying the glucoamylase variant, is provided. In one aspect, the glucoamylase variant is purified.

In the expression and production methods of the present disclosure the host cells are cultured under suitable conditions in shake flask cultivation, small scale or large scale fermentations (including continuous, batch and fed batch fermentations ) in laboratory or industrial fermentors, with suitable medium containing physiological salts and nutrients (see, e.g. , Pourquie, J. et al., Biochemistry And Genetics Of Cellulose Degradation, eds. Aubert, J. P. et al., Academic Press, pp. 71-86, 1988 and Ilmen, M . et al., Appl. Environ. Microbiol. 63 : 1298-1306 (1997)). Common commercially prepared media (e.g. , Yeast Malt Extract (YM) broth, Luria Bertani (LB) broth and Sabouraud Dextrose (SD) broth) find use in the present disclosure. Culture conditions for bacterial and filamentous fungal cells are known in the art and may be found in the scientific literature and/or from the source of the fungi such as the American Type Culture Collection and Fungal Genetics Stock Center. In cases where a glucoamylase coding sequence is under the control of an inducible promoter, the inducing agent (e.g. , a sugar, metal salt or antimicrobial), is added to the medium at a concentration effective to induce glucoamylase expression.

In some embodiments, the present disclosure relates to methods of producing the variant glucoamylase in a plant host comprising transforming a plant cell with a vector comprising a polynucleotide encoding a glucoamylase variant according to the disclosure and growing the plant cell under conditions suitable for the expression and production of the variant.

In some embodiments, assays are carried out to evaluate the expression of a variant glucoamylase by a cell line that has been transformed with a polynucleotide encoding a variant glucoamylase encompassed by the disclosure. The assays can be carried out at the protein level, the NA level and/or by use of functional bioassays particular to glucoamylase activity and/or production. Some of these assays include Northern blotting, dot blotting (DNA or RNA analysis), RT-PCR (reverse transcriptase polymerase chain reaction), in situ hybridization using an appropriately labeled probe (based on the nucleic acid coding sequence) and conventional Southern blotting and autoradiography. In addition, the production and/or expression of a variant glucoamylase may be measured in a sample directly, for example, by assays directly measuring reducing sugars such as glucose in the culture medium and by assays for measuring glucoamylase activity, expression and/or production. In particular, glucoamylase activity may be assayed by the 3,5-dinitrosalicylic acid (DNS) method (see Goto et al., Biosci. Biotechnol. Biochem. 58:49-54 ( 1994)) . In additional embodiments, protein expression, is evaluated by immunological methods, such as immunohistochemical staining of cells, tissue sections or immunoassay of tissue culture medium, (e.g., by Western blot or ELISA). Such immunoassays can be used to qualitatively and quantitatively evaluate expression of a glucoamylase. The details of such methods are known to those of skill in the art and many reagents for practicing such methods are commercially available.

The glucoamylases of the present disclosure may be recovered or purified from culture media by a variety of procedures known in the art including centrifugation, filtration, extraction, precipitation and the like.

8. Compositions and Uses In one aspect, the use of a glucoamylase variant as described herein for the preparation of an enzymatic composition, is provided.

The variant glucoamylases of the disclosure may be used in enzyme compositions including but not limited to starch hydrolyzing and saccharifying compositions, cleaning and detergent compositions (e.g. , laundry detergents, dish washing detergents, and hard surface cleaning compositions), alcohol fermentation compositions, and in animal feed compositions. Further, the variant glucoamylases may be used in, for example, brewing, healthcare, textile, environmental waste conversion processes, biopulp processing, and biomass conversion applications. The variant glucoamylases of the disclosure may be used in enzyme

compositions including a starch hydrolyzing composition, a saccharifying composition, a detergent, an alcohol fermentation enzymatic composition, and an animal feed. In one aspect, the composition is a starch hydrolyzing composition.

In some embodiments, an enzyme composition comprising a variant glucoamylase encompassed by the disclosure will be optionally used in combination with any one or combination of the following enzymes - alpha-amylases, proteases, pullulanases,

isoamylases, cellulases, hemicellulases, xylanases, cyclodextrin glycotransferases, lipases, phytases, laccases, oxidases, esterases, cutinases, xylanases, granular starch hydrolyzing enzymes and other glucoamylases. In some embodiments, an enzyme composition comprising a variant glucoamylase encompassed by the disclosure will be optionally used in combination with any one or combination of the following enzymes - amylase, protease, pullulanase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase and a further glucoamylase. In some embodiments, an enzyme composition comprising a variant glucoamylase encompassed by the disclosure will be optionally used in combination with any one or combination of the following enzymes - amylase, pullulanase and a further glucoamylase. In some embodiments, an enzyme composition comprising a variant glucoamylase encompassed by the disclosure will be optionally used in combination with any one or combination of the following enzymes - amylase and pullulanase. In a further aspect, the amylase is alpha-amylase and/or isoamylase. In a further aspect, the glucanase is exoglucanase and/or endoglucanase.

In some embodiments, the enzyme composition will include an alpha-amylase such as fungal alpha-amylases (e.g., Aspergillus sp. ) or bacterial alpha-amylases (e.g. , Bacillus sp. such as B. stearothermophilus, B. amyloliquefaciens and B. licheniformis) and variants and hybrids thereof. In the present context, an alpha-amylase (EC. 3.2.1.1) catalyses the endohydrolysis of (l->4)-alpha-D-glucosidic linkages in oligosaccharides and polysaccharides. An alpha- amylase acts on starch, glycogen and related polysaccharides and oligosaccharides in a random manner; reducing groups are liberated in the alpha-configuration. In some embodiments, the alpha-amylase is an acid stable alpha-amylase. In some embodiments, the alpha-amylase is Aspergillus kawachi alpha-amylase (AkAA), see U.S. Patent No.

7,037,704. Other alpha-amylases contemplated for use in the compositions of the disclosure include, but are not limited to, bacterial alpha-amylases such as those from Bacillus subtilis (AmyE), and Bacillus licheniformis (AmyL) and Geobacillus stearothermophilus (AmyS) as described by Gray et al. ( 1986) (Gray GL, Mainzer SE, Rey MW, Lamsa MH, Kindle KL, Carmona C and Requadt C "Structural genes encoding the thermophilic alpha-amylases of Bacillus stearothermophilus and Bacillus licheniformis" Journal of Bacteriology ( 1986) 166(2) p635-643) along with variants and combinations, including combinations of variants of the above. Variants of AmyE, AmyL and AmyS are well known and examples are described in US Patent Application 20100015686 Al ("Variant Alpha-Amylases from Bacillus subtilis and Methods of Uses, Thereof"), US Patent Application 20090314286 Al ("Geobacillus

stearothermophilus Alpha-Amylase (AmyS) Variants with Improved Properties"),

WO/2006/066594) ("Alpha-Amylase Variants"), US 20090238923 Al ("Variants Of Bacillus Licheniformis Alpha-Amylase With Increased Thermostability And/Or Decreased Calcium Dependence"). Commercially available alpha-amylases contemplated for use in the compositions of the disclosure are known and include GZYME G997, SPEZYME® FRED, SPEZYME® XTRA AMYLEX® 4T, AMYLEX® 3T and AMYLEX® XT (Danisco US, Inc, Genencor Division), TERMAMYL® 120-L and SUPRA® (Novozymes, A/S).

In some embodiments, the enzyme composition will include a pullulanase (EC 3.2.1.41). In one aspect, the pullulanases used herein is pullulanase from e.g. Pyrococcus or Bacillus sp, such as Bacillus acidopullulyticus (e.g., the one described in FEMS Microbiol. Letters 115: 97- 106) or Bacillus deramificans, or Bacillus naganoencis. In one aspect, the pullulanase is the Bacillus acidopullulyticus PulB enzyme, described in the paper by Kelly et al . FEMS

Microbiology Letters 115 ( 1994) 97- 106. The pullulanase may also be an engineered pullulanases from, e.g., a Bacillus strain. Other pullulanases which are preferably used in the processes according to the invention include: Bacillus deramificans (U.S. Patent No.

5,736,375), or the pullulanase may be derived from Pyrococcus woesei described in

PCT/DK91/00219, or the pullulanase may be derived from Fervidobacterium sp. Ven 5 described in PCT/DK92/00079, or the pullulanase may be derived from Thermococcus celer described in PCT/DK95/00097, or the pullulanase may be derived from Pyrodictium abyssei described in PCT/DK95/00211 , or the pullulanase may be derived from Fervidobacterium pennavorans described in PCT/DK95/00095, or the pullulanase may be derived from

Desulforococcus mucosus described in PCT/DK95/00098. The pullulanase (EC 3.2.1.41) may also be derived from, but not limited to, Klebsiella (Aerobacter) spp. (PulA); for example Klebsiella planticola, Klebsiella (Aerobacter) aerogenes and Klebsiella pneumoniae (see: Katsuragi et a/. Journal of Bacteriology ( 1987) 169(5) p2301-2306; Fouts et a/. PLoS Genetics (2008) 4(7), E1000141). These pullulanases, along with those from, for example, Bacillus acidopullulyticus are members of Glycoside Hydrolase Family 13. In some

embodiments, the enzyme composition will include an acid fungal protease. In a further embodiment, the acid fungal protease is derived from a Trichoderma sp. and may be any one of the proteases disclosed in US Patent No. 7,563,607 (published as US 2006/0154353 July 13, 2006), incorporated herein by reference. In a further embodiment, the enzyme composition will include a phytase from Buttiauxiella spp. (e.g. , BP-17, see also variants disclosed in PCT patent publication WO 2006/043178).

In other embodiments, the variant glucoamylases of the disclosure may be combined with other glucoamylases. In some embodiments, the glucoamylases of the disclosure will be combined with one or more glucoamylases derived from strains of Aspergillus or variants thereof, such as A. oryzae, A. niger, A. kawachi, and A. awamori; glucoamylases derived from strains of Humicola or variants thereof, particularly H. grisea, such as the glucoamylase having at least about 90%, about 93%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 3 disclosed in WO 05/052148; glucoamylases derived from strains of Talaromyces or variants thereof, particularly T. emersonii;

glucoamylases derived from strains of Athelia and particularly A. rolfsii; glucoamylases derived from strains of Penicillium, particularly P. chrysogenum. In particular, the variant glucoamylases may be used for starch conversion processes, and particularly in the production of dextrose for fructose syrups, specialty sugars and in alcohol and other end-product (e.g. , organic acid, ascorbic acid, and amino acids) production from fermentation of starch containing substrates (G.M.A. van Beynum et al., Eds. ( 1985) Starch Conversion Technology, Marcel Dekker Inc. NY). Dextrins produced using variant glucoamylase compositions of the disclosure may result in glucose yields of at least 80%, at least 85%, at least 90% and at least 95%. Production of alcohol from the fermentation of starch substrates using glucoamylases encompassed by the disclosure may include the production of fuel alcohol or potable alcohol. In some embodiments, the production of alcohol will be greater when the variant glucoamylase is used under the same conditions as the parent glucoamylase. In some embodiments, the production of alcohol will be between about 0.5% and 2.5% better, including but not limited to about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%. about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, and about 2.4% more alcohol than the parent glucoamylase. In some embodiments, the variant glucoamylases of the disclosure will find use in the hydrolysis of starch from various plant-based substrates, which are used for alcohol production. In some embodiments, the plant-based substrates will include corn, wheat, barley, rye, milo, rice, sugar cane, potatoes and combinations thereof. In some

embodiments, the plant-based substrate will be fractionated plant material, for example a cereal grain such as corn, which is fractionated into components such as fiber, germ, protein and starch (endosperm) (U.S. Patent No. 6,254,914 and U.S. Patent No. 6,899,910).

Methods of alcohol fermentations are described in The Alcohol Textbook, K.A. Jacques et al., Eds. 2003, Nottingham University Press, UK.

In certain embodiments, the alcohol will be ethanol. In particular, alcohol fermentation production processes are characterized as wet milling or dry milling processes. In some embodiments, the variant glucoamylase will be used in a wet milling fermentation process and in other embodiments the variant glucoamylase will find use in a dry milling process.

Dry grain milling involves a number of basic steps, which generally include: grinding, cooking, liquefaction, saccharification, fermentation and separation of liquid and solids to produce alcohol and other co-products. Plant material and particularly whole cereal grains, such as corn, wheat or rye are ground. In some cases, the grain may be first fractionated into component parts. The ground plant material may be milled to obtain a coarse or fine particle. The ground plant material is mixed with liquid (e.g. , water and/or thin stillage) in a slurry tank. The slurry is subjected to high temperatures (e.g. , about 90°C to about 105°C or higher) in a jet cooker along with liquefying enzymes (e.g. , alpha-amylases) to solublize and hydrolyze the starch in the grain to dextrins. The mixture is cooled down and further treated with saccharifying enzymes, such as glucoamylases encompassed by the instant disclosure, to produce glucose. The mash containing glucose may then be fermented for approximately 24 to 120 hours in the presence of fermentation microorganisms, such as ethanol producing microorganism and particularly yeast (Saccharomyces spp). The solids in the mash are separated from the liquid phase and alcohol such as ethanol and useful co- products such as distillers' grains are obtained.

In some embodiments, the saccharification step and fermentation step are combined and the process is referred to as simultaneous saccharification and fermentation or simultaneous saccharification, yeast propagation and fermentation.

In other embodiments, the variant glucoamylase is used in a process for starch hydrolysis wherein the temperature of the process is between about 30°C and about 75°C, in some embodiments, between about 40°C and about 65°C. In some embodiments, the variant glucoamylase is used in a process for starch hydrolysis at a pH between about 3.0 and about 6.5. The fermentation processes in some embodiments include milling of a cereal grain or fractionated grain and combining the ground cereal grain with liquid to form a slurry that is then mixed in a single vessel with a variant glucoamylase according to the disclosure and optionally other enzymes such as, but not limited to, alpha-amylases, other glucoamylases, phytases, proteases, pullulanases, isoamylases or other enzymes having granular starch hydrolyzing activity and yeast to produce ethanol and other co-products (see e.g. , U.S. Patent No. 4,514,496, WO 04/081193, and WO 04/080923).

In some embodiments, the disclosure pertains to a method of saccharifying a liquid starch solution, which comprises an enzymatic saccharification step using a variant glucoamylase of the disclosure. The liquid starch solution may be produced by solubilising starch in water or an aqueous buffer and optionally heating to gelatinize the starch. Further partial degradation of the starch by amylases may be applied.

The present invention provides a method of using glucoamylase variants of the invention for producing glucose and the like from starch. Generally, the method includes the steps of partially hydrolyzing precursor starch in the presence of alpha-amylase and then further hydrolyzing the release of D-glucose from the non-reducing ends of the starch or related oligo- and polysaccharide molecules in the presence of glucoamylase by cleaving alpha-( l-4) and alpha-(l-6) glucosidic bonds. The partial hydrolysis of the precursor starch utilizing alpha-amylase provides an initial breakdown of the starch molecules by hydrolyzing internal alpha-(l-4)-linkages. In commercial applications, the initial hydrolysis using alpha-amylase is run at a temperature of approximately 105°C. A very high starch concentration is processed, usually 30% to 40% solids. The initial hydrolysis is usually carried out for five minutes at this elevated temperature. The partially hydrolyzed starch can then be transferred to a second tank and incubated for approximately one hour at a temperature of 85° to 90°C to derive a dextrose equivalent (D.E.) of 10 to 15. The step of further hydrolyzing the release of D- glucose from the non-reducing ends of the starch or related oligo- and polysaccharides molecules in the presence of glucoamylase is normally carried out in a separate tank at a reduced temperature between 30° and 60°C. Often the temperature of the substrate liquid is dropped to between 55°C and 60°C. The pH of the solution is dropped from 6 to 6.5 to a range between 3 and 5.5. Often, the pH of the solution is 4 to 4.5. The glucoamylase is added to the solution and the reaction is carried out for 24-72 hours, such as 36-48 hours.

Examples of saccharification processes wherein the glucoamylase variants of the invention may be used include the processes described in JP 3-224493; JP 1- 191693; JP 62-272987; and EP 452,238. The glucoamylase variant(s) described herein may be used in combination with an enzyme that hydrolyzes only alpha-( l-6)-glucosidic bonds in molecules with at least four glucosyl residues. Preferentially, the glucoamylase variant can be used in combination with pullulanase or alpha-amylase. The use of alpha-amylase and pullulanase for

debranching, the molecular properties of the enzymes, and the potential use of the enzymes with glucoamylase is set forth in G.M.A. van Beynum et al., Starch Conversion Technology, Marcel Dekker, New York, 1985, 101-142. In one embodiment, the use of a glucoamylase variant as described herein in a starch conversion process, such as in a continuous saccharification step, is provided. The glucoamylase variants described herein may also be used in immobilised form. This is suitable and often used for producing maltodextrins or glucose syrups or speciality syrups, such as maltose syrups and further for the raffinate stream of oligosaccharides in connection with the production of fructose syrups.

When the desired final sugar product is, e.g., high fructose syrup the dextrose syrup may be converted into fructose. After the saccharification process the pH is increased to a value in the range of 6-8, such as pH 7.5, and the calcium is removed by ion exchange. The dextrose syrup is then converted into high fructose syrup using, e.g., an immobilized glucose isomerase (such as Sweetzyme™ IT). In other embodiments, the variant glucoamylase is used in a process for beer brewing.

Brewing processes are well-known in the art, and generally involve the steps of malting, mashing, and fermentation. Mashing is the process of converting starch from the milled barley malt and solid adjuncts into fermentable and un-fermentable sugars to produce wort. Traditional mashing involves mixing milled barley malt and adjuncts with water at a set temperature and volume to continue the biochemical changes initiated during the malting process. The mashing process is conducted over a period of time at various temperatures in order to activate the endogenous enzymes responsible for the degradation of proteins and carbohydrates. After mashing, the wort is separated from the solids (spent grains).

Following wort separation, the wort may be fermented with brewers' yeast to produce a beer. The short-branched glucose oligomers formed during mashing may be further hydrolyzed by addition of exogenous enzymes like glucoamylases and/or alpha-amylases, beta-amylases and pullulanase, among others. The wort may be used as it is or it may be concentrated and/or dried. The concentrated and/or dried wort may be used as brewing extract, as malt extract flavoring, for non-alcoholic malt beverages, malt vinegar, breakfast cereals, for confectionary etc. The wort is fermented to produce an alcoholic beverage, typically a beer, e.g. , ale, strong ale, bitter, stout, porter, lager, export beer, malt liquor, barley wine, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer, or light beer. In another typical embodiment, the wort is fermented to produce potable ethanol. In some embodiments, the disclosure pertains to a method of hydrolyzing and saccharifying gelatinised and liquefied (typically) grist starch to be used in brewing, whereby an enzymatic composition comprising one or more glucoamylases as contemplated herein, is used to enhance the amount of brewers' yeast fermentable sugars obtained from the starch. A brewing process is used to produce the potable product, beer, where fermentable sugars are converted to ethanol and C0 2 by fermentation with brewers' yeast. The fermentable sugars are traditionally derived from starch in cereal grains, optionally supplemented with fermentable sugar sources such as glucose and maltose syrups and cane sugar. Briefly, beer production, well-known in the art, typically includes the steps of malting, mashing, and fermentation. Historically the first step in beer production is malting - steeping, germination and drying of cereal grain (e.g. barley). During malting enzymes are produced in the germinating cereal (e.g. barley) kernel and there are certain changes in its chemical constituents (known as modification) including some degradation of starch, proteins and beta-glucans.

The malted cereal is milled to give a grist which may be mixed with a milled adjunct (e.g. non-germinated cereal grain) to give a mixed grist. The grist is mixed with water and subjected to mashing; a previously cooked (gelatinised and liquefied) adjunct may be added to the mash. The mashing process is conducted over a period of time at various temperatures in order to hydrolyse cereal proteins, degrade beta-glucans and solubilise and hydrolyse the starch. The hydrolysis of the grist starch in the malt and adjunct in traditional mashing is catalysed by two main enzymes endogenous to malted barley. Alpha-amylase, randomly cleaves alpha-1,4 bonds in the interior of the starch molecule fragmenting them into smaller dextrins. Beta-amylase sequentially cleaves alpha-1,4 bonds from the non-reducing end of the these dextrins producing mainly maltose. Both alpha- and beta-amylase are unable to hydrolyse the alpha- 1,6 bonds which forms the branching points of the starch chains in the starch molecule, which results in the accumulation of limit dextrins in the mash. Malt does contain an enzyme, limit dextrinase, which catalyses the hydrolysis of alpha-1,6 bonds but it only shows weak activity at mashing temperatures due to its thermolability. After mashing, the liquid extract (wort) is separated from the spent grain solids (i .e. the insoluble grain and husk material forming part of grist). The objectives of wort separation include: · to obtain good extract recovery, · to obtain good filterability, and · to produce clear wort. Extract recovery and filterability of the wort are important in the economics of the brewing process.

The composition of the wort depends on the raw materials, mashing process and profiles and other variables. A typical wort comprises 65-80% fermentable sugars (glucose, maltose and maltotriose, and 20-35% non-fermentable limit dextrins (sugars with a higher degree of polymerization than maltotriose). An insufficiency of starch hydrolytic enzymes during mashing can arise when brewing with high levels of adjunct unmalted cereal grists. A source of exogenous enzymes, capable of producing fermentable sugars during the mashing process is thus needed. Furthermore, such exogenous enzymes are also needed to reduce the level of non-fermentable sugars in the wort, with a corresponding increase in fermentable sugars, in order to brew highly attenuated beers with a low carbohydrate content. Herein disclosed is a enzyme composition for hydrolysis of starch comprising at least one glucoamylase as contemplated herein, which can be added to the mash or used in the mashing step of a brewing process, in order to cleave alpha-1,4 bonds and/or alpha-1,6 bonds in starch grist and thereby increase the fermentable sugar content of the wort and reduce the residue of non-fermentable sugars in the finished beer. In addition, the wort, so produced may be dried (by for example spray drying) or concentrated (e.g. boiling and evaporation) to provide a syrup or powder.

The grist, as contemplated herein, may comprise any starch and/or sugar containing plant material derivable from any plant and plant part, including tubers, roots, stems, leaves and seeds. Often the grist comprises grain, such as grain from barley, wheat, rye, oat, corn, rice, milo, millet and sorghum, and more preferably, at least 10%, or more preferably at least 15%, even more preferably at least 25%, or most preferably at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w/w) of the grist of the wort is derived from grain. Most preferably the grist comprises malted grain, such as barley malt. Preferably, at least 10%, or more preferably at least 15%, even more preferably at least 25%, or most preferably at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w/w) of the grist of the wort is derived from malted grain. Preferably the grist comprises adjunct, such as non-malted grain from barley, wheat, rye, oat, corn, rice, milo, millet and sorghum, and more preferably, at least 10%, or more preferably at least 15%, even more preferably at least 25%, or most preferably at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w/w) of the grist of the wort is derived from non-malted grain or other adjunct. Adjunct comprising readily fermentable carbohydrates such as sugars or syrups may be added to the malt mash before, during or after the mashing process of the invention but is preferably added after the mashing process. A part of the adjunct may be treated with an alpha-amylase, and/or endopeptidase (protease) and/or a endoglucanase, and/or heat treated before being added to the mash. The enzyme composition, as contemplated herein, may include additional enzyme(s), preferably an enzyme selected from among an alpha-amylase, protease, pullulanase, isoamylase, cellulase, glucanase such as exoglucanase or endoglucanase, xylanase, arabinofuranosidase, feruloyl esterase, xylan acetyl esterase, phytase and glucoamylase. During the mashing process, starch extracted from the grist is gradually hydrolyzed into fermentable sugars and smaller dextrins.

Preferably the mash is starch negative to iodine testing, before wort separation. In one aspect, a pullulanase (E. C. 3.2.1 .41 ) enzyme activity is exogenously supplied and present in the mash. The pullulanase may be added to the mash ingredients, e.g., the water and/or the grist before, during or after forming the mash.

In another aspect, an alpha-amylase enzyme activity is exogenously supplied and present in the mash. The alpha-amylase may be added to the mash ingredients, e.g., the water and/or the grist before, during or after forming the mash.

In a further aspect, both pullulanase and alpha-amylase enzyme activities are exogenously supplied and present in the mash. The alpha-amylase and pullulanase may be added to the mash ingredients, e.g., the water and/or the grist before, during or after forming the mash.

A further enzyme may be added to the mash, said enzyme being selected from the group consisting of among amylase, protease, pullulanase, isoamylase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, phytase and a further glucoamylase.

Prior to the third step of the brewing process, fermentation, the wort is typically transferred to a brew kettle and boiled vigorously for 50 - 60 minutes. A number of important processes occur during wort boiling (further information may be found in "Technology Brewing and Malting" by Wolfgang Kunze of the Research and Teaching Institute of Brewing, Berlin (VLB), 3rd completely updated edition, 2004, ISBN 3-921690-49-8) including inactivation of the endogenous malt enzymes and any exogenous enzyme added to the mash or adjunct. The boiled wort is then cooled, pitched with brewers' yeast and fermented at temperatures typically ranging from 8-16 °C to convert the fermentable sugars to ethanol. A low-alcohol beer can be produced from the final beer, by a process of vacuum evaporation that serves to selectively remove alcohol.

In an alternative embodiment, the disclosure pertains to a method of enhancing the amount of fermentable sugars in the wort, using an enzymatic composition comprising one or more glucoamylases as contemplated herein (e.g. thermolabile glucoamylase), whereby the enzymatic composition is added to the wort after it has been boiled, such that the one or more glucoamylases are active during the fermentation step. The enzymatic composition can be added to the boiled wort either before, simultaneously, or after the wort is pitched with the brewers' yeast. At the end of the fermentation and maturation step the beer, which may optionally be subjected to vacuum evaporation to produce a low-alcohol beer, is then pasteurized. An inherent advantage of this method lies in the duration of the fermentation process, which is about 6-15 days (depending on pitching rate, fermentation, temperature, etc), which allows more time for the enzymatic cleavage of non-fermentable sugars, as compared to the short mashing step (2-4 h duration). A further advantage of this method lies in the amount of the enzymatic composition needed to achieve the desired decrease in non- fermentable sugars (and increase in fermentable sugars), which corresponds to a significantly lower number of units of enzymatic activity (e.g. units of glucoamylase activity) than would need to be added to the mash to achieve a similar decrease in non-fermentable sugars. In addition, it removes the difficulties often seen during wort separation, especially by lautering, when high dose rates of glucoamylase are added in the mash.

In one aspect, the disclosure pertains to an enzymatic composition comprising at least one additional enzyme selected among amylase, protease, pullulanase, isoamylase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, phytase and a further glucoamylase.

In a further aspect, the disclosure pertains to an enzymatic composition, wherein the composition comprises at least one additional enzyme selected among alpha-amylase and/or pullulanase.

In a further aspect, the disclosure pertains to an enzymatic composition, wherein the composition further comprises alpha-amylase and pullulanase. In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XU of xylanase activity per GAU of a glucoamylase variant as described herein. If desired, xylanase activity may be reduced by different methods known to the skilled person such as e.g. heat treatment, passing through wheat bran, or other materials, which may selectively adsorb xylanase activity.

In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises less than 400, less than 200, less than 50, less than 20, or less than 2 XU of xylanase activity per gram of the composition.

In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.1-20, 0.1-10, 0.1-5 or 0.2-3 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein.

In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.30-10, 1-8, 3-10 or 5-9 PU of pullulanase activity per GAU of a glucoamylase variant as described herein.

In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.95-20 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein and between 0.30-10 PU of pullulanase activity per GAU of a glucoamylase variant as described herein.

In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.95 - 20 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein and between 0.30 - 10 PU of pullulanase activity per GAU of a glucoamylase variant as described herein and less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XU of xylanase activity per GAU of a glucoamylase variant as described herein.

In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.05-10, 0.1-10, 0.1-8, 0.1-5, 0.1 -3, 0.2-3, 0.2-2 PU of pullulanase activity per GAU of a glucoamylase variant as described herein. In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.1-20, 1-15, 2- 10, 3-10 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein.

In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.05-10 PU of pullulanase activity per GAU of a

glucoamylase variant as described herein and between 0.1-20 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein.

In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.1-5 PU of pullulanase activity per GAU of a glucoamylase variant as described herein and between 1-15 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein.

In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.2-2 PU of pullulanase activity per GAU of a glucoamylase variant as described herein and between 2-10 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein.

In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.05-10 PU of pullulanase activity per GAU of a

glucoamylase variant as described herein and between 0.1-20 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein and less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XU of xylanase activity per GAU of a glucoamylase variant as described herein.

In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.1-5 PU of pullulanase activity per GAU of a glucoamylase variant as described herein and between 1-15 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein and less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XU of xylanase activity per GAU of a glucoamylase variant as described herein. In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.2-2 PU of pullulanase activity per GAU of a glucoamylase variant as described herein and between 2-10 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein and less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XU of xylanase activity per GAU of a glucoamylase variant as described herein.

In one aspect, the glucoamylase variant as described herein is added in an amount of 500 - 20000 GAU/kg grist. In another aspect, the glucoamylase variant as described herein is added in an amount of 750 - 10000 GAU/kg grist. In a further aspect, the glucoamylase variant as described herein is added in an amount of 1000 - 7500 GAU/kg grist.

The present disclosure also provides an animal feed composition or formulation comprising at least one variant glucoamylase encompassed by the disclosure. Methods of using a glucoamylase enzyme in the production of feeds comprising starch are provided in WO 03/049550 (herein incorporated by reference in its entirety). Briefly, the glucoamylase variant is admixed with a feed comprising starch. The glucoamylase is capable of degrading resistant starch for use by the animal. In some embodiments a glucoamylase variant as described herein is used in processes in the generation of fuels based on starch feed stocks. Other objects and advantages of the present disclosure are apparent from the present specification.

Further embodiments according to the invention :

Embodiment 1. Use of a glucoamylase variant comprising two or more amino acid substitutions relative to interconnecting loop 2' with the amino acid sequence from position 518 to position 543 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO: 2 or equivalent sequence of residues in a parent glucoamylase for reducing the synthesis of condensation products during hydrolysis of starch.

Embodiment 2. Use of a glucoamylase variant, which when in its crystal, form has a crystal structure for which the atomic coordinates of the main chain atoms have a root- mean-square deviation from the atomic coordinates of the equivalent main chain atoms of TrGA (as defined in Table 20 in WO2009/067218) of less than 0.13 nm following alignment of equivalent main chain atoms, and which have a linker region, a starch binding domain and a catalytic domain, said variant comprising two or more amino acid substitutions relative to the amino acid sequence of the parent glucoamylase in interconnecting loop 2' of the starch binding domain, and/or in loop 1, and/or in helix 2, and/or in loop 11, and/or in helix 12 of the catalytic domain for reducing the synthesis of condensation products during hydrolysis of starch. Embodiment 3. The use of a glucoamylase variant according to any one of the embodiments 1-2, wherein said two or more amino acid substitutions are relative to the interconnecting loop 2' with the amino acid sequence from position 518 to position 543 of SEQ ID NO: 2, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO: 2, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO: 2, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO: 2, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO: 2.

Embodiment 4. The use of a glucoamylase variant according to any one of the embodiments 1-3, wherein the two or more amino acid substitutions are at least one amino acid substitution in the interconnecting loop 2' and at least one amino acid substitution in loop 1 and/or helix 2 and/or loop 11 and/or helix 12.

Embodiment 5. The use of a glucoamylase variant according to any one of the embodiments 1-4, wherein the two or more amino acid substitutions are 1, 2, 3 or 4 amino acid substitutions in the interconnecting loop 2' and 1, 2, 3 or 4 amino acid substitutions in loop 1 and/or helix 2 and/or loop 11 and/or helix 12.

Embodiment 6. The use of a glucoamylase variant according to any one of the embodiments 1-5, wherein the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in loop 1.

Embodiment 7. The use of a glucoamylase variant according to any one of the embodiments 1-6, wherein the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in helix 2.

Embodiment 8. The use of a glucoamylase variant according to any one of the embodiments 1-7, wherein the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in loop 11.

Embodiment 9. The use of a glucoamylase variant according to any one of the embodiments 1-8, wherein the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in helix 12.

Embodiment 10. The use of a glucoamylase variant according to any one of the embodiments 1-9, wherein the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2' and at least one amino acid substitution in loop 1 and at least one amino acid substitution in helix 2.

Embodiment 11. The use of a glucoamylase variant according to any one of embodiments 1-10, wherein the glucoamylase variant has at least one amino acid

substitution within position 520-543, 530-543, or 534-543 of interconnecting loop 2', the positions corresponding to the respective position in SEQ ID NO: 2 or equivalent positions in a parent glucoamylase.

Embodiment 12. The use of a glucoamylase variant according to any one of embodiments 1-11, wherein the glucoamylase variant has at least one amino acid

substitution within the amino acid sequence of position 30-50, 35-48, or 40-46 of loop 1, the positions corresponding to the respective position in SEQ ID NO: 2 or equivalent positions in a parent glucoamylase.

Embodiment 13. The use of a glucoamylase variant according to any one of embodiments 1-12, wherein the glucoamylase variant has at least one amino acid

substitution within the amino acid sequence of position 50-66, 55-64, or 58-63 of helix 2, the positions corresponding to the respective position in SEQ ID NO: 2 or equivalent positions in a parent glucoamylase.

Embodiment 14. The use of a glucoamylase variant according to any one of embodiments 1-13, wherein the glucoamylase variant has at least one amino acid

substitution within the amino acid sequence of position 405-420, 410-420, or 415-420 of loop 11, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase.

Embodiment 15. The use of a glucoamylase variant according to any one of embodiments 1-14, wherein the glucoamylase variant has at least one amino acid

substitution within the amino acid sequence of position 421-434, 425-434, or 428-434 of helix 12, the positions corresponding to the respective position in SEQ ID NO: 2 or equivalent positions in a parent glucoamylase. Embodiment 16. The use of a glucoamylase variant according to any one of embodiments 1-15, wherein the glucoamylase variant has at least 80%, 85%, 90%, 95%, 98%, or 99.5% sequence identity to the parent glucoamylase.

Embodiment 17. The use of a glucoamylase variant according to any one of embodiments 1-16, wherein the glucoamylase variant has at least 80%, 85%, 90%, 95%, 98%, or 99.5% sequence identity to SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9.

Embodiment 18. The use of a glucoamylase variant to any one of the embodiments

1-17, wherein the glucoamylase variant has a starch binding domain that has at least 96%, 97%, 98%, 99%, or 99.5% sequence identity with the starch binding domain of SEQ ID NO: 1, 2, 11, 385, 386, 387, 388, 389, or 390.

Embodiment 19. The use of a glucoamylase variant according to any one of the embodiments 1-18, wherein the glucoamylase variant has a catalytic domain that has at least 80%, 85%, 90%, 95%, or 99.5% sequence identity with the catalytic domain of SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9. Embodiment 20. The use of a glucoamylase variant according to any one of embodiments 1-19, wherein the glucoamylase variant has at least 80%, 85%, 90%, 95%, 98%, or 99.5% sequence identity to SEQ ID NO:2.

Embodiment 21. The use of a glucoamylase variant according to any one of embodiments 1-20, wherein the condensation product is isomaltose. Embodiment 22. The use of a glucoamylase variant according to any one of embodiments 1-21, wherein the hydrolysis of starch is in a brewing process.

Embodiment 23. The use of a glucoamylase variant according to any one of embodiments 1-22, wherein the glucoamylase exhibit an enhanced production of fermentable sugar(s) as compared to the parent glucoamylase, such as TrGA. Embodiment 24. The use of a glucoamylase variant according to any one of embodiments 1-23, wherein the glucoamylase exhibit an enhanced production of fermentable sugars in a mashing step of the brewing process as compared to the parent glucoamylase, such as TrGA.

Embodiment 25. The use of a glucoamylase variant according to any one of embodiments 1-24, wherein the glucoamylase exhibit an enhanced production of fermentable sugars in a fermentation step of the brewing process as compared to the parent glucoamylase, such as TrGA.

Embodiment 26. The use of a glucoamylase variant according to any one of embodiments 1-25, wherein the fermentable sugar is glucose. Embodiment 27. The use of a glucoamylase variant according to any one of embodiments 1-26, wherein the hydrolysis of starch is in a process for producing glucose syrup.

Embodiment 28. The use of a glucoamylase variant according to any one of embodiments 1-27, wherein the glucoamylase exhibit a reduced ratio between isomaltose synthesis (IS) and starch hydrolysis activity (SH) as compared to the parent glucoamylase, such as TrGA.

Embodiment 29. The use of a glucoamylase variant according to any one of embodiments 1-28, wherein the glucoamylase exhibit a reduced starch hydrolysis activity, which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase, such as TrGA.

Embodiment 30. The use of a glucoamylase variant according to any one of embodiments 1-29, wherein the glucoamylase exhibit an enhanced real degree of

fermentation as compared to the parent glucoamylase such as TrGA.

Embodiment 31. The use of a glucoamylase variant according to any one of embodiments 1-30, wherein the glucoamylase forms a lower amount of condensation products than the amount of condensation products formed by the glucoamylase Aspergillus niger (AnGA) (SEQ ID NO: 6) under comparable conditions.

Embodiment 32. The use of a glucoamylase variant according to any one of embodiments 1-31, wherein the glucoamylase forms an amount of condensation products which amount is essentially the same as, not more than 5% higher, not more than 8% higher or not more than 10% higher than the amount of condensation products formed by

Aspergillus niger (AnGA) (SEQ ID NO: 6) under comparable conditions.

Embodiment 33. The use of a glucoamylase variant according to any one of embodiments 31-32, wherein dosing of the glucoamylases are the same based on protein concentration. Embodiment 34. The use of a glucoamylase variant according to any one of embodiments 31-33, wherein dosing of the glucoamylases are the same based on

measurement of activity in activity assays.

Embodiment 35. The use of a glucoamylase variant according to any one of embodiments 1-34, which glucoamylase variant has an amino acid substitution in position

539 and one or more amino acid substitutions in a position selected from position 44, 61, 417 and 431, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.

Embodiment 36. The use of a glucoamylase variant according to any one of embodiments 1-35, which glucoamylase variant has an amino acid substitution in position 539 and a) an amino acid substitution in position 44 and/or b) amino acid substitutions in both positions 417 and 431, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.

Embodiment 37. The use of a glucoamylase variant according to any one of embodiments 1-36, which glucoamylase variant has an amino acid substitution in position 539 and an amino acid substitution in position 44, the positions corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase.

Embodiment 38. The use of a glucoamylase variant according to any one of embodiments 1-37, which glucoamylase variant has an amino acid substitution in position 539 and amino acid substitutions in positions 417 and 431, the positions corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase.

Embodiment 39. The use of a glucoamylase variant according to any one of embodiments 1-38, which glucoamylase variant has an amino acid substitution in position 539 and amino acid substitutions in positions 44 and 61, the positions corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase.

Embodiment 40. The use of a glucoamylase variant according to any one of embodiments 1-39, which glucoamylase variant has an amino acid substitution in position 43, the position corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase. Embodiment 41. The use of a glucoamylase variant according to any one of embodiments 1-40, which glucoamylase variant has an amino acid substitution in position 61, the position corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase.

Embodiment 42. The use of a glucoamylase variant according to any one of embodiments 1-41, wherein the amino acid substitution in position 539 is 539R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.

Embodiment 43. The use of a glucoamylase variant according to any one of embodiments 1-42, wherein the amino acid substitution in position 44 is 44R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.

Embodiment 44. The use of a glucoamylase variant according to any one of embodiments 1-43, wherein the amino acid substitution in position 417 is 417R/V, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. Embodiment 45. The use of a glucoamylase variant according to any one of embodiments 1-44, wherein the amino acid substitution in position 417 is 417R, the position corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase.

Embodiment 46. The use of a glucoamylase variant according to any one of embodiments 1-45, wherein the amino acid substitution in position 417 is 417V, the position corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase.

Embodiment 47. The use of a glucoamylase variant according to any one of embodiments 1-46, wherein the amino acid substitution in position 431 is 431L, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.

Embodiment 48. The use of a glucoamylase variant according to any one of embodiments 1-47, wherein the amino acid substitution in position 43 is 43R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. Embodiment 49. The use of a glucoamylase variant according to any one of embodiments 1-48, wherein the amino acid substitution in position 61 is 611, the position corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase. Embodiment 50. A glucoamylase variant as defined in any one of embodiments 1-

49.

Embodiment 51. A glucoamylase variant comprising two or more amino acid substitutions, wherein an amino acid substitution is in position 539 and an amino acid substitution is in position 44, the positions corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase, and which sequence has at least 80% sequence identity to the parent glucoamylase, and wherein the amino acid substitution in position 44 is not 44C.

Embodiment 52. The glucoamylase variant according to embodiment 51 comprising two or more amino acid substitutions, wherein an amino acid substitution is in position 539 and an amino acid substitution is 44R, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.

Embodiment 53. The glucoamylase variant according to any one of embodiments

51-52 comprising an amino acid substitution in position 61, the position corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase. Embodiment 54. The glucoamylase variant according to any one of embodiments

51-53, wherein the glucoamylase variant has at least 85%, 90%, 95%, 98%, or 99.5% sequence identity to the parent glucoamylase.

Embodiment 55. The glucoamylase variant according to any one of embodiments

51-54, wherein the glucoamylase variant has at least 85%, 90%, 95%, 98%, or 99.5% sequence identity to SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9.

Embodiment 56. The glucoamylase variant according to any one of embodiments

51-55, wherein the glucoamylase variant has at least 85%, 90%, 95%, 98%, or 99.5% sequence identity to SEQ ID NO:2.

Embodiment 57. The glucoamylase variant according to any one of embodiments 51-56, wherein the amino acid substitution in position 539 is 539R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.

Embodiment 58. The glucoamylase variant according to any one of embodiments

51-57, wherein the amino acid substitution in position 44 is 44R, the position corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase.

Embodiment 59. The glucoamylase variant according to any one of embodiments

51-58, wherein the amino acid substitution in position 61 is 611, the position corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase.

Embodiment 60. The glucoamylase variant according to any one of embodiments 51-59 comprising the following amino acid substitutions: a. D44R and A539R; or b. D44R, N61I and A539R, the positions corresponding to the respective position in SEQ ID NO: 2 or an equivalent position in a parent glucoamylase. Embodiment 61. The glucoamylase variant according to any one of embodiments

51-60 consisting of SEQ ID NO: 2 and having the following amino acid substitutions: a. D44R and A539R; or b. D44R, N61I and A539R, the positions corresponding to the respective position in SEQ ID NO: 2. Embodiment 62. The glucoamylase variant according to any one of embodiments

51-61, wherein the glucoamylase variant has a starch binding domain that has at least 96%, 97%, 98%, 99%, or 99.5% sequence identity with the starch binding domain of SEQ ID NO: 1, 2, 11, 385, 386, 387, 388, 389, or 390.

Embodiment 63. The glucoamylase variant according to any one of embodiments 51-62, wherein the glucoamylase variant has a catalytic domain that has at least 80%, 85%, 90%, 95%, or 99.5% sequence identity with the catalytic domain of SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9. Embodiment 64. The glucoamylase variant according to any one of embodiments

50-63, wherein the parent glucoamylase is selected from a glucoamylase obtained from a Trichoderma spp., an Aspergillus spp., a Humicola spp., a Penicillium spp., a Talaromyces spp., or a Schizosaccharmyces spp. Embodiment 65. The glucoamylase variant according to any one of embodiments

50-64, wherein the parent glucoamylase is obtained from a Trichoderma spp. or an

Aspergillus spp.

Embodiment 66. The glucoamylase variant according to any one of embodiments

50-65, which glucoamylase exhibit an enhanced production of fermentable sugar(s) as compared to the parent glucoamylase such as TrGA.

Embodiment 67. The glucoamylase variant according to any one of embodiments

50-66, which glucoamylase exhibit an enhanced production of fermentable sugars in the mashing step of the brewing process as compared to the parent glucoamylase such as TrGA.

Embodiment 68. The glucoamylase variant according to any one of embodiments 50-67, which glucoamylase exhibit an enhanced production of fermentable sugars in the fermentation step of the brewing process as compared to the parent glucoamylase such as TrGA.

Embodiment 69. The glucoamylase variant according to embodiment 68, wherein the fermentable sugar is glucose. Embodiment 70. The glucoamylase variant according to any one of embodiments

50-69, which glucoamylase exhibit a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase such as TrGA.

Embodiment 71. The glucoamylase variant according to any one of embodiments

50-70, which glucoamylase exhibit a reduced starch hydrolysis activity which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase such as TrGA.

Embodiment 72. The glucoamylase variant according to any one of embodiments

50-71, which glucoamylase exhibit an enhanced real degree of fermentation as compared to the parent glucoamylase such as TrGA. Embodiment 73. The glucoamylase variant according to any one of embodiments

50-72, which glucoamylase forms a lower amount of condensation products than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under the same conditions. Embodiment 74. The glucoamylase variant according to any one of embodiments

50-73, which glucoamylase forms an amount of condensation products which amount is essentially the same as, not more than 5%, not more than 8%, or not more than 10% higher than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under the same conditions. Embodiment 75. The glucoamylase variant according to any one of embodiments

73-74, wherein the dosing of the glucoamylases are the same based on protein

concentration.

Embodiment 76. The glucoamylase variant according to any one of embodiments

73-74, wherein the dosing of the glucoamylases are the same based on measurement of activity in activity assays.

Embodiment 77. The glucoamylase variant according to any one of embodiments

50-76, which glucoamylase has been purified.

Embodiment 78. A polynucleotide encoding a glucoamylase variant according to any of embodiments 50-77. Embodiment 79. A vector comprising the polynucleotide according to embodiment

78, or capable of expressing a glucoamylase variant according to any of embodiments 50-77.

Embodiment 80. A host cell comprising a vector according to embodiment 79.

Embodiment 81. A host cell which has stably integrated into the chromosome a nucleic acid encoding the variant glucoamylase according to any of embodiments 50-80. Embodiment 82. A cell capable of expressing a glucoamylase variant according to any one of embodiments 50-76.

Embodiment 83. The host cell according to any one of embodiments 78-81, or the cell according to embodiment 81, which is a bacterial, fungal or yeast cell. Embodiment 84. The host cell according to embodiment 83, which is Trichoderma spp. such as Trichoderma reesei.

Embodiment 85. The host cell according to any one of embodiments 83-84, which is a protease deficient and/or xylanase deficient and/or native glucanase deficient host cell. Embodiment 86. A method of expressing a glucoamylase variant, the method comprising obtaining a host cell or a cell according to any one of embodiments 80-85 and expressing the glucoamylase variant from the cell or host cell, and optionally purifying the glucoamylase variant.

Embodiment 87. The method according to embodiment 86 comprising purifying the glucoamylase variant.

Embodiment 88. Use of a glucoamylase variant according to any one of embodiments 50-76 for the preparation of an enzymatic composition.

Embodiment 89. An enzymatic composition comprising at least one glucoamylase variant according to any one of embodiments 50-77. Embodiment 90. The enzymatic composition according to embodiment 89 comprising at least one glucoamylase variant according to any one of embodiments 50-77, wherein the composition is selected from among a starch hydrolyzing composition, a saccharifying composition, a detergent, an alcohol fermentation enzymatic composition, and an animal feed. Embodiment 91. The enzymatic composition according to embodiment 90, which is a starch hydrolyzing composition.

Embodiment 92. The enzymatic composition according to any one of embodiments

89-91 comprising at least one additional enzyme selected among amylase, protease, pullulanase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, and a further glucoamylase.

Embodiment 93. The enzymatic composition according to embodiment 89-92, wherein the at least one additional enzyme is selected among amylase, pullulanase, and a further glucoamylase. Embodiment 94. The enzymatic composition according to embodiment 89-93, wherein the at least one additional is selected among amylase and pullulanase.

Embodiment 95. The enzymatic composition according to any one of embodiments

89-94, wherein the amylase is selected among alpha-amylase, and isoamylase. Embodiment 96. A method for converting starch or partially hydrolyzed starch into a syrup containing glucose, said process including saccharifying a liquid starch solution in the presence of at least one glucoamylase variant according to any one of embodiments 50-77 or an enzymatic composition according to any one of embodiments 89-95.

Embodiment 97. The method according to embodiment 96 of saccharifying a liquid starch solution, which comprises an enzymatic saccharification step using a glucoamylase variant according to embodiment 50-77 or an enzymatic composition according to any one of embodiments 89-95.

Embodiment 98. The method according to any one of embodiments 96-97, further comprising contacting the liquid starch solution with at least one additional enzyme. Embodiment 99. The method according to embodiment 98, wherein the additional enzyme is selected among amylase, protease, pullulanase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, and glucoamylase.

Embodiment 100. The method according to embodiment 96-99, wherein the additional enzyme is amylase and pullulanase. Embodiment 101. The method according to embodiment any one of embodiments

96-100, wherein the amylase is selected among alpha-amylase, and isoamylase.

Embodiment 102. Use of a glucoamylase variant according to any one of

embodiments 50-77 in a starch conversion process, such as a in a continuous starch conversion process.

Embodiment 103. Use of a glucoamylase variant according to any one of

embodiments 50-77 in a process for producing oligosaccharides, maltodextrins, or glucose syrups.

Embodiment 104. Use of a glucoamylase variant according to any one of

embodiments 50-77 in a process for producing high fructose corn syrup. Embodiment 105. A method for producing a wort for brewing comprising forming a mash from a grist, and contacting the mash with a glucoamylase variant according to any one of embodiments 50-77 or an enzymatic composition according to any one of

embodiments 89-95. Embodiment 106. The method of embodiment 105, further comprising contacting the mash with one or more additional enzyme(s)

Embodiment 107. The method according to embodiment 106, wherein the one or more enzyme(s) is selected among amylase, protease, pullulanase, cellulase, endoglucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, and glucoamylase. Embodiment 108. The method according to embodiment 107, wherein the one or more enzyme(s) is amylase and/or pullulanase.

Embodiment 109. The method according to embodiment any one of embodiments

107-108, wherein the amylase is alpha-amylase and/or isoamylase.

Embodiment 110. The method according to any one of embodiments 105-109, wherein the grist comprises one ore more of malted grain, unmalted grain, adjunct, and any combination thereof.

Embodiment 111. The method of any one of embodiments 105-110, further comprising fermenting the wort to obtain a fermented beverage.

Embodiment 112. The method of any one of embodiments 105-111, further comprising fermenting the wort to obtain a beer.

Embodiment 113. A method for production of a beer which comprises: a. preparing a mash, b. filtering the mash to obtain a wort, and c. fermenting the wort to obtain a beer, wherein a glucoamylase variant according to any one of embodiments 50-77 is added to: step (a) and/or step (b) and/or step (c). Embodiment 114. The method of embodiment 113, wherein the beer is subjected to a pasteurization step.

Embodiment 115. Use of a glucoamylase variant according to any one of embodiments 50-77 to enhance the production of fermentable sugars in either the mashing step or the fermentation step of a brewing process.

Embodiment 116. A beer, wherein the beer is produced by the steps of: a. preparing a mash, b. filtering the mash to obtain a wort, c. fermenting the wort to obtain a beer, and d. pasteurizing the beer, wherein a glucoamylase variant according to any one of embodiments 50-77 is added to: step (a) and/or step (b) and/or step (c).

Embodiment 117. The beer of embodiment 116, wherein the pasteurized beer is further characterized as being : a. essentially without glucoamylase activity; and/or b. a low-calorie beer and/or a low-alcohol beer.

Embodiment 118. Use of a glucoamylase variant according to any one of embodiments 50-77 in an alcohol fermentation process.

Embodiment 119. A screening method for identification of a glucoamylase variant having a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase.

Embodiment 120. A screening method for identification of a glucoamylase variant having the same or increased starch hydrolysis activity and reduced isomaltose synthesis, which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase and having a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase. Embodiment 121. A screening method for identification of a glucoamylase variant having a reduced synthesis of condensation products during hydrolysis of starch, the method comprising the steps of measuring the isomaitose synthesis and starch hydrolysis activity of glucoamylase variants and selecting the variants having a reduced starch hydrolysis activity which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase and having a reduced ratio between isomaitose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase.

Embodiment 122. The glucoamylase variant obtained by the method according to any one of embodiments 119-121. Further embodiments also part of the invention:

Further embodiment 1. A glucoamylase variant comprising the following amino acid substitutions: a. 44R and 539R; or b. 44R, 611 and 539R, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase, wherein the glucoamylase variant has at least 80% sequence identity with SEQ ID NO: 1 or 2, or the parent glucoamylase.

Further embodiment 2. The glucoamylase variant according to further embodiment 1 comprising the following amino acid substitutions: a. D44R and A539R; or b. D44R, N61I and A539R, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase, wherein the glucoamylase variant has at least 80% sequence identity with SEQ ID NO: 1 or 2, or the parent glucoamylase. Further embodiment 3. The glucoamylase variant according to any one of further embodiments 1-2 comprising the following amino acid substitutions: a. D44R, N61I and A539R, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase, wherein the glucoamylase variant has at least 80% sequence identity with SEQ ID NO: 1 or 2, or the parent glucoamylase.

Further embodiment 4. The glucoamylase variant according to any one of further embodiments 1-2 comprising the following amino acid substitutions: a. D44R and A539 , the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase, wherein the glucoamylase variant has at least 80% sequence identity with SEQ ID NO: 1 or 2, or the parent glucoamylase. Further embodiment 5. The glucoamylase variant of any one of further embodiments 1-4, wherein the glucoamylase variant has at least 85% or 90% sequence identity with SEQ ID NO: 1 or 2.

Further embodiment 6. The glucoamylase variant of further embodiment 5, wherein the glucoamylase variant has at least 95% sequence identity with SEQ ID NO: 1 or 2. Further embodiment 7. The glucoamylase variant of further embodiment 6, wherein the glucoamylase variant has at least 99.5% sequence identity with SEQ ID NO: 1 or 2.

Further embodiment 8. The glucoamylase variant of any one of further embodiments 1-7, wherein the parent glucoamylase comprises SEQ ID NO: 1 or 2.

Further embodiment 9. The glucoamylase variant of further embodiment 8, wherein the parent glucoamylase consists of SEQ ID NO: 1 or 2.

Further embodiment 10. The glucoamylase variant according to any one of further embodiments 1-9, wherein the glucoamylase variant has a starch binding domain that has at least 96%, 97%, 98%, 99%, or 99.5% sequence identity with the starch binding domain of SEQ ID NO: 1, 2, 11, 385, 386, 387, 388, 389, or 390. Further embodiment 11. The glucoamylase variant according to any one of further embodiments 1-10, wherein the glucoamylase variant has a catalytic domain that has at least 80%, 85%, 90%, 95%, or 99.5% sequence identity with the catalytic domain of SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9. Further embodiment 12. The glucoamylase variant according to any one of further embodiments 1-11, wherein the parent glucoamylase is selected from a glucoamylase obtained from a Trichoderma spp., an Aspergillus spp., a Humicola spp., a Penicillium spp., a Talaromyces spp., or a Schizosaccharmyces spp. Further embodiment 13. The glucoamylase variant according to any one of further embodiments 1-12, wherein the parent glucoamylase is obtained from a Trichoderma spp. or an Aspergillus spp.

Further embodiment 14. The glucoamylase variant according to any one of further embodiments 1-13, which glucoamylase exhibit an enhanced production of fermentable sugar(s) as compared to the parent glucoamylase.

Further embodiment 15. The glucoamylase variant according to any one of further embodiments 1-14, which glucoamylase exhibit an enhanced production of fermentable sugars in the mashing step of the brewing process as compared to the parent glucoamylase.

Further embodiment 16. The glucoamylase variant according to any one of further embodiments 1-15, which glucoamylase exhibit an enhanced production of fermentable sugars in the fermentation step of the brewing process as compared to the parent glucoamylase.

Further embodiment 17. The glucoamylase variant according to further embodiment 16, wherein the fermentable sugar is glucose. Further embodiment 18. The glucoamylase variant according to any one of further embodiments 1-17, which glucoamylase exhibit a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase.

Further embodiment 19. The glucoamylase variant according to any one of further embodiments 1-18, which glucoamylase exhibit a reduced starch hydrolysis activity which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase.

Further embodiment 20. The glucoamylase variant according to any one of further embodiments 1-19, which glucoamylase exhibit an enhanced real degree of fermentation as compared to the parent glucoamylase. Further embodiment 21. The glucoamylase variant according to any one of further embodiments 1-20, which glucoamylase forms a lower amount of condensation products than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under the same conditions. Further embodiment 22. The glucoamylase variant according to any one of further embodiments 1-21, which glucoamylase forms an amount of condensation products which amount is essentially the same as, not more than 5%, not more than 8%, or not more than 10% higher than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under the same conditions. Further embodiment 23. The glucoamylase variant according to any one of further embodiments 18-21, wherein the dosing of the glucoamylases are the same based on protein concentration.

Further embodiment 24. The glucoamylase variant according to any one of further embodiments 18-23, wherein the dosing of the glucoamylases are the same based on measurement of activity in activity assays.

Further embodiment 25. The glucoamylase variant according to any one of further embodiments 1-24, which glucoamylase has been purified.

Further embodiment 26. A polynucleotide encoding a glucoamylase variant according to any of further embodiments 1-25. Further embodiment 27. A vector comprising the polynucleotide according to further embodiment 26, or capable of expressing a glucoamylase variant according to any of further embodiments 1-25.

Further embodiment 28. A host cell comprising a vector according to further embodiment 27. Further embodiment 29. A host cell which has stably integrated into the chromosome a nucleic acid encoding the variant glucoamylase according to any of further embodiments 1- 25.

Further embodiment 30. A cell capable of expressing a glucoamylase variant according to any one of further embodiments 1-25. Further embodiment 31. The host cell according to any one of further embodiments 28-29, or the cell according to further embodiment 30, which is a bacterial, fungal or yeast cell.

Further embodiment 32. The host cell according to further embodiment 31, which is Trichoderma spp. such as Trichoderma reesei.

Further embodiment 33. The host cell according to any one of further embodiments 28-29 and 31-32, which is a protease deficient and/or xylanase deficient and/or glucanase deficient host cell.

Further embodiment 34. A method of expressing a glucoamylase variant, the method comprising obtaining a host cell or a cell according to any one of further embodiments 28-33 and expressing the glucoamylase variant from the cell or host cell, and optionally purifying the glucoamylase variant.

Further embodiment 35. The method according to further embodiment 34 comprising purifying the glucoamylase variant.

Further embodiment 36. Use of a glucoamylase variant according to any one of further embodiments 1-25 for the preparation of an enzymatic composition.

Further embodiment 37. An enzymatic composition comprising at least one glucoamylase variant according to any one of further embodiments 1-25.

Further embodiment 38. An enzymatic composition comprising at least one glucoamylase variant according to any one of embodiments 1-25, said enzyme composition comprising one or more further enzymes.

Further embodiment 39. The enzymatic composition according to any one of further embodiments 37-38 comprising at least one glucoamylase variant according to any one of further embodiments 1-25, wherein the composition is selected from among a starch hydrolyzing composition, a saccharifying composition, a detergent composition, an alcohol fermentation enzymatic composition, and an animal feed composition.

Further embodiment 40. An enzymatic composition according to any one of further embodiments 36-39 comprising at least one additional enzyme selected among amylase, protease, pullulanase, isoamylase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, phytase and a further glucoamylase. Further embodiment 41. The enzymatic composition according to any one of further embodiments 36-40, wherein the composition comprises at least one additional enzyme selected among alpha-amylase and/or pullulanase.

Further embodiment 42. The enzymatic composition according to any one of further embodiments 36-41, wherein the composition comprises alpha-amylase and pullulanase.

Further embodiment 43. The enzymatic composition according to any one of further embodiments 36-42, which enzymatic composition comprises less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XU of xylanase activity per GAU of a glucoamylase variant according to any one of further embodiments 1-25.

Further embodiment 44. The enzymatic composition according to any one of further embodiments 36-43, which enzymatic composition comprises less than 400, less than 200, less than 50, less than 20, or less than 2 XU of xylanase activity per gram of the composition.

Further embodiment 45. The enzymatic composition according to any one of further embodiments 36-44, which enzymatic composition comprises between 0.1 - 20, 1-15, 2-10, or 3-10 SSU of alpha-amylase activity per GAU of a glucoamylase variant according to any one of further embodiments 1-25.

Further embodiment 46. The enzymatic composition according to any one of further embodiments 36-45, which enzymatic composition comprises between 0.05 - 10, 0.1 - 10, 0.1-8, 0.1-5, 0.1 -3, 0.2-3, or 0.2-2 PU of pullulanase activity per GAU of a glucoamylase variant according to any one of further embodiments 1-25.

Further embodiment 47. The enzymatic composition according to any one of further embodiments 36-46, which enzymatic composition comprises between 0.05 - 10 PU of pullulanase activity per GAU of a glucoamylase variant according to any one of further embodiments l-25and between 0.1 - 20 SSU of alpha-amylase activity per GAU of a glucoamylase variant according to any one of further embodiments 1-25.

Further embodiment 48. The enzymatic composition according to any one of further embodiments 36-47, which enzymatic composition comprises between 0.05 - 10 PU of pullulanase activity per GAU of a glucoamylase variant according to any one of further embodiments l-25and between 0.1 - 20 SSU of alpha-amylase activity per GAU of a glucoamylase variant according to any one of further embodiments 1-25 and less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XU of xylanase activity per GAU of a glucoamylase according to any one of further embodiments 1-25.

Further embodiment 49. A method for producing a wort for brewing comprising forming a mash from a grist, and contacting the mash with a glucoamylase variant according to any one of further embodiments 1-25 or an enzymatic composition according to any one of further embodiments 36-48.

Further embodiment 50. The method of further embodiment 49, further comprising contacting the mash with one or more additional enzyme(s)

Further embodiment 51. The method according to further embodiment 50, wherein the one or more enzyme(s) is selected among amylase, protease, pullulanase, isoamylase, cellulase, endoglucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, phytase and glucoamylase.

Further embodiment 52. The method according to further embodiment 51, wherein the one or more enzyme(s) is/are alpha-amylase and/or pullulanase. Further embodiment 53. The method according to any one of further embodiments 49-52, wherein the grist comprises one ore more of malted grain, unmalted grain, adjunct, and any combination thereof.

Further embodiment 54. The method of any one of further embodiments 49-53, further comprising fermenting the wort to obtain a fermented beverage. Further embodiment 55. The method of any one of further embodiments 49-54, further comprising fermenting the wort to obtain a beer.

Further embodiment 56. A method for production of a beer which comprises: a. preparing a mash, b. filtering the mash to obtain a wort, and c. fermenting the wort to obtain a beer, wherein a glucoamylase variant according to any one of further embodiments 1-25 or an enzymatic composition according to any one of further embodiments 36-48 is added to: step (a) and/or step (b) and/or step (c).

Further embodiment 57. The method of further embodiment 55, wherein the beer is subjected to a pasteurization step.

Further embodiment 58. Use of a glucoamylase variant according to any one of further embodiments 1-25 or an enzymatic composition according to any one of further

embodiments 36-48 to enhance the production of fermentable sugars in either the mashing step or the fermentation step of a brewing process.

Further embodiment 59. A beer, wherein the beer is produced by the steps of: a. preparing a mash, b. filtering the mash to obtain a wort, c. fermenting the wort to obtain a beer, and d. pasteurizing the beer, e. wherein a glucoamylase variant according to any one of further embodiments 1- 25 or an enzymatic composition according to any one of further embodiments 36- 48 is added to: step (a) and/or step (b) and/or step (c).

Further embodiment 60. The beer of further embodiment 59, wherein the pasteurized beer is further characterized as being: a. essentially without glucoamylase activity; and/or b. a low-calorie beer and/or a low-alcohol beer.

The invention will now be further described by way of the following non-limiting examples. EXAMPLES

Assays and Methods

The following assays and nnethods are used in the examples provided below. The methods used to provide variants are described below. However, it should be noted that different methods may be used to provide variants of a parent enzyme and the invention is not limited to the methods used in the examples. It is intended that any suitable means for making variants and selection of variants may be used. pNPG glucoamylase activity assay for 96-well microtiter plates

The reagent solutions were: NaAc buffer: 200 mM sodium acetate buffer pH 4.5; Substrate: 50 mM p-nitrophenyl-a-D-glucopyranoside (Sigma N-1377) in NaAc buffer (0.3 g/20 ml) and stop solution : 800 mM glycine-NaOH buffer pH 10. 30 μΙ filtered supernatant was placed in a fresh 96-well flat bottom micro titer plate (MTP). To each well 50 μΙ NaAc buffer and 120 μΙ substrate was added and incubated for 30 minutes at 50°C (Thermolab systems iEMS Incubator/shaker HT). The reaction was terminated by adding 100 μΙ stop solution. The absorbance was measured at 405 nm in a MTP-reader (Molecular Devices Spectramax 384 plus) and the activity was calculated using a molar extinction coefficient of 0.011 μΜ/cm.

Thermal stability assay 1

With a stock dilution of 150 ppm of purified enzyme (in 50 mM NaAc pH 4.0), a 3 ppm dilution was made by adding 6 μΙ to 294 μΙ 50 mM NaAc buffer pH 4.5. The diluted sample was equally divided over 2 MTPs. One MTP (initial plate) was incubated for 1 hr at 4°C and the other MTP (residual plate) was incubated at 64°C (Thermolab systems iEMS

Incubator/Shaker HT) for 1 hr. The residual plate was chilled for 10 min on ice. 60 μΙ of both the initial plate and the residual plate was added to 120 μΙ 4% soluble corn starch pH 3.7 and incubated for 2 hrs at 32°C, 900 rpm in 2 separate MTPs (Thermolabsystems iEMS

Incubator/Shaker HT). Activity of both plates was measured in the Hexokinase activity assay, using the ethanol application assay described below.

Thermal stability was calculated as % residual activity as follows: ABS 340 residual - blank

X IUU 0

A BS . n initial - blank Hexokinase activity assay

Hexokinase cocktail : 10 - 15 minutes prior to use, 90 ml water was added to a BoatIL container glucose HK Rl (IL test glucose (HK) kit, Instrument Laboratory # 182507-40) and gently mixed. 100 μΙ of Hexokinase cocktail was added to 85 μΙ of dH 2 0. 15 μΙ of sample was added to the mixtures and incubated for 10 minutes in the dark at room temperature. Absorbance was read at 340 nm in a MTP-reader after 10 minutes. Glucose concentrations were calculated according to a glucose (0 - 1.6 mg/ml) standard curve.

Ethanol application— glucose release from corn starch

8% stock solution : 8 g of soluble corn starch (Sigma #S4180) was suspended in 40 ml dH 2 0 at room temperature. The slurry was added in portions to 50 ml of boiling dH 2 0 in a 250 ml flask and cooked for 5 minutes. The starch solution was cooled to 25°C while stirring and the volume adjusted with remain 10 ml of dH 2 0.

Stop solution : 800 mM Glycine-NaOH buffer, pH 10.

4% (m/v) soluble starch working solution : stock solution was diluted (1 : 1) with 100 mM sodium acetate buffer pH 4.0.

6 μΙ of 150 ppm purified enzyme was diluted with 294 μΙ 50mM NaAc buffer pH 4.0 in a flat bottom 96-well MTP. 60 μΙ of this dilution was added to 120 μΙ 4% soluble corn starch pH 4.0 and incubated for 2 hrs at 32°C 900 rpm (Thermolabsystems iEMS Incubator/Shaker HT). The reaction was stopped by adding 90 μΙ 4°C-cold Stop Solution. The sample was placed on ice for 20 minutes. Starch was spun down at 1118 χ g at 10°C for 5 minutes (SIGMA 6K15) and 15 μΙ supernatant was used in the Hexokinase activity assay described above to determine the glucose content.

Data analysis and calculation of performance index of ethanol screening assay

Protein levels were measured using a microfluidic electrophoresis instrument (Caliper Life Sciences, Hopkinton, MA, USA). The microfluidic chip and protein samples were prepared according to the manufacturer's instructions (LabChip® HT Protein Express, P/N 760301). Culture supernatants were prepared and stored in 96-well microtiter plates at -20°C until use, when they were thawed by warming in a 37°C incubator for 30 minutes. After shaking briefly, 2 μΙ of each culture sample was transferred to a 96-well PCR plate (Bio-Rad, Hercules, CA,USA) containing 7 μΙ samples buffer (Caliper) followed by heating the plate to 90°C for 5 minutes on a thermostatically controlled plate heater. The plate was allowed to cool before adding 40 μΙ water to each sample. The plate was then placed in the instrument along with a protein standard supplied and calibrated by the manufacturer. As the proteins move past a focal point in the chip, the fluorescence signal is recorded and the protein concentration is determined by quantitating the signal relative to the signal generated by the calibrated set of protein standards.

After the Caliper protein determination the data is processed in the following way.

The calibration ladders are checked for correctness of the peak pattern. If the calibration ladder that is associated with the run does not suffice, it is replaced by a calibration ladder of an adjacent run. For peak detection, the default settings of the global peak find option of the caliper software are used . The peak of interest is selected at 75 kDA +/-10%. The result is exported to a spreadsheet program and the peak area is related to the corresponding activity (ABS340-blank measurement) in the ethanol screening assay.

With the area and activity numbers of 12 Wild Type samples, a calibration line is made using the "Enzyme Kinetics" equation of the program Grafit Version 5 (Erithacus Software, Horley, UK) in combination with a non-linear fit function. The default settings are used to calculate the Km and Vmax parameters. Based on these two parameters, a Michaelis-Menten reference line is made and the specific activity of each variant is calculated.

Based on the specific activity the performance index (PI) is calculated. The PI of a variant is the quotient "Variant-specific activity/WT-specific activity." The PI of WT is 1.0 and a variant with a PI > 1.0 has a specific activity that is greater than WT.

Purification of TrGA variants

Culture supernatants of expressed TrGA variants were purified in one step by affinity chromatography using an AKTA explorer 100 FPLC system (Amersham Biosciences,

Piscataway, NJ). β-cyclodextrin (Sigma-Aldrich, Zwijndrecht, The Netherlands; 85.608-8) was coupled to epoxy activated Sepharose beads (GE Healthcare, Diegem, Belgium; 17- 0480-01) and employed for purification. The column was equilibrated with 25 mM sodium acetate buffer pH 4.3 followed by application of concentrated enzyme sample. Bound variants were eluted from the column with 25 mM sodium acetate buffer pH 4.3 containing 10 mM a-cyclodextrin (Sigma, 28705). Purified samples were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Protein quantification of purified TrGA variants The protein concentration of purified TrGA variants was determined by anion exchange chromatography using an AKTA explorer 100 FPLC system. Purified sample was injected onto a ResourceQ_lml column (GE Healthcare) and a linear gradient of 0 to 500 mM NaCI in 25 mM sodium acetate buffer pH 4.3 was applied to elute bound protein. The peak area was determined and the protein concentration was calculated relative to a TrGA standard with know concentration.

Liquefact assay

Glucose release of the variants was determined on corn mash liquefact from a local ethanol producer in a 6-well plate. Each well of the plate was filled with 6 g of 26% DS liquefact pH 4.3. Subsequently, 300 ppm enzyme and 400 ppm urea was added and 250 μΙ sample was collected after 2, 4 and 6 hr incubation at 32°C. The sample was centrifuged for 5 minutes at 14.000 x g and 50 μΙ of the supernatant was transferred to an eppendorf tube containing 50 μΙ of kill solution ( 1.1 N sulfuric acid) and allowed to stand for 5 minutes. 250 μΙ of water was added to the tube and then filtered with a 0.22 μηι filter plate and injected onto an HPX- 87H column as described below.

Evaluation of performance of TrGA variant in ethanol fermentations

A sample of corn mash liquefact from a local ethanol producer was obtained and diluted in some cases to 26% DS using thin stillage. The pH of the slurry was adjusted to pH 4.3 using 4 N sulfuric acid. A lOOg or 50g aliquot of mash was put into a 125 ml shake flask and placed into a 32°C incubator and allowed to equilibrate. After addition of 100 μΙ 400 ppm urea, 1 ml purified variant at intended concentration or purified TrGA at 2 different concentrations was added to the shake flasks. Finally, 333 μΙ of a solution of Red Star Red yeast ( 15 g hydrated for 30 minutes in 45 ml DI water; Lesaffre yeast Corp. Milwaukee, WI) was added to each sample. Samples were collected at 5, 21, 28, 48 and 52 hours and analyzed by HPLC (Agilent 1200 series) using an Aminex HPX-87H column (Bio-Rad).

Ethanol and carbohydrate determinations

A 2 ml eppendorf centrifuge tube was filled with fermentor beer and cooled on ice for 10 minutes. The sample was centrifuged for 3 minutes at 14.000 χ g and 500 μΙ of the supernatant was transferred to a test tube containing 50 μΙ of kill solution (1.1 N sulfuric acid) and allowed to stand for 5 minutes. 5.0 ml of water was added to the test tube and then filtered into a 0.22 μσι filter plate (multiscreen, Millipore, Amsterdam, the Netherlands) and run on HPLC. Column Temperature: 60°C; mobile phase: 0.01 N sulfuric acid; flow rate 0.6 ml/min; detector: RI; injection volume: 20 μΙ. The column separates molecules based on charge and molecular weight; DPI (monosaccharides); DP2 (disaccharides); DP3 (trisaccharides); DP>3 (oligosaccharides sugars having a degree of polymerization greater than 3); succinic acid; lactic acid; glycerol; methanol; ethanol.

Determination of GAU activity Substrate: p-Nitrophenyl-p-maltoside (4 mM), plus thermostable β-glucosidase (5 U/ml) (from assay R-A GR3 05/04; Megazyme International Wicklow, Ireland) was freshly prepared

Buffer. 200 mM Sodium acetate buffer (pH 4.5).

Enzyme samples were diluted by at least a factor 10 in sodium acetate buffer In a 96 well plate: 20 μΙ_ substrate was mixed with 20 μΐ enzyme solution and incubate at 40°C with agitation for 10 minutes. 300 μΙ_ 2% Trizma base was added to terminate reaction and develop the colour. Absorbance at 400nm was measured against a reagent blank.

Blanks were prepared by adding 300 μΙ_ of Trizma base solution (2%) to 20 pL of substrate with vigorous stirring, followed by the enzyme solution (20 μ1_). Activity was calculated as follows:

Δ 400 340 1 1 _ .. .

Activitv (GAU/mL) = — Dilution

LU Ly ' 10 20 18 .1 0,88

Where: GAU = International units of enzyme activity. One Unit is the amount of enzyme which release one pmole of p-nitrophenol from the substrate per minute at the defined pH and temperature. ΔΑ, 00 = absorbance (reaction) - Absorbance (blank). 10 = incubation time (min). 340 = final reaction volume (μί). 20 = volume of enzyme assayed (μί.) 18.1 = fmM p-nitrophenol in 2% trizma base (pH ~ 8.5) at 400 nm (unit: μΜ ' ^ατ 1 ). 0.88 = Light path (cm)

Starch hydrolysis activity (SH activity):

Buffer. 0.1 M Citrate buffer pH 5.4 (made from 0.1 M citric acid and 0.1 M Tri-sodium citrate) Substrate: 30% soluble starch (Merck, v.nr 1.01257.1000) in buffer (heat slightly until all starch is in solution)

Enzyme: Glucoamylase standardised to 3GAU/ml on basis of above assay.

60 pL 30% starch was transferred to a 96 well PCR plate. 60 L enzyme sample or standard was added and mixed by pumping a couple of times with the pipette. Following steps until incubation wes carried uot as fast as possible.

The PCR plate was covered with sealing tape and following PCR programme was run : 6 min at 63°C, 6 min at 99°C and 10 min at 4°C. Lid was not heated . After the temperature cycle the PCR plate was centrifuged (app 1 min at 300 rpm) to collect all liquid in the bottom of the wells. Plates were stored at 4°C until further analysis. Glucose concentration was measured according to method below and the hydrolysis activity was calculated as follows:

[glu cose]—

Starch hydrolysis activity (M/min) = —

180 J>- x 6 min

mol

Isomaltose hydrolysis activity Same as for starch hydrolysis activity except that substrate is 2% iso-maltose (Sigma 17253) and the first step in the PCR programme is 10 min at 63°C instead of 6 min.

Determination of glucose concentration

Modified from the Megazyme© D-glucose assay (KGLUC 04/06) and used to determine the amount of glucose released from starch and isomaltose hydrolysis reactions. The contents of bottle 1 [Reagent Buffer: Potassium phosphate buffer ( 1.0 M, pH 7.4), p- hydroxybenzoic acid (0.22 M) and sodium azide (0.4 % w/v)] was diluted tto 1 L with distilled water. The contents of bottle 2 [Reagent Enzymes: Glucose oxidase (> 12,000 U) plus peroxidase (> 650 U) and 4-aminoantipyrine (80 mg). Freeze-dried powder] was diluted in approx. 20 itiL of solution 1 and quantitatively transfered to the bottle containing the remainder of solution 1. This is Glucose Determination Reagent (GOPOD Reagent). It was either used fresh or stored frozen and dark. Before use it was cheked that the absorbance ( 510) of this solution was less than 0.05 when read against distilled water.

In a 96 well plate, add 250 μΙ_ of GOPOD reagent to 10 μΙ_ of sample solution. Cover the plate with sealing tape and incubate in an Eppendorf thermomixer at 40°C, 700 rpm for 20 min. Read absorbances at 510 nm. A glucose standard curve is made from solutions of 1.4; 1.2; 0.8; 0.4; 0.2 and 0 mg/ml glucose in milli-q water and used for calculation of the sample glucose concentrations.

Determination of maltose and isomaltose synthesis by TLC Substrate: 30% glucose in 0.1 citric acid buffer, pH 5.4 (heated slightly to bring all glucose into solution) .

Enzyme: Glucoamylase standardized to 3 GAU/ml Aspergillus niger glucoamylase product (AnGA; Diazyme®X4, Danisco, Denmark) and Trichoderma reesei glucoamylase product (TrGA Diazyme TR8 Danisco, Denmark) were always run as references . Reference: Heat inactivated enzyme sample and/or buffer solution (not used in all cases).

Standards: 1) 0.3% Maltose and 0.1% isomaltose in demineralised water. 2) 0.2% Maltose and 0.05% isomaltose in demineralised water. 3) 0.3% Maltose and 0.1% isomaltose in demineralised water.

Reaction conditions: 60 μΙ substrate was mixed with 60 μΙ_ enzyme solution in wells of a PCR- plate. The plate with was covered with sealing tape and following temperature was run : 120 min at 63°C, 6 min at 99°C, 10 min at 4°C. Lid heated to 70°C. After incubation the plates were giveen a moderate centrifugation ( lmin at 300 rpm), and they were store at 4°C until further analysis. All samples were run in duplicate.

Quantification of maltose and iso-maltose: Preheat TLC-Plate at 167°C for 10 min prior to sample application. Dilute all samples and standards 20 times in demineralised water. An automatic TLC sampler (ATS4, CAMAG, Muttenz, Switzerland ) was used for accurately transferring 4 pL samples to the TLC plate. Each plate could contain 20 samples, placed in 4 mm wide bands. Plates were heated for 10 min at 40°C to let bands dry out. The TLC-plates were eluted in AcN, EtAc, 1-propanol, H20 (85: 20 : 50:40), whereafter the plates were heated 5 min at 167 °C to remove excess solvent. The plate was dipped up side down (i .e. by hold the plate on the edge near where the samples were applied) in 5% H2S04: EtOH (95: 5). The dipping solution was made daily. The plates were heated 3 min at 167°C to visualize spots. Determination of spot intensity was done by scanning in a TLC scanner (CAMAG scanner 3, Muttenz, Switzerland) and quantification was done by drawing a standard-curve based on all maltose and isomaltose concentrations vs. spot intensities. Both maltose and isomaltose concentrations were calculated from this curve, using the fact that the spot intensity vs. concentration is equal for the two compounds.

Figure 10 depicts an example of a TLC plate with standards containing different

concentrations of glucose, maltose and isomaltose and samples containing reaction products from glucose incubated with Aspergillus niger glucoamylase product (AnGA; Diazyme®X4, Danisco, Denmark) and Trichoderma reesei glucoamylase product (TrGA Diazyme TR8 Danisco, Denmark). Blind is glucose incubated without enzyme.

The isomaltose synthesis activity (IS activity) is calculated on basis of the isomaltose concentrations determined by TLC according to the following formula :

Isomaltose synthesis activity (M/min) =

[isomaltostyox W

342^x l20min

mol

Thermal stability assay 2

As a measure of thermostability of the enzymes under the conditions used in the present experiments, the GAU activity was determined according to the above assay before and after incubation of enzymes in 15% glucose, O. IM citrate buffer, pH 5.4 at 63°C for 120 min. Data is presented as % activity lost. Production of GA by fermentation

400x Trace element solution: Dilute in 1000 ml of demi water: Anhudrous Citric Acid (175 g), FeS0 4 *7 H 2 0 (200g), ZnS0 4 *7 H 2 0 ( 16g), CuS0 4 *5 H 2 0 (3.2g), MnS0 4 *H 2 0 ( 1.4g), H 3 B0 3 (0.8g). It may be helpful to acidify this to get all components into solution. The solution was filtered and sterilized. LD-medium: Add to ~ 800 ml of demi water: Casamino acids (9g), MgS0 4 *7H 2 0 ( lg),

(NH 4 ) 2 S0 4 (5g), KH 2 P0 4 (4.5g), CaCI 2 *2H 2 0 ( lg), Piperazine-l,4-bis(propanesulfonic acid (PIPPS) buffer (33g), 400x T. reesei trace elements (2.5ml), Adjust pH to 5.5 with NaOH 4N. Adjust final volume to 920 ml.

2xAmd S Base ager (1 litre): Mix KH 2 PO 4 (30 g), 1M Acetamide (20 ml), 1M CsCI (20 ml), 20% MgS04.7H 2 0 (6 ml), 20% CaCI2.2H 2 0 (6 ml), T. reesei spore elements 400x (2 ml), 50% glucose. H 2 0 (80 ml). Adjust pH to 4.5 with 4N NaOH Make up to 1 L and filter sterilize. Store at 4°C.

Initial culture: Strains were grown on AmdS-Base agar plates. To produce agar plates minimal media agar was boiled and after cooling down to app. 50°C it was diluted with 2x AmdS Base 1 : 1 and poured on petri dishes. After sporulation (app. 6-7 days) the plates were scraped with 2 ml saline 0.015% Tween 80. Approx 1 ml was added to glycerol tubes containing 500-600 μΙ 35% glycerol and stored at -80°C. The pre-culture fermentations were started directly from this spore suspension.

Pre culture: The medium is made by adding 2.5% glucose to the LD-medium, which is subsequently made up to 1 L. To produce biomass 50 μΙ spore suspension is added to 100 ml medium (sterilised in 500 ml shake flask). The flasks are incubated on a rotary shaker at 30°C, 180 rpm for 2 days, then 10 ml suspension is used to inoculate a new shake flask, which is incubated under similar conditions for 1 day. The content of this flask is used to inoculate a fermentor.

Main culture: To make 1 L of medium, 40 ml glucose/sophorose mix (Danisco, Jamsa, Finland) was added to the LD-medium and mede up to 1 L. 6 L fermentors containing 4 L of medium were inoculated with the pre-culture, and grown at pH 3.5 for approximately 16 hours at 34°C, until CER/OU (Carbondioxide Excretion Rate/Oxygen Uptake Rate) started falling. Then temperature was lowered to 28°C, pH was raised to 4.0 and the fermentation was continued for approximately 80 hours. Brew analysis with determination of real degree of fermentation (RDF)

Pure malt brew analysis

Analysis was carried out at using the following procedure: 70 g milled pilsner malt

(Weyermann, Bamberg, Germany) was mashed with 266 ml water. The temperature cycle after mashing in (mixing malt and water) was: 140 minutes at 63.9°C, increasing to 73.9°C over 10 minutes, 5 minutes at 73.9°C. At the end of mashing, the mashes were cooled, made up to 350 g and filtered. Filtrate volumes were measured after 30 minutes. The filtrated worts were sampled for specific gravity determination, then heated to 99°C for 10 minutes in a water bath in order to destroy any residual glucoamylase activity. The heat treatment results in a loss of 1.5 g per 200 ml wort. The worts were fermented at 18°C and 100 rpm in 500 ml conical flasks after yeast addition for at least 88 hours and no more than 120 hours. Specific gravity was determined on the ferments. Malt-adiunct brew analysis

A modified decoction mashing, using corn grist as adjunct was employed. The brewing protocol was modified from US 2009014247. 40% of the malt was substituted with corn grist with a moisture content of 12.6% (Benntag Nordic; Nordgetreide GmBH Liibec, Germany). All corn grist was heated to 100°C at 2°C /min, together with 54% of the water and 5% of the malt (well modified Pilsner malt; Fuglsang Denmark). 5 min rests were held at 72°C and 80°C and a 10 min rest was held at 100°C. Hereafter the adjunct was cooled to 64°C and combined with the main mash, also at 64°C. Enzymes were added at this stage, and the 64°C rest was extended to 250min. After fermentation the RDF values were determined.

Real degree of fermentation (RDF) value may be calculated according to the equation below:

RE

RDF(%) ■ o p x lOO

Where: RE = real extract = (0.1808 x "Pmmai) + (0.8192 x °Ρ ίίη3 ι), "Pinmai is the specific gravity of the standardised worts before fermentation and °Ρ ηη3 ι is the specific gravity of the fermented worts expressed in degree plato.

In the present context, Real degree of fermentation (RDF) was determined as follows: After fermentation samples were filtered and degassed. Specific gravity and alcohol concentration was determined on the ferments using a Beer Alcolyzer Plus and a DMA 5000 Density meter (both from Anton Paar, Graz, Austria). Based on these measurements, the real degree of fermentation (RDF) value is calculated, by the instrument, according to the equation below:

RDF(%) = OE E r

OE Where: E(r) is the real extract in degree Plato (°P) and OE is the original extract in °P. Xylanase assay method

Samples were diluted in citric acid (0.1 M) - di-sodium-hydrogen phosphate (0.2 M) buffer, pH 5.0, to obtain approx. OD 59 o = 0.7 in this assay. Three different dilutions of the sample were pre-incubated for 5 minutes at 40°C. At time = 5 minutes, 1 Xylazyme tablet

(crosslinked, dyed xylan substrate, Megazyme, Bray, Ireland) was added to the enzyme solution in a reaction volume of lml. At time = 15 minutes the reaction was terminated by adding 10 ml of 2% TRIS/NaOH, pH 12. Blanks were prepared using ΙΟΟΟμΙ buffer instead of enzyme solution. The reaction mixture was centrifuged ( 1500 x g, 10 minutes, 20°C) and the OD of the supernatant was measured at 590 nm. One xylanase unit (XU) is defined as the xylanase activity increasing OD 590 with 0.025 per minute.

Pullulanase assay method

Principle:

On incubation of Red-Pullulan (partially depolymerised pullulan, which is dyed with Procion Red MX-5B, from assay kit S-RPUL, Megazyme Int., Bray, Ireland) with pullulanase or limit- dextrinase the substrate is depolymerised by an en fo-mechanism to produce low molecular- weight dyed fragments which remain in solution on addition of ethanol to the reaction mixture. High-molecular weight material is removed by centrifugation, and the colour of the supernatant is measured at 510 nm. Pullulanase in the assay solution is determined by reference to a standard curve.

Substrate:

0.5 g of powdered substrate was dissolved in to 25 mL of 0.5 M potassium chloride solution. Buffer: Sodium Acetate, 200 mM, pH 5.0 Enzyme Preparation : Enzyme is diluted at least 10 times in buffer. If the resulting A 5 i 0 reading was above 1,0 the enzyme was further diluted.

Assay Procedure: In a test tube 1.0 mL of enzyme solution, pre-equilibrated to 40°C, was mixed with 0.5 mL substrate solution, pre-equilibrated to 40°C. The mix was incubated at 40°C for 10 min. Reaction was terminated and high-molecular weight substrate was precipitated by the adding 2.5 mL ethanol (95 % v/v). The tubes were equilibrate to room temperature for 10 min, then stirred for lOsec on a vortex mixer and centrifuged at l,000g for 10 min. Supernatants were transferred to a 96 well plate and the absorbance of blank and reaction solutions was measured at 510nm against distilled water. The blank reading was subtracted from the sample reading to obtain the A 5 i 0 used in the formula below.

Activity is determined from the formula : milli-PU/mL = 360*A 510 + 11. The blank is prepared by adding ethanol to the Red-Pullulan substrate before addition of the enzyme.

One Unit of activity is defined as the amount of enzyme required to release one mole of D- glucose reducing sugar equivalents per minute from borohydride reduced pullulan, under the defined assay conditions (see Megazyme method S-RPUL 10/08, Megazyme Int., Bray, Ireland).

Alpha-amylase assay (SSU) method

Principle:

Method is based on the release of reducing groups measured, by reference to a standard curve, as glucose from a 20 minute hydrolysis of 4 % potato soluble starch at pH 4.5 and 50°C. One Soluble Starch Unit (SSU) is the activity which liberates 1 milligram of glucose equivalents per minute.

Substrate:

4 %(w/v) potato soluble starch (Sigma, S 2630) solution in 0.05 M sodium acetate buffer, pH 4.5. The potato soluble starch is slurried in deionised water then added to vigoously boiling water in a flask. The starch solution is boiled, with stirring, for three minutes then cooled to 25°C and acetic acid and sodium hydroxide solution added to give a 0.05 M sodium acetate buffer, pH 4.5 , when made up to the final concentration.

DNS Solution: 1 L of DNS solution is made up by adding to water: 16 g sodium hydroxide followed by 10 g 3-5 dinitro salicylic acid and then 300 g sodium potassium tartrate, each component being dissolved before the next is added. The solution is made up to volume and stored in the dark.

Assay Procedure: Add 0.4 ml starch substrate and 0.1 ml 0.5M sodium acetate buffer, pH 4.5 to a test tube which is capped and equilibrated to temperature in a water bath set at 50°C. Add 0.1 ml of diluted enzyme solution and after 20 minutes stop the reaction by adding 0.1 ml 2 %(w/v) NaOH. Run each enzyme assay in duplicate along with an enzyme blank.

Color Development: To each tube (assay and blank) add 1.5 ml water and 2.0 ml DNS solution, mix and place in a boiling water bath for 5 minutes then cool the tubes in an ice bath for 10 minutes. Let the tubes stand at room temperature for 20 minutes and read the absorbance at 543 nm.

Standard Curve:

Construct a glucose standard curve corresponding to 0.0 - 1.0 mg glucose per 2.2 ml water, then adding 2.0 ml of DNS reagent and proceeding as described.

Calculation Of Enzyme Activity (SSU)

Convert absorbance values (A 5 3 )of samples to mg of glucose using the standard curve, and calculate the ΔΑ 543 mg glucose values [sample - blank] (ΔΑ 543 must be between 0.2 - 0.4). Activity is determined from the formula: SSU/ml or g = average (ΔΑ 543 mg glucose values [sample - blank]) x (1/0.1) x ( 1/20) x Enzyme Dilution

Example 1: Construction of TrGA site evaluation libraries (SELs) in the pTTT vector for expression in Trichoderma reesei

A Trichoderma reesei cDNA sequence (SEQ ID NO: 4) was cloned into pDONR™201 via the Gateway® BP recombination reaction (Invitrogen, Carlsbad, CA, USA) resulting in the entry vector pDONR-TrGA (FIG. 2). The cDNA sequence (SEQ ID NO: 4) encodes the TrGA signal peptide, the pro-sequence, and the mature protein, including the catalytic domain, linker region and starch binding domain (SEQ ID NO: 1). SEQ ID NO: 4 and SEQ ID NO: 1 are shown in FIGs. IB and IB. FIG. 1C illustrates the precursor and mature protein TrGA domains.

To express the TrGA protein in Trichoderma reesei, the TrGA coding sequence (SEQ ID NO: 4) was cloned into the Gateway compatible destination vector pTTT-Dest (FIG. 3) via the GATEWAY® LR recombination reaction. The expression vector contained the T. reesei cbhl- derived promoter and terminator regions that allowed for strong inducible expression of a gene of interest. The vector also contained the Aspergillus nidulans amdS selective marker that allowed for growth of the transformants on acetamide as a sole nitrogen source. The expression vector also contained T. reesei telomere regions that allowed for non- chromosomal plasmid maintenance in a fungal cell. On the destination pTTT-Dest plasmid, the cbhl promoter and terminator regions were separated by the chloramphenicol resistance gene, Cm R , and the lethal E. coli gene, ccdB, flanked by the bacteriophage lambda-based specific recombination sites attRl, attR2. This configuration allowed for direct selection of recombinants containing the TrGA gene under control of the cbhl regulatory elements in the right orientation via the GATEWAY® LR recombination reaction. The final expression vector pTTT-TrGA is shown in FIG. 4.

SELs were constructed using the pDONR-TrGA entry vector (FIG. 2) as a template and the primers listed in Table 2. All primers used in the mutagenesis experiments contained the triplet NNS (N = A,C,T,G, and S = C or G) at the position that aligns with the codon of the TrGA sequence designed to be mutated (SEQ ID NO: 1), allowing for a random incorporation of nucleotides at the preselected position. Construction of each SEL library started with two independent PCR amplifications on the pDONR-TrGA entry vector: one using the Gateway F (pDONR201 - FW) and a specific mutagenesis primer R (Table 2), and the other - the Gateway primer R (pDONR201 - RV) and a specific mutagenesis primer F (Table 2). High fidelity PHUSION DNA polymerase (Finnzymes OY, Espoo, Finland) was used in a PCR amplification reaction including 0.2 μ primers. The reactions were carried out for 25 cycles according to the protocol provided by Finnzymes. 1 μΙ aliquots of the PCR fragments obtained were used as templates for a subsequent fusion PCR reaction together with the Gateway FW and Gateway RV primers (Invitrogen). This PCR amplification, after 22 cycles, produced a population of the full-length linear TrGA DNA fragments randomly mutated at the specific codon position. The fragments were flanked by the Gateway-specific attLl, attL2 recombination sites on both ends. The DNA fragments were purified with a

CHARGESWITCH® PCR clean-up kit (Invitrogen, Carlsbad USA) and then recombined with 100 ng of the pTTT- destination vector (FIG. 3) using the LR CLONASE™ II enzyme mix according to the protocol supplied by Invitrogen. The recombination products that were generated were transformed into E.coli Max Efficiency DH5a, as described by the supplier (Invitrogen). The final expression constructs pTTT-TrGA with mutations at the desired position were selected by plating bacteria on 2 χ YT agar plates (16 g/L Bacto Tryptone (Difco), 10 g/L Bacto Yeast Extract (Difco), 5 g/L NaCI, 16 g/L Bacto Agar (Difco)) with 100 pg/ml ampicillin.

96 single colonies from each library were grown for 24 hrs at 37°C in MTP containing 200 pL 2 x YT medium with 100pg/ml ampicillin. Cultures were used directly to amplify PCR fragments encompassing the region where a specific mutation was introduced. The specific PCR products obtained were sequenced using an ABI3100 sequence analyzer (Applied Biosystems). Each library contained from 15 to 19 different TrGA variants in the final expression vector. These variants were individually transformed into T. reesei, as described below. Libraries are numbered from 1 to 182 referencing the specific amino acid residue in the TrGA sequence that was randomly mutated.

Table 2: Primers used to generate TrGA SELs

GGACGCCGTCAACTATGCCGATNNSCACCCCCTGTGGA

541 F

TTGGG (SEQ ID NO:358)

541 R ATCGGCATAGTTGACGGCGTCCAGAGC (SEQ ID NO: 359)

CTATGCCGATAACCACCCCCTGNNSATTGGGACGGTC

545 F

AACCTC (SEQ ID NO: 360)

545 R CAGGGGGTGGTTATCGGCATAGTTGAC (SEQ ID NO: 361)

TGCCGATAACCACCCCCTGTGGNNSGGGACGGTCAA

546 F

CCTCGAG (SEQ ID NO:362)

546 R CCACAGGGGGTGGTTATCGGCATAGTT (SEQ ID NO: 363)

547 CGATAACCACCCCCTGTGGATTNIMSACGGTCAACCTC

F GAGGCT (SEQ ID NO: 364)

547 R A ATCC AC AGGG GGTG GTTATCG G C ATA (SEQ ID NO: 365)

CCACCCCCTGTGGATTGGGACG N NSAACCTCGAGGC

549 F

TGGAGAC (SEQ ID NO:366)

549 R CGTCCCAATCCACAGGGGGTGGTTATC (SEQ ID NO: 367)

CCTGTGGATTGGGACGGTCAACNNSGAGGCTGGAGA

551 F

CGTCGTG (SEQ ID NO: 368)

551 R GTTGACCGTCCCAATCCACAGGGGGTG (SEQ ID NO: 369)

TGGAGACGTCGTGGAGTACAAGNNSATCAATGTGGG

561 F

CCAAGAT (SEQ ID NO: 370)

561 R CTTGTACTCCACGACGTCTCCAGCCTC (SEQ ID NO: 371)

CGTCGTGGAGTACAAGTACATCNNSGTGGGCCAAG

563 F

ATGGCTCC (SEQ ID NO: 372)

563 R GATGTACTTGTACTCCACGACGTCTCC (SEQ ID NO: 373)

CAAGTACATCAATGTGGGCCAANNSGGCTCCGTGAC

567 F

CTGGGAG (SEQ ID NO:374)

567 R TTG G CCC AC ATTG ATGTACTTGTACTC (SEQ ID NO: 375)

CATCAATGTGGGCCAAGATGGCNNSGTGACCTGGGA

569 F

GAGTGAT (SEQ ID NO: 376)

569 R GCCATCTTGGCCCACATTGATGTACTTG (SEQ ID NO:377)

CGTGACCTGGGAGAGTGATCCCNNSCACACTTACAC

577 F

GGTTCCT (SEQ ID NO:378)

577 R GGGATCACTCTCCCAGGTCACGGAGCC (SEQ ID NO: 379)

CTGGGAGAGTGATCCCAACCACNNSTACACGGTTCC

579 F

TGCGGTG (SEQ ID NO:380)

579 R GTGGTTGGGATCACTCTCCCAGGTCAC (SEQ ID NO:381)

TCCCAACCACACTTACACG GTTN NSGCGGTGG CTTG

583 F

TGTGACG (SEQ ID NO:382)

583 R AACCGTGTAAGTGTGGTTGGGATCACT (SEQ ID NO: 383)

Example 2: Transformation of TrGA SELs into Trichoderma reesei

The SELs were transformed into T. reesei using the PEG protoplast method. The E.coli clones of the SELs confirmed by sequence analysis were grown overnight at 37°C in deep well microtiter plates (Greiner Art. No. 780271) containing 1200 μΙ of 2 χ YT medium with ampicillin (100 pg/ml) and kanamycin (50 g/ml). Plasmid DNAs were isolated from the cultures using CHEMAGIC® Plasmid Mini Kit (Chemagen - Biopolymer Technologie AG, Baesweiler, Germany) and were transformed individually into a T. reesei host strain derived from L-P37 bearing four gene deletions {Acbhl, Acbh2, Aegll , Aegl2, i.e. , "quad-deleted; " see U .S. Patent No. 5,847,276, WO 92/06184, and WO 05/001036) using the PEG-Protoplast method (Penttila et al. ( 1987) Gene 61 : 155-164) with the following modifications.

For protoplast preparation, spores were grown for 16-24 hours at 24°C in Trichoderma Minimal Medium ( MM) (20 g/L glucose, 15 g/L KH 2 P0 4 , pH 4.5, 5 g/L (NH 4 ) 2 S0 4 , 0.6 g/L MgS0 4 -7H 2 0, 0.6 g/L CaCI 2 -2H 2 0, 1 ml of 1000 χ T. reesei Trace elements solution {5 g/L FeS0 4 -7H 2 0, 1.4 g/L ZnS0 4 -7H 2 0, 1.6 g/L MnS0 4 H 2 0, 3.7 g/L CoCI 2 -6H 2 0 }) with shaking at 150 rpm . Germinating spores were harvested by centrifugation and treated with 15mg/ml of β-D-glucanase-G (Interspex - Art. o. 0439-1) solution to lyse the fungal cell walls. Further preparation of protoplasts was performed by a standard method, as described by Penttila et al . ( 1987 supra) .

The transformation method was scaled down 10 fold. In general, transformation mixtures containing up to 600 ng of DNA and 1-5 χ 10 s protoplasts in a total volume of 25 μΙ were treated with 200 ml of 25% PEG solution, diluted with 2 volumes of 1.2 M sorbitol solution, mixed with 3% selective top agarose MM with acetamide (the same Minimal Medium as mentioned above but (N H 4 ) 2 S0 4 was substituted with 20 mM acetamide) and poured onto 2% selective agarose with acetamide either in 24 well microtiter plates or in a 20 χ20 cm Q-tray divided in 48 wells. The plates were incubated at 28°C for 5 to 8 days. Spores from the total population of transformants regenerated on each individual well were harvested from the plates using a solution of 0.85% NaCI, 0.015% Tween 80. Spore suspensions were used to inoculate fermentations in 96 wells MTPs. In the case of 24 well MTPs, an additional plating step on a fresh 24 well MTP with selective acetamide MM was introduced in order to enrich the spore numbers.

Example 3: Fermentation of T. reesei transformants expressing TrGA variants in a MTP format The tranformants were fermented and the supernatants containing the expressed variant TrGA proteins were tested for various properties. In brief, 96-well filter plates (Corning Art.No. 3505) containing in each well 200 μΙ of LD-GSM medium (5.0 g/L (N H 4 ) 2 S0 4 , 33 g/L l,4-Piperazinebis(propanesulfonic acid), pH 5.5, 9.0 g/L Casamino acids, 1.0 g/L KH 2 P0 4 , 1.0 g/L CaCI 2 -2H 2 0, 1.0 g/L MgS0 4 -7H 2 0, 2.5 ml/L of 1000 χ T. reesei trace elements, 20 g/L Glucose, 10 g/L Sophorose) were inoculated in quadruplicate with spore suspensions of T. reesei transformants expressing TrGA variants (more than 10 4 spores per well) . The plates were incubated at 28°C with 230 rpm shaking and 80% humidity for 6 days. Culture supernatants were harvested by vacuum filtration . The supernatants were used in different assays for screening of variants with improved properties. Example 4: Preparation of the whole broth samples from GA-producing transformants

TrGA producing transformants were initially pre-grown in 250 ml shake flasks containing 30 ml of ProFlo medium. Proflo medium contained : 30 g/L a-lactose, 6.5 g/L (NH 4 ) 2 S0 4 , 2 g/L KH 2 P0 4 , 0.3 g/L MgS0 4 -7H 2 0, 0.2 g/L CaCI 2 -2H 2 0, 1 ml/L 1000 χ trace element salt solution as mentioned above, 2 ml/L 10% Tween 80, 22.5 g/L ProFlo cottonseed flour (Traders protein, Memphis, TN), 0.72 g/L CaC0 3 . After two days of growth at 28°C and 140 rpm, 10% of the Proflo culture was transferred into a 250 ml shake flask containing 30 ml of Lactose Defined Medium. The composition of the Lactose Defined Medium was as follows: 5 g/L (NH 4 ) 2 S0 4 , 33 g/L 1,4-Piperazinebis (propanesulfonic acid) buffer, pH 5.5, 9 g/L casamino acids, 4.5 g/L KH 2 P0 4 , 1.0 g/L MgS0 4 -7H 2 0, 5 ml/L Mazu DF60-P antifoam (Mazur Chemicals, IL), lml/L of 1000 χ trace element solution. 40 ml/L of 40% (w/v) lactose solution was added to the medium after sterilization. Shake flasks with the Lactose Defined Medium were incubated at 28°C, 140 rpm for 4 - 5 days. Mycelium was removed from the culture samples by centrifugation and the supernatant was analyzed for total protein content (BCA Protein Assay Kit, Pierce Cat. No.23225) and GA activity, as described above in the Assays and Methods section.

The protein profile of the whole broth samples was determined by SDS-PAGE electrophoresis. Samples of the culture supernatant were mixed with an equal volume of 2 χ sample loading buffer with reducing agent and separated on NUPAGE® Novex 10% Bis-Tris Gel with MES SDS Running Buffer (Invitrogen, Carlsbad, CA, USA). Polypeptide bands were visualized in the SDS gel with SIMPLYBLUE SafeStain (Invitrogen, Carlsbad, CA, USA).

Example 5: Thermal stability of the variants

The thermal stability was measured according to above assay "Thermal stability assay 2". The parent molecule under the conditions described had a residual activity of 87.2%, Table 3 shows the residual activity for the variants, which were selected from an initial screen for fermentation in large scale and further analysis. The material used was crude fermentation broth from shake flasks. Residual activity was calculated on basis of GAU activity before and after 120min incubation at 63°C in 0.1M citrate buffer pH 5.4, containing 15% glucose. Table 3: Thermostability for selected TrGA variants, shown as residual activity after incubation for 120m in at 63°C in 0.1M citrate buffer pH 5.4, containing 15% glucose.

Example 6: Determination of isomaltose synthesis and starch hydrolysis and ratio thereof

Variants were tested according to above assays: "Starch hydrolysis activity" and

"Determination of maltose and isomaltose synthesis by TLC". The IS/SH ratio was calculated from the results of these analysis as described. Table 4 summarises the data for for the variants selected for fermentation in large scale and further analysis. The material used was crude fermentation broth from shake flasks. Table 4: Isomaltose synthesis activity (IS), starch hydrolysis activity (SH) and IS/SH ratio of selected TrGA variants

Example 7: Brew analysis with determinaiton of real degree of fermentation (RDF) All the variants shown in table 3 and 4 were grown in fermentors and GA enzyme was collected and purified (as described above under "Purification of TrGA variants"). The purified enzymes were reanalysed for IS/SH ratio as described above in Example 6 and

thermostability was measured as described in Example 5. Brew analysis with determination of RDF value was carried out on the four variants which showed the best combination of IS/SH ratio and thermostability (Brewl l, Brewl, Varl6 and Varl3) as described above under "Brew analysis with determinaiton of real degree of fermentation (RDF)". RDF values are listed in Table 5. Table 5. RDF values of selected purified TrG A- variants, purified wild type TrGA and purified AnGA.

Below values were obtained using the above described "Pure malt brew analysis"-method.

Example 8: Construction and characterization of combinatorial variants

Based on data a selected set of variants with single substitutions were further characterized. These variants have single substitution at positions: 43, 44, 61, 73, 294, 417, 430, 431, 503, 511, 535, 539, and 563. Among these sites, 43, 44, and 294 were identified in a previous screening experiment in Schizosaccharomyces pombe. See WO 08/045489, which is incorporated herein by reference. Variants were purified from large-scale fermentation, and Pis of thermal stability and specific activities were determined. Specifically, specific activities were determined using various substrates, including DP7, cornstarch, and liquefact. The results are shown in Table 6. Table 6: Pis of a selected set of single site variants, each of which is obtained from a 500 ml fermentation.

P.I. DP7- P.I. CornStarch- P.I. Thermal P.I. Liquefact-

Variants FPLC FPLC Stability FPLC

N61I 1.16 1.35 1.00 1.66

A431L 1.15 1.38 1.18 1.51

L 17V 1.18 1.32 1.02 1.40

A431Q 1.06 1.20 0.92 1.24

G294C 1.01 0.84 0.94 1.23

N563K 1.07 1.12 1.97 1.15

Q511H 1.05 1.09 1.52 1.13

T430M 1.05 1.15 0.89 1.09 P.I. DP7- P.I. CornStarch- P.I. Thermal P.I. Liquefact-

Variants FPLC FPLC Stability FPLC

E503A 1.08 1.16 1.40 1.09

I43Q 1.11 1.24 0.94 1.08

A539R 1.15 1.37 1.43 1.08

I43R 1.03 1.07 1.41 1.07

L417R 1.23 1.27 1.51 1.04

T430A 1.13 1.35 1.23 1.04

G73F 1.06 1.06 1.45 1.03

D44R 0.97 1.06 1.46 0.98

N563I 1.09 1.22 2.06 0.92

D44C 0.80 0.82 0.96 0.91

E503V 1.17 1.07 1.66 0.88

A535R 1.09 1.44 1.47 0.85

Additionally, combinatorial variants were constructed using the PCR method with

substitutions among : 43, 44, 61, 73, 294, 417, 430, 431, 503, 511, 535, 539, and 563. Briefly, the combinatorial variants were constructed by using plasmid pDONR-TrGA (FIG. 2) as the backbone. The methodology to construct combinatorial variants is based on the Gateway technology (Invitrogen, Carlsbad, CA). The primers used to create the

combinatorial variants are shown in Tables 2 and 7. The following synthetic construct approach was chosen for the construction of all combinatorial variants.

CTCTCT [ Xbal site] [MF] GAGAGGGG [attBl] [GAP combinatorial variant][attB2 sites] CCCCAGAG [MR][HindIII] AGAGAG

This construct was treated with restriction enzymes Xba-I and Hindlll. The digested fragments were ligated into Xba-I/ Hindlll treated pBC (a pUC19 derived vector). The ligation mixture was transformed to E. coli DH IOB (Invitrogen, Carlsbad, CA) and plated onto selective agar supplemented with 100 pg/ml ampicillin. The plates were incubated for 16 h at 37°C. Colonies from the selective plates were isolated and inoculated into selective liquid medium. After 16 h incubation at 37°C and 250 rpm the plasmids were isolated using a standard plasmid isolation kit and combined with pDONR 2.21 (Invitrogen, Carlsbad, CA) to create a Gateway entry vector with the specific combinatorial variants. The reaction mixture was transformed into E. coli Max efficiency DH5a (Invitrogen, Carlsbad, CA) and plated on selective agar (2 χ ΤΎ supplemented with 50 g kanamycin/ml). After overnight incubation at 37°C, single colonies were picked for sequence analysis (BaseClear B.V., Leiden,

Netherlands). The combinatorial variants were subcloned in pTrexTrTel and expressed in a T. reesei host strain as described in WO 06/060062. Table 7: Primers used to construct combinatorial variants

SEQ ID

Primer DNA sequence

NO:

Varl Basic-1 GAGAGAGTGCGGGCCTCTTCGCTATTT TAGA 391

Varl Basic-2 CAAAATAAAATCATTATTTGTCTAGAAATAGCGAAGAGGC 392

Varl Basic-3 CAAATAATGA 1 1 1 I A I 1 1 1 GACTGATAGTGACCTGTTCGT 393

Varl Basic-4 TTGCTCATCAATGTGTTGCAACGAACAGGTCACTATCAGT 394

Varl Basic-5 TGCAACACATTGATGAGCAATGC 1 1 1 1 1 1 ATAATGCCAAC 395

Varl Basic-6 AG CCTGCTTTTTTGTAC AAAGTTG G C ATTATAAAAAAG C A 396

Varl Basic-7 TTTGTACAAAAAAGCAGGCTATGCACGTCCTGTCGACTGC 397

Varl Basic-8 CAACGGAGCCGAGCAGCACCGCAGTCGACAGGACGTGCAT 398

Varl Basic-9 GGTGCTGCTCGGCTCCGTTGCCGTTCAAAAGGTCCTGGGA 399

Varl Basic- 10 AGACCGCTTGATCCTGGTCTTCCCAGGAC 1 1 1 1 GAACGG 400

Varl Basic- 11 AGACCAGGATCAAGCGGTCTGTCCGACGTCACCAAGAGGT 401

Varl Basic-12 GCTGATGAAGTCGTCAACAGACCTCTTGGTGACGTCGGAC 402

Varl Basic-13 CTGTTGACGACTTCATCAGCACCGAGACGCCTATTGCACT 403

Varl Basic-14 CATTGCAAAGAAGATTGTTCAGTGCAATAGGCGTCTCGGT 404

Varl Basic-15 GAACAAT I I U 1 1 GCAATGTTGGTCCTGATGGATGCCGT 405

Varl Basic- 16 CCAGCTGATGTGCCGAATGCACGGCATCCATCAGGACCAA 406

Varl Basic-17 GCATTCGGCACATCAGCTGGTGCGGTGATTGCATCTCCCA 407

Varl Basic-18 GTAGTCCGGGTCAATTGTGCTGGGAGATGCAATCACCGCA 408

Varl Basic-19 GCACAATTGACCCGGACTACTATTACATGTGGACGCGAGA 409

Varl Basic-20 TCTTGAAGACAAGAGCGCTATCTCGCGTCCACATGTAATA 410

Varl Basic-21 TAGCGCTCTTGTCTTCAAGAACCTCATCGACCGCTTCACC 411

Varl Basic-22 AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGGT 412

Varl Basic-23 GAAACGTACGATGCGGGCCTGCAGCGCCGCATCGAGCAGT 413

Varl Basic-24 AGTGACCTGGGCAGTAATGTACTGCTCGATGCGGCGCTGC 414

Varl Basic-25 ACATTACTGCCCAGGTCACTCTCCAGGGCCTCTCTAACCC 415

Varl Basic-26 CGTCCGCGAGGGAGCCCGAGGGGTTAGAGAGGCCCTGGAG 416

Varl Basic-27 CTCGGGCTCCCTCGCGGACGGCTCTGGTCTCGGCGAGCCC 417

Varl Basic-28 TTCAGGGTCAACTCAAACTTGGGCTCGCCGAGACCAGAGC 418 SEQ ID

Primer DNA sequence

NO:

Varl Basic-29 AAGTTTGAGTTGACCCTGAAGCC 1 1 1 CACCGGCAACTGGG 419

Varl Basic-30 GCCATCCCGCTGCGGTCGACCCCAGTTGCCGGTGAAAGGC 420

Varl Basic-31 GTCGACCGCAGCGGGATGGCCCAGCTCTGCGAGCCATTGC 421

Varl Basic-32 AC I 1 I GAGTATCCAATCAAGGCAATGGCTCGCAGAGCTGG 422

Varl Basic-33 CTTGATTGGATACTCAAAGTGGCTCATCAACAACAACTAT 423

Varl Basic-34 ACGTTGGACACAGTCGACTGATAGTTGTTGTTGATGAGCC 424

Varl Basic-35 CAGTCGACTGTGTCCAACGTCATCTGGCCTATTGTGCGCA 425

Varl Basic-36 GGCAACATAGTTGAGGTCGTTGCGCACAATAGGCCAGATG 426

Varl Basic-37 ACGACCTCAACTATGTTGCCCAGTACTGGAACCAAACCGG 427

Varl Basic-38 C 1 1 C 1 1 CCCAGAGGTCAAAGCCGGTTTGGTTCCAGTACTG 428

Varl Basic-39 C I 1 1 A JU U GGAAGAA 1 CAA 1 GG AGU CA 1 I C I 1 1 429

Varl Basic-40 CGGTGCTGGTTGGCAACAGTAAAGAATGAGCTCCCATTGA 430

Varl Basic-41 ACTGTTGCCAACCAGCACCGAGCACTTGTCGAGGGCGCCA 431

Varl Basic-42 GCCAAGAGTGGCAGCAAGAGTGGCGCCCTCGACAAGTGCT 432

Varl Basic-43 CTCTTGCTGCCACTCTTGGCCAGTCGGGAAGCGCTTATTC 433

Varl Basic-44 AAACCTGGGGAGCAACAGATGAATAAGCGCTTCCCGACTG 434

Varl Basic-45 ATCTGTTGCTCCCCAGG 1 1 1 I G I GU 1 1 (_ 1 CCAACGATTC 435

Varl Basic-46 TATCCACCAGACGACACCCAGAATCGTTGGAGAAAGCACA 436

Varl Basic-47 TGGGTGTCGTCTGGTGGATACGTCGACTCCAACATCAACA 437

Varl Basic-48 GCCAGTCCTGCCCTCGTTGGTGTTGATGTTGGAGTCGACG 438

Varl Basic-49 CCAACGAGGGCAGGACTGGCAAGGATGTCAACTCCGTCCT 439

Varl Basic-50 CGAAGGTGTGGATGGAAGTCAGGACGGAGTTGACATCCTT 440

Varl Basic-51 GACTTCCATCCACACCTTCGATCCCAACCTTGGCTGTGAC 441

Varl Basic-52 CATGGCTGGAAGGTGCCTGCGTCACAGCCAAGGTTGGGAT 442

Varl Basic-53 GCAGGCACCTTCCAGCCATGCAGTGACAAAGCGCTCTCCA 443

Varl Basic-54 GTCGACAACAACCTTGAGGTTGGAGAGCGCTTTGTCACTG 444

Varl Basic-55 ACCTCAAGGTTGTTGTCGACTCCTTCCGCTCCATCTACGG 445

Varl Basic-56 CAGGAATGCCCTTGTTCACGCCGTAGATGGAGCGGAAGGA 446

Varl Basic-57 CGTGAACAAGGGCATTCCTGCCGGTGCTGCCGTCGCCATT 447

Varl Basic-58 ACATCCTCTGCATACCGGCCAATGGCGACGGCAGCACCGG 448 SEQ ID

Primer DNA sequence

NO:

Varl Basic-59 GGCCGGTATGCAGAGGATGTGTACTACAACGGCAACCCTT 449

Varl Basic-60 AGCAAATGTAGCAAGATACCAAGGGTTGCCGTTGTAGTAC 450

Varl Basic-61 GGTATCTTGCTACATTTGCTGCTGCCGAGCAGCTGTACGA 451

Varl Basic-62 TCTTCCAGACGTAGATGGCATCGTACAGCTGCTCGGCAGC 452

Varl Basic-63 TGCCATCTACGTCTGGAAGAAGACGGGCTCCATCACGGTG 453

Varl Basic-64 AAGGCCAGGGAGGTGGCGGTCACCGTGATGGAGCCCGTCT 454

Varl Basic-65 ACCGCCACCTCCCTGGCCTTCTTCCAGGAGCTTGTTCCTG 455

Varl Basic-66 GTAGGTCCCGGCCGTCACGCCAGGAACAAGCTCCTGGAAG 456

Varl Basic-67 GCGTGACGGCCGGGACCTACTCCAGCAGCTCTTCGACCTT 457

Varl Basic-68 CGGCGTTGATGATGTTGGTAAAGGTCGAAGAGCTGCTGGA 458

Varl Basic-69 TACCAACATCATCAACGCCGTCTCGACATACGCCGATGGC 459

Varl Basic-70 TTGGCAGCCTCGCTGAGGAAGCCATCGGCGTATGTCGAGA 460

Varl Basic-71 TTCCTCAGCGAGGCTGCCAAGTACGTCCCCGCCGACGGTT 461

Varl Basic-72 GTCAAACTGCTCGGCCAGCGAACCGTCGGCGGGGACGTAC 462

Varl Basic-73 CGCTGGCCGAGCAGTTTGACCGCAACAGCGGCACTCCGCT 463

Varl Basic-74 ACGTCAGGTGAACCGCAGACAGCGGAGTGCCGCTGTTGCG 464

Varl Basic-75 GTCTGCGGTTCACCTGACGTGGTCGTACGCCTCGTTCTTG 465

Varl Basic-76 GCCCGACGAAGCGTGGCTGTCAAGAACGAGGCGTACGACC 466

Varl Basic-77 ACAGCCACGCTTCGTCGGGCTGGCATCGTGCCCCCCTCGT 467

Varl Basic-78 GCTAGCGCTGCTGTTGGCCCACGAGGGGGGCACGATGCCA 468

Varl Basic-79 GGGCCAACAGCAGCGCTAGCACGATCCCCTCGACGTGCTC 469

Varl Basic-80 ATCCGACCACGGACGCGCCGGAGCACGTCGAGGGGATCGT 470

Varl Basic-81 CGGCGCGTCCGTGGTCGGATCCTACTCGCGTCCCACCGCC 471

Varl Basic-82 TGCGACGGAGGGAATGACGTGGCGGTGGGACGCGAGTAGG 472

Varl Basic-83 ACGTCATTCCCTCCGTCGCAGACGCCCAAGCCTGGCGTGC 473

Varl Basic-84 CGTGTAGGGAGTACCGGAAGGCACGCCAGGCTTGGGCGTC 474

Varl Basic-85 CTTCCGGTACTCCCTACACGCCCCTGCCCTGCGCGACCCC 475

Varl Basic-86 AGGTGACGGCCACGGAGGTTGGGGTCGCGCAGGGCAGGGG 476

Varl Basic-87 AACCTCCGTGGCCGTCACCTTCCACGAGCTCGTGTCGACA 477

Varl Basic-88 TTGACCGTCTGGCCAAACTGTGTCGACACGAGCTCGTGGA 478 SEQ ID

Primer DNA sequence

NO:

Varl Basic-89 CAGTTTGGCCAGACGGTCAAGGTGGCGGGCAACGCCGCGG 479

Varl Basic-90 CGTGCTCCAGTTGCCCAGGGCCGCGGCGTTGCCCGCCACC 480

Varl Basic-91 CCCTGGGCAACTGGAGCACGAGCGCCGCCGTGGCTCTGGA 481

Varl Basic-92 TATCACGATAGTTGACGGCGTCCAGAGCCACGGCGGCGCT 482

Varl Basic-93 CGCCGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG 483

Varl Basic-94 CCAGCCTCGAGGTTGACCGTCCCAATCCACAGGGGGTGGT 484

Varl Basic-95 ACGGTCAACCTCGAGGCTGGAGACGTCGTGGAGTACAAGT 485

Varl Basic-96 ATCTTGGCCCACATTGATGTACTTGTACTCCACGACGTCT 486

Varl Basic-97 ACATCAATGTGGGCCAAGATGGCTCCGTGACCTGGGAGAG 487

Varl Basic-98 TGTAAGTGTGGTTGGGATCACTCTCCCAGGTCACGGAGCC 488

Varl Basic-99 TGATCCCAACCACACTTACACGGTTCCTGCGGTGGCTTGT 489

Varl Basic-100 TCCTTGACAACCTGCGTCACACAAGCCACCGCAGGAACCG 490

Varl Basic-101 GTGACGCAGGTTGTCAAGGAGGACACCTGGCAGTCGTAAA 491

Varl Basic-102 CTTTGTACAAGAAAGCTGGGTTTACGACTGCCAGGTGTCC 492

Varl Basic-103 CCCAGC I M C I 1 GTACAAAGTTGGCATTATAAGAAAGCAT 493

Varl Basic-104 TTG CAAC AAATTG ATAAG C AATG CTTTCTTATAATG CCAA 494

Varl Basic-105 TGCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTC 495

Varl Basic-106 TCAAATAATGA 1 1 1 I A I 1 1 1 GACTGATAGTGACCTGTTCG 496

Varl Basic-107 AAAATAAAATCATTATTTGAAGCTTAAGCCTGGGGTGCCT 497

Varl Basic-108 AGAGAGTCATTAGGCACCCCAGGCTTAAGCT 498

Var2 -18 GTAGTCCGGGTCTTGTGTGCTGGGAGATGCAATCACCGCA 499

Var2 -19 GCACACAAGACCCGGACTACTATTACATGTGGACGCGAGA 500

Var3 -21 TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC 501

Var3 -22 AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA 502

Var4 -92 TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT 503

Var4 -93 CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG 504

Var5 -18 GTAGTCCGGGTCTTGTGTGCTGGGAGATGCAATCACCGCA 505

Var5 -19 GCACACAAGACCCGGACTACTATTACATGTGGACGCGAGA 506

Var5 -21 TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC 507

Var5 -22 AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA 508 SEQ ID

Primer DNA sequence

NO:

Var6 -21 TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC 509

Var6 -22 AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA 510

Var6 -92 TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT 511

Var6 -93 CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG 512

Var7 -92 TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT 513

Var7 -93 CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG 514

Var7 -18 GTAGTCCGGGTCTTGTGTGCTGGGAGATGCAATCACCGCA 515

Var7 -19 GCACACAAGACCCGGACTACTATTACATGTGGACGCGAGA 516

Var8 -18 GTAGTCCGGGTCTTGTGTGCTGGGAGATGCAATCACCGCA 517

Var8 -19 GCACACAAGACCCGGACTACTATTACATGTGGACGCGAGA 518

Var8 -21 TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC 519

Var8 -22 AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA 520

Var8 -92 TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT 521

Var8 -93 CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG 522

Var9 -18 GTAGTCCGGGTCTTGTGTGCTGGGAGATGCAATCACCGCA 523

Var9 -19 GCACACAAGACCCGGACTACTATTACATGTGGACGCGAGA 524

Var9 -21 TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC 525

Var9 -22 AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA 526

Var9 -92 TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT 527

Var9 -93 CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG 528

Var9 -76 GCCCGACGAAGAGCGGCTGTCAAGAACGAGGCGTACGACC 529

Var9 -77 ACAGCCGCTCTTCGTCGGGCTGGCATCGTGCCCCCCTCGT 530

VarlO -76 GCCCGACGAAGAGCGGCTGTCAAGAACGAGGCGTACGACC 531

VarlO -77 ACAGCCGCTCTTCGTCGGGCTGGCATCGTGCCCCCCTCGT 532

VarlO -88 TTGACCGTATGGCCAAACTGTGTCGACACGAGCTCGTGGA 533

VarlO -89 CAGTTTGGCCATACGGTCAAGGTGGCGGGCAACGCCGCGG 534

VarlO -92 TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT 535

VarlO -93 CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG 536

VarlO -96 ATCTTGGCCCACAATGATGTACTTGTACTCCACGACGTCT 537

VarlO -97 ACATCATTGTGGGCCAAGATGGCTCCGTGACCTGGGAGAG 538 Primer SEQ ID

DNA sequence

NO:

VarlO -18 GTAGTCCGGGTCTTGTGTGCTGGGAGATGCAATCACCGCA 539

VarlO -19 GCACACAAGACCCGGACTACTATTACATGTGGACGCGAGA 540

Varll -76 GCCCGACGAAGAGCGGCTGTCAAGAACGAGGCGTACGACC 541

Varll -77 ACAGCCGCTCTTCGTCGGGCTGGCATCGTGCCCCCCTCGT 542

Varll -88 TTGACCGTATGGCCAAACTGTGTCGACACGAGCTCGTGGA 543

Varll -89 CAGTTTGGCCATACGGTCAAGGTGGCGGGCAACGCCGCGG 544

Varll -92 TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT 545

Varll -93 CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG 546

Varll -96 ATCTTGGCCCACAATGATGTACTTGTACTCCACGACGTCT 547

Varl l -97 ACATCATTGTGGGCCAAGATGGCTCCGTGACCTGGGAGAG 548

Varll -21 TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC 549

Varll -22 AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA 550

Varl2 -76 GCCCGACGAAGAGCGGCTGTCAAGAACGAGGCGTACGACC 551

Varl2 -77 ACAGCCGCTCTTCGTCGGGCTGGCATCGTGCCCCCCTCGT 552

Varl2 -88 TTGACCGTATGGCCAAACTGTGTCGACACGAGCTCGTGGA 553

Varl2 -89 CAGTTTGGCCATACGGTCAAGGTGGCGGGCAACGCCGCGG 554

Varl2 -92 TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT 555

Varl2 -93 CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG 556

Varl2 -96 ATCTTGGCCCACAATGATGTACTTGTACTCCACGACGTCT 557

Varl2 -97 ACATCATTGTGGGCCAAGATGGCTCCGTGACCTGGGAGAG 558

Varl2 -18 GTAGTCCGGGTCTTGTGTGCTGGGAGATGCAATCACCGCA 559

Varl2 -19 GCACACAAGACCCGGACTACTATTACATGTGGACGCGAGA 560

Varl2 -21 TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC 561

Varl2 -22 AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA 562

Varl3 -18 GTAGTCCGGGTCACGTGTGCTGGGAGATGCAATCACCGCA 563

Varl3 -19 GCACACGTGACCCGGACTACTATTACATGTGGACGCGAGA 564

Varl4 -18 GTAGTCCGGGTCACGTGTGCTGGGAGATGCAATCACCGCA 565

Varl4 -19 GCACACGTGACCCGGACTACTATTACATGTGGACGCGAGA 566

Varl4 -21 TAGCGCTC TGTCTTCAAGATTCTCATCGACCGCTTCACC 567

Varl4 -22 AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA 568 SEQ ID

Primer DNA sequence

NO:

Varl5 -18 GTAGTCCGGGTCACGTGTGCTGGGAGATGCAATCACCGCA 569

Varl5 -19 GCACACGTGACCCGGACTACTATTACATGTGGACGCGAGA 570

Varl5 -21 TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC 571

Varl5 -22 AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA 572

Varl 5 -92 TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT 573

Varl 5 -93 CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG 574

Varl 5 -76 GCCCGACGAAGAGCGGCTGTCAAGAACGAGGCGTACGACC 575

Varl5 -77 ACAGCCGCTCTTCGTCGGGCTGGCATCGTGCCCCCCTCGT 576

Varl6 -74 ACGTCAGGTGACGCGCAGACAGCGGAGTGCCGCTGTTGCG 577

Varl6 -75 GTCTGCGCGTCACCTGACGTGGTCGTACGCCTCGTTC TG 578

Varl 7 -74 ACGTCAGGTGACCCGCAGACAGCGGAGTGCCGCTGTTGCG 579

Varl7 -75 GTCTGCGGGTCACCTGACGTGGTCGTACGCCTCGTTCTTG 580

Varl8 -22 AGAAACGCATCGTACGTTTCGGTGAAGCGGTCGATGAGGT 581

Varl8 -23 GAAACGTACGATGCGTTTCTGCAGCGCCGCATCGAGCAGT 582

Varl8 -74 ACGTCAGGTGACGCGCAGACAGCGGAGTGCCGCTGTTGCG 583

Varl8 -75 GTCTGCGCGTCACCTGACGTGGTCGTACGCCTCGTTCTTG 584

Varl8 -76 GCCCGACGGGCCGTGGCTGTCAAGAACGAGGCGTACGACC 585

Varl8 -77 ACAGCCACGGCCCGTCGGGCTGGCATCGTGCCCCCCTCGT 586

Varl8 -87 AACCTCCGTGGCCGTCACCTTCCACGTTCTCGTGTCGACA 587

Varl 8 -88 TTGACCGTCTGGCCAAACTGTGTCGACACGAGAACGTGGA 588

Varl 8 -96 ATCTTGGCCCAC 1 1 1 GATGTACTTGTACTCCACGACGTCT 589

Varl8 -97 ACATCAAAGTGGGCCAAGATGGCTCCGTGACCTGGGAGAG 590

Varl9 -18 GTAGTCCGGGTCACGTGTGCTGGGAGATGCAATCACCGCA 591

Varl9 -19 GCACACGTGACCCGGACTACTATTACATGTGGACGCGAGA 592

Varl 9 -22 AGAAACGCATCGTACGTTTCGGTGAAGCGGTCGATGAGGT 593

Varl9 -23 GAAACGTACGATGCGTTTCTGCAGCGCCGCATCGAGCAGT 594

Varl 9 -74 ACGTCAGGTGACGCGCAGACAGCGGAGTGCCGCTGTTGCG 595

Varl9 -75 GTCTGCGCGTCACCTGACGTGGTCGTACGCCTCGTTCTTG 596

Varl9 -76 GCCCGACGGGCCGTGGCTGTCAAGAACGAGGCGTACGACC 597

Varl9 -77 ACAGCCACGGCCCGTCGGGCTGGCATCGTGCCCCCCTCGT 598 SEQ ID

Primer DNA sequence

NO:

Varl9 -87 AACCTCCGTGGCCGTCACCTTCCACGTTCTCGTGTCGACA 599

Varl9 -88 TTGACCGTCTGGCCAAACTGTGTCGACACGAGAACGTGGA 600

Varl9 -96 ATCTTGGCCCACTTTGATGTACTTGTACTCCACGACGTCT 601

Varl9 -97 ACATCAAAGTGGGCCAAGATGGCTCCGTGACCTGGGAGAG 602

Var20 -18 GTAGTCCGGGTCACGTGTGCTGGGAGATGCAATCACCGCA 603

Var20 -19 GCACACGTGACCCGGACTACTATTACATGTGGACGCGAGA 604

Var20 -22 AGAAACGCATCGTACGTTTCGGTGAAGCGGTCGATGAGGT 605

Var20 -23 GAAACGTACGATGCGTTTCTGCAGCGCCGCATCGAGCAGT 606

Var20 -74 ACGTCAGGTGAAGCGCAGACAGCGGAGTGCCGCTGTTGCG 607

Var20 -75 GTCTGCGCTTCACCTGACGTGGTCGTACGCCTCGTTCTTG 608

Var20 -76 GCCCGACGGGCCGTGGCTGTCAAGAACGAGGCGTACGACC 609

Var20 -77 ACAGCCACGGCCCGTCGGGCTGGCATCGTGCCCCCCTCGT 610

Var20 -87 AACCTCCGTGGCCGTCACCTTCCACGTTCTCGTGTCGACA 611

Var20 -88 TTGACCGTATGGCCAAACTGTGTCGACACGAGAACGTGGA 612

Var20 -89 CAGTTTGGCCATACGGTCAAGGTGGCGGGCAACGCCGCGG 613

Var20 -93 TATCGGCATAGTTGACGGCGTCCAGAGCCACGGCGGCGCT 614

Var20 -94 CGCCGTCAACTATGCCGATAACCACCCCCTGTGGATTGGG 615

Var20 -96 ATCTTGGCCCAL 1 1 1 GATGTACTTGTACTCCACGACGTCT 616

Var20 -97 ACATCAAAGTGGGCCAAGATGGCTCCGTGACCTGGGAGAG 617

GAV Basic-1 ACAAGTTTGTACAAAAAAGCAGGCT 618

GAV Basic-2 GCAGTCGACAGGACGTGCATAGCCTGU 1 1 1 1 I GTACAAA 619

GAV Basic-3 ATGCACGTCCTGTCGACTGCGGTGCTGCTCGGCTCCGTTG 620

GAV Basic-4 TCCCAGGACC I 1 1 1 GAACGGCAACGGAGCCGAGCAGCACC 621

GAV Basic-5 CCGTTCAAAAGGTCCTGGGAAGACCAGGATCAAGCGGTCT 622

GAV Basic-6 ACCTCTTGGTGACGTCGGACAGACCGCTTGATCCTGGTCT 623

GAV Basic-7 GTCCGACGTCACCAAGAGGTCTGTTGACGACTTCATCAGC 624

GAV Basic-8 AGTGCAATAGGCGTCTCGGTGCTGATGAAGTCGTCAACAG 625

GAV Basic-9 ACCGAGACGCCTATTGCACTGAACAATCT 1 C 1 1 1 GCAATG 626

GAV Basic- 10 ACGGCATCCATCAGGACCAACATTGCAAAGAAGATTGTTC 627

GAV Basic- 11 TTGGTCCTGATGGATGCCGTGCATTCGGCACATCAGCTGG 628 SEQ ID

Primer DNA sequence

NO:

GAV Basic- 12 TGGGAGATGCAATCACCGCACCAGCTGATGTGCCGAATGC 629

GAV Basic- 13 TGCGGTGATTGCATCTCCCAGCACAATTGACCCGGACTAC 630

GAV Basic-14 TCTCGCGTCCACATGTAATAGTAGTCCGGGTCAATTGTGC 631

GAV Basic- 15 TATTACATGTGGACGCGAGATAGCGCTCTTGTCTTCAAGA 632

GAV BasiC-16 GGTGAAGCGGTCGATGAGGTTCTTGAAGACAAGAGCGCTA 633

GAV Basic- 17 ACCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT 634

GAV Basic- 18 ACTGCTCGATGCGGCGCTGCAGGCCCGCATCGTACGTTTC 635

GAV Basic-19 GCAGCGCCGCATCGAGCAGTACATTACTGCCCAGGTCACT 636

GAV Basic-20 GGGTTAGAGAGGCCCTGGAGAGTGACCTGGGCAGTAATGT 637

GAV Basic-21 CTCCAGGGCCTCTCTAACCCCTCGGGCTCCCTCGCGGACG 638

GAV Basic-22 GGGCTCGCCGAGACCAGAGCCGTCCGCGAGGGAGCCCGAG 639

GAV Basic-23 GCTCTGGTCTCGGCGAGCCCAAGTTTGAGTTGACCCTGAA 640

GAV BasiC-24 CCCAGTTGCCGGTGAAAGGCTTCAGGGTCAACTCAAACTT 641

GAV Basic-25 GCC 1 1 1 CACCGGCAACTGGGGTCGACCGCAGCGGGATGGC 642

GAV Basic-26 GCAATGGCTCGCAGAGCTGGGCCATCCCGCTGCGGTCGAC 643

GAV Basic-27 CCAGCTCTGCGAGCCATTGCCTTGATTGGATACTCAAAGT 644

GAV BasiC-28 ATAGTTGTTGTTGATGAGCCAL 1 1 1 GAGTATCCAATCAAG 645

GAV Basic-29 GGCTCATCAACAACAACTATCAGTCGACTGTGTCCAACGT 646

GAV Basic-30 TGCGCACAATAGGCCAGATGACGTTGGACACAGTCGACTG 647

GAV Basic-31 CATCTGGCCTATTGTGCGCAACGACCTCAACTATGTTGCC 648

GAV Basic-32 CCGGTTTGGTTCCAGTACTGGGCAACATAGTTGAGGTCGT 649

GAV Basic-33 CAGTACTGGAACCAAACCGGCTTTGACCTCTGGGAAGAAG 650

GAV Basic-34 AAAGAATGAGCTCCCATTGACTTCTTCCCAGAGGTCAAAG 651

GAV Basic-35 TCAATGGGAGCTCATTCTTTACTGTTGCCAACCAGCACCG 652

GAV BasiC-36 TGGCGCCCTCGACAAGTGCTCGGTGCTGGTTGGCAACAGT 653

GAV Basic-37 AGCACTTGTCGAGGGCGCCACTCTTGCTGCCACTCTTGGC 654

GAV Basic-38 GAATAAGCGCTTCCCGACTGGCCAAGAGTGGCAGCAAGAG 655

GAV Basic-39 CAGTCGGGAAGCGCTTATTCATCTGTTGCTCCCCAGGTTT 656

GAV Basic-40 GAATCGTTGGAGAAAGCACAAAACCTGGGGAGCAACAGAT 657

GAV Basic-41 TGTGCTTTCTCCAACGATTCTGGGTGTCGTCTGGTGGATA 658 SEQ ID

Primer DNA sequence

NO:

GAV Basic-42 TGTTGATGTTGGAGTCGACGTATCCACCAGACGACACCCA 659

GAV Basic-43 CGTCGACTCCAACATCAACACCAACGAGGGCAGGACTGGC 660

GAV Basic-44 AGGACGGAGTTGACATCCTTGCCAGTCCTGCCCTCGTTGG 661

GAV Basic-45 AAGGATGTCAACTCCGTCCTGACTTCCATCCACACCTTCG 662

GAV BasiC-46 GTCACAGCCAAGGTTGGGATCGAAGGTGTGGATGGAAGTC 663

GAV BasiC-47 ATCCCAACCTTGGCTGTGACGCAGGCACCTTCCAGCCATG 664

GAV Basic-48 TGGAGAGCGCTTTGTCACTGCATGGCTGGAAGGTGCCTGC 665

GAV BasiC-49 CAGTGACAAAGCGCTCTCCAACCTCAAGGTTGTTGTCGAC 666

GAV Basic-50 CCGTAGATGGAGCGGAAGGAGTCGACAACAACCTTGAGGT 667

GAV Basic-51 TCCTTCCGCTCCATCTACGGCGTGAACAAGGGCATTCCTG 668

GAV Basic-52 AATGGCGACGGCAGCACCGGCAGGAATGCCCTTGTTCACG 669

GAV Basic-53 CCGGTGCTGCCGTCGCCATTGGCCGGTATGCAGAGGATGT 670

GAV Basic-54 AAG GGTTG CCGTTGTAGTAC ACATCCTCTG CATACCGG CC 671

GAV Basic-55 GTACTAC AACGG CAACCCTTG GTATCTTG CTAC ATTTG CT 672

GAV BasiC-56 TCGTACAGCTGCTCGGCAGCAGCAAATGTAGCAAGATACC 673

GAV Basic-57 GCTGCCGAGCAGCTGTACGATGCCATCTACGTCTGGAAGA 674

GAV Basic-58 CACCGTGATGGAGCCCGTC I TC 1 1 CCAGACGTAGATGGCA 675

GAV BasiC-59 AGACGGGCTCCATCACGGTGACCGCCACCTCCCTGGCCTT 676

GAV Basic-60 CAGGAACAAGCTCCTGGAAGAAGGCCAGGGAGGTGGCGGT 677

GAV Basic-61 CTTCCAGGAGCTTGTTCCTGGCGTGACGGCCGGGACCTAC 678

GAV Basic-62 AAGGTCGAAGAGCTGCTGGAGTAGGTCCCGGCCGTCACGC 679

GAV Basic-63 TCCAGCAGCTCTTCGACCTTTACCAACATCATCAACGCCG 680

GAV Basic-64 GCCATCGGCGTATGTCGAGACGGCGTTGATGATGTTGGTA 681

GAV Basic-65 TCTCGACATACGCCGATGGCTTCCTCAGCGAGGCTGCCAA 682

GAV Basic-66 AACCGTCGGCGGGGACGTACTTGGCAGCCTCGCTGAGGAA 683

GAV Basic-67 GTACGTCCCCGCCGACGGTTCGCTGGCCGAGCAGTTTGAC 684

GAV Basic-68 AGCGGAGTGCCGCTGTTGCGGTCAAACTGCTCGGCCAGCG 685

GAV Basic-69 CGCAACAGCGGCACTCCGCTGTCTGCGCTTCACCTGACGT 686

GAV Basic-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAAGCGCAGAC 687

GAV Basic-71 GGTCGTACGCCTCGTTCTTGACAGCCACGGCCCGTCGGGC 688 Primer SEQ ID

DNA sequence

NO:

GAV Basic-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGTGGCTGT 689

GAV Basic-73 TGGCATCGTGCCCCCCTCGTGGGCCAACAGCAGCGCTAGC 690

GAV Basic-74 GAGCACGTCGAGGGGATCGTGCTAGCGCTGCTGTTGGCCC 691

GAV Basic-75 ACGATCCCCTCGACGTGCTCCGGCGCGTCCGTGGTCGGAT 692

GAV Basic-76 GGCGGTGGGACGCGAGTAGGATCCGACCACGGACGCGCCG 693

GAV Basic-77 CCTACTCGCGTCCCACCGCCACGTCATTCCCTCCGTCGCA 694

GAV Basic-78 GCACGCCAGGCTTGGGCGTCTGCGACGGAGGGAATGACGT 695

GAV Basic-79 GACGCCCAAGCCTGGCGTGCCTTCCGGTACTCCCTACACG 696

GAV Basic-80 GGGGTCGCGCAGGGCAGGGGCGTGTAGGGAGTACCGGAAG 697

GAV Basic-81 CCCCTGCCCTGCGCGACCCCAACCTCCGTGGCCGTCACCT 698

GAV Basic-82 TGTCGACACGAGCTCGTGGAAGGTGACGGCCACGGAGGTT 699

GAV Basic-83 TCCACGAGCTCGTGTCGACACA 1 1 1 GGCCAGACGGTCAA 700

GAV Basic-84 CCGCGGCGTTGCCCGCCACCTTGACCGTCTGGCCAAACTG 701

GAV Basic-85 GGTGGCGGGCAACGCCGCGGCCCTGGGCAACTGGAGCACG 702

GAV Basic-86 TCCAGAGCCACGGCGGCGCTCGTGCTCCAGTTGCCCAGGG 703

GAV Basic-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATGCCGATA 704

GAV Basic-88 CCCAATCCACAGGGGGTGGTTATCGGCATAGTTGACGGCG 705

GAV Basic-89 ACCACCCCCTGTGGATTGGGACGGTCAACCTCGAGGCTGG 706

GAV Basic-90 ACTTGTACTCCACGACGTCTCCAGCCTCGAGGTTGACCGT 707

GAV Basic-91 AGACGTCGTGGAGTACAAGTACATCAATGTGGGCCAAGAT 708

GAV Basic-92 CTCTCCCAG GTCACGGAG CCATCTTG G CCCAC ATTG ATGT 709

GAV Basic-93 GGCTCCGTGACCTGGGAGAGTGATCCCAACCACACTTACA 710

GAV Basic-94 ACAAGCCACCGCAGGAACCGTGTAAGTGTGGTTGGGATCA 711

GAV Basic-95 CGGTTCCTGCGGTGGCTTGTGTGACGCAGGTTGTCAAGGA 712

GAV Basic-96 TTTACGACTGCCAGGTGTCCTCCTTGACAACCTGCGTCAC 713

GAV Basic-97 GGACACCTGGCAGTCGTAAACCCAGL 1 M C I 1 GTACAAAG 714

GAV Basic-98 ACCACTTTGTACAAGAAAGCTGGG 715

Alll-13 TGCGGTGATTGCATCTCCCAGCACACTTTGCCCGGACTAC 716

Alll-14 TCTCGCGTCCACATGTAATAGTAGTCCGGGCAAAGTGTGC 717

Alll-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 718 SEQ ID

Primer DNA sequence

NO:

Alll-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 719

Alll-82 TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT 720

Alll-83 TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 721

Alll-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 722

Alll-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA 723

Alll-88 CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG 724

AII2-13 TGCGGTGATTGCATCTCCCAGCACACTTGACCCGGACTAC 725

AII2-14 TCTCGCGTCCACATGTAATAGTAGTCCGGGTCAAGTGTGC 726

AII2-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 727

AII2-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 728

AII2-82 TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT 729

AII2-83 TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 730

AII2-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 731

AII2-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA 732

AII2-88 CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG 733

AII3-13 TG CG GTG ATTG CATCTCCC AG CAC AC 1 1 1 GCCCGGACTAC 734

AII3-14 TCTCGCGTCCACATGTAATAGTAGTCCGGGCAAAGTGTGC 735

AII3-16 GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA 736

AII3-17 TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT 737

AII3-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 738

AII3-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 739

AII3-82 TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT 740

AII3-83 TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 741

AII3-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 742

AII3-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA 743

AII3-88 CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG 744

AII4-13 TGCGGTGATTGCATCTCCCAGCACACTTGACCCGGACTAC 745

AII4-14 TCTCGCGTCCACATGTAATAGTAGTCCGGGTCAAGTGTGC 746

AII4-16 GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA 747

AII4-17 TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT 748 SEQ ID

Primer DNA sequence

NO:

AII4-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 749

AII4-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 750

AII4-82 TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT 751

AII4-83 TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 752

AII4-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 753

AII4-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA 754

AII4-88 CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG 755

AII5-13 TGCGGTGATTGCATCTCCCAGCACAAGAGACCCGGACTAC 756

AII5-14 TCTCGCGTCCACATGTAATAGTAGTCCGGGTCTCTTGTGC 757

AII5-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 758

AII5-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 759

AII5-82 TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT 760

AII5-83 TCCACGCGCTCGTGTCGACACAG 1 1 1 GGCCACACGGTCAA 761

AII5-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 762

AII5-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA 763

AII5-88 CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG 764

AII6-13 TGCGGTGATTGCATCTCCCAGCACAAGAGACCCGGACTAC 765

AII6-14 TCTCG CGTCCACATGTAATAGTAGTCCG G GTCTCTTGTG C 766

AII6-16 GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA 767

AII6-17 TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT 768

AII6-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 769

AII6-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 770

AII6-82 TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT 771

AII6-83 TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 772

AII6-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 773

AII6-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA 774

AII6-88 CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG 775

AII7-13 TGCGGTGATTGCATCTCCCAGCACAAGAGACCCGGACTAC 776

AII7-14 TCTCGCGTCC ACATGTAATAGTAGTCCG G GTCT CTTGTG C 777

AII7-69 CGCAACAGCGGCACTCCGCTGTCTGCGAGACACCTGACGT 778 SEQ ID

Primer DNA sequence

NO:

AII7-70 CAAGAACGAGGCGTACGACCACGTCAGGTGTCTCGCAGAC 779

AII7-82 TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT 780

AII7-83 TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 781

AII7-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 782

AII7-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA 783

AII7-88 CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG 784

AII8-13 TGCGGTGATTGCATCTCCCAGCACAAGAGACCCGGACTAC 785

AII8-14 TCTCG CGTCC ACATGTAATAGTAGTCCG G GTCTCTTGTG C 786

AII8-16 GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA 787

AII8-17 TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT 788

AII8-69 CGCAACAGCGGCACTCCGCTGTCTGCGAGACACCTGACGT 789

AII8-70 CAAGAACGAGGCGTACGACCACGTCAGGTGTCTCGCAGAC 790

AII8-82 TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT 791

AII8-83 TCCACGCGCTCGTGTCGACACA 1 1 1 GGCCACACGGTCAA 792

AII8-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 793

AII8-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA 794

AII8-88 CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG 795

CSl-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 796

CSl-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 797

CSl-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGCTCCGTCGGGC 798

CSl-72 ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGCGGCTGT 799

CSl-83 TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 800

CSl-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 801

CSl-87 AGCGCCGCCGTGGCTCTGGACCGCGTCAACTATCGCGATA 802

CSl-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGCGG 803

CSl-91 AGACGTCGTGGAGTACAAGTACATCATTGTGGGCCAAGAT 804

CSl-92 CTCTCCCAGGTCACGGAGCCATCTTGGCCCACAATGATGT 805

CS2-69 CG CAACAGCG G C ACTCCG CTGTCTG CG GTTC ACCTG ACGT 806

CS2-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 807

CS2-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGCAACGTCGGGC 808 SEQ ID

Primer DNA sequence

NO:

CS2-72 ACGAGGGGGGCACGATGCCAGCCCGACGTTGCGCGGCTGT 809

CS2-83 TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 810

CS2-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 811

CS2-87 AGCGCCGCCGTGGCTCTGGACCGCGTCAACTATCGCGATA 812

CS2-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGCGG 813

CS2-91 AGACGTCGTGGAGTACAAGTACATCATTGTGGGCCAAGAT 814

CS2-92 CTCTCCCAGGTCACGGAGCCATCTTGGCCCACAATGATGT 815

CS3-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 816

CS3-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 817

CS3-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC 818

CS3-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT 819

CS3-83 TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 820

CS3-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 821

CS3-87 AGCGCCGCCGTGGCTCTGGACCGCGTCAACTATGCCGATA 822

CS3-88 CCCAATCCACAGGGGGTGGTTATCGGCATAGTTGACGCGG 823

CS3-91 AGACGTCGTGGAGTACAAGTACATCATTGTGGGCCAAGAT 824

CS3-92 CTCTCCCAGGTCACGGAGCCATCTTGGCCCACAATGATGT 825

CS4-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 826

CS4-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 827

CS4-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC 828

CS4-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT 829

CS4-83 TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 830

CS4-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 831

CS4-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA 832

CS4-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG 833

CS4-91 AGACGTCGTGGAGTACAAGTACATCATTGTGGGCCAAGAT 834

CS4-92 CTCTCCCAGGTCACGGAGCCATCTTGGCCCACAATGATGT 835

LQ1-50 CAGTAGATGGAGCGGAAGGAGTCGACAACAACCTTGAGGT 836

LQ1-51 TCCTTCCGCTCCATCTACTGCGTGAACAAGGGCATTCCTG 837

LQ1-69 CGCAACAGCGGCACTCCGCTGTCTGCGAGACACCTGACGT 838 SEQ ID

Primer DNA sequence

NO:

LQ1-70 CAAGAACGAGGCGTACGACCACGTCAGGTGTCTCGCAGAC 839

LQ1-71 GGTCGTACGCCTCGTTCTTGACAGCCACGCTCCGTCGGGC 840

LQ1-72 ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGTGGCTGT 841

LQ2-50 CAGTAGATGGAGCGGAAGGAGTCGACAACAACCTTGAGGT 842

LQ2-51 TCCTTCCGCTCCATCTACTGCGTGAACAAGGGCATTCCTG 843

LQ2-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTACACCTGACGT 844

LQ2-70 CAAGAACGAGGCGTACGACCACGTCAGGTGTACCGCAGAC 845

LQ2-71 GGTCGTACGCCTCGTTCTTGACAGCCACGCAGCGTCGGGC 846

LQ2-72 ACGAGGGGGGCACGATGCCAGCCCGACGCTGCGTGGCTGT 847

LQ3-50 CAGTAGATGGAGCGGAAGGAGTCGACAACAACCTTGAGGT 848

LQ3-51 TCCTTCCGCTCCATCTACTGCGTGAACAAGGGCATTCCTG 849

LQ3-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTACACCTGACGT 850

LQ3-70 CAAGAACGAGGCGTACGACCACGTCAGGTGTACCGCAGAC 851

LQ3-71 GGTCGTACGCCTCGTTCTTGACAGCCACGTTACGTCGGGC 852

LQ3-72 ACGAGGGGGGCACGATGCCAGCCCGACGTAACGTGGCTGT 853

LQ3-83 TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 854

LQ3-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 855

LQ4-50 CAGTAGATGGAGCGGAAGGAGTCGACAACAACCTTGAGGT 856

LQ4-51 TCCTTCCGCTCCATCTACTGCGTGAACAAGGGCATTCCTG 857

LQ4-69 CGCAACAGCGGCACTCCGCTGTCTGCGAGACACCTGACGT 858

LQ4-70 CAAGAACGAGGCGTACGACCACGTCAGGTGTCTCGCAGAC 859

LQ4-71 GGTCGTACGCCTCGTTCTTGACAGCCACGCAGCGTCGGGC 860

LQ4-72 ACGAGGGGGGCACGATGCCAGCCCGACGCTGCGTGGCTGT 861

LQ4-83 TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 862

LQ4-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 863

LQ5-69 CGCAACAGCGGCACTCCGCTGTCTGCGCGTCACCTGACGT 864

LQ5-70 CAAGAACGAGGCGTACGACCACGTCAGGTGACGCGCAGAC 865

LQ5-71 GGTCGTACGCCTCGTTCTTGACAGCCACGCTCCGTCGGGC 866

LQ5-72 ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGTGGCTGT 867

LQ5-83 TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 868 SEQ ID

Primer DNA sequence

NO:

LQ5-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 869

LQ6-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 870

LQ6-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 871

LQ6-71 GGTCGTACGCCTCGTTCTTGACAGCCACGCTTCGTCGGGC 872

LQ6-72 ACGAGGGGGGCACGATGCCAGCCCGACGAAGCGTGGCTGT 873

LQ6-83 TCCACGAGCTCGTGTCGACACAGTTTGGCCATACGGTCAA 874

LQ6-84 CCGCGGCGTTGCCCGCCACCTTGACCGTATGGCCAAACTG 875

TS1-13 TGCGGTGATTGCATCTCCCAGCACAAGAGACCCGGACTAC 876

TS1-14 TCTCGCGTCCACATGTAATAGTAGTCCGGGTCTCTTGTGC 877

TS1-71 GGTCGTACGCCTCGTTCTTGACAGCCGCAGCCCGTCGGGC 878

TS1-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCTGCGGCTGT 879

TS1-82 TGTCGACACGAGCACGTGGAAGGTGACGGCCACGGAGGTT 880

TS1-83 TCCACGTGCTCGTGTCGACACAGTTTGGCCAGACGGTCAA 881

TS1-87 AGCGCCGCCGTGGCTCTGGACCGCGTCAACTATGCCGATA 882

TS1-88 CCCAATCCACAGGGGGTGGTTATCGGCATAGTTGACGCGG 883

TS1-91 AGACGTCGTGGAGTACAAGTACATCAAAGTGGGCCAAGAT 884

TS1-92 CTCTCCCAGGTCACGGAGCCATCTTGGCCCAU 1 I GATGT 885

TS2-13 TGCGGTGATTGCATCTCCCAGCACAATTAGACCGGACTAC 886

TS2-14 TCTCGCGTCCACATGTAATAGTAGTCCGGTCTAATTGTGC 887

TS2-82 TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT 888

TS2-83 TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 889

TS2-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 890

TS2-91 AGACGTCGTGGAGTACAAGTACATCATTGTGGGCCAAGAT 891

TS2-92 CTCTCCCAGGTCACGGAGCCATCTTGGCCCACAATGATGT 892

TS3-82 TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT 893

TS3-83 TCCACGCGCTCGTGTCGACACAGTTTGGCCAGACGGTCAA 894

TS3-91 AGACGTCGTGGAGTACAAGTACATCATTGTGGGCCAAGAT 895

TS3-92 CTCTCCCAGGTCACGGAGCCATCTTGGCCCACAATGATGT 896

TS4-13 TGCGGTGATTGCATCTCCCAGCACAAGAGACCCGGACTAC 897

TS4-14 TCTCG CGTCC ACATGTAATAGTAGTCCG G GTCT CTTGTG C 898 SEQ ID

Primer DNA sequence

NO:

TS4-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC 899

TS4-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT 900

TS4-82 TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT 901

TS4-83 TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 902

TS4-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 903

TS4-91 AGACGTCGTGGAGTACAAGTACATCATTGTGGGCCAAGAT 904

TS4-92 CTCTCCCAGGTCACGGAGCCATCTTGGCCCACAATGATGT 905

TS5-13 TG CG GTGATTG C ATCTCCC AG CAC AATTCG CCCG GACTAC 906

TS5-14 TCTCG CGTCC ACATGTAATAGTAGTCCG G G CG AATTGTG C 907

TS5-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC 908

TS5-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT 909

TS5-83 TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 910

TS5-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 911

TS5-87 AGCGCCGCCGTGGCTCTGGACGCGGTCAACTATGCCGATA 912

TS5-88 CCCAATCCACAGGGGGTGGTTATCGGCATAGTTGACCGCG 913

GAV st-1 GAGAGGGGACAAGTTTGTACAAAAAAGCAGGCT 914

GAV st- 2 GCAGTCGACAGGACGTGCATAGCCTGC 1 1 1 1 1 1 GTACAAA 915

GAV st-3 ATGCACGTCCTGTCGACTGCGGTGCTGCTCGGCTCCGTTG 916

GAV st-4 TCCCAGGACCTTTTGAACGGCAACGGAGCCGAGCAGCACC 917

GAV St- 5 CCGTTCAAAAGGTCCTGGGAAGACCAGGATCAAGCGGTCT 918

GAV st-6 ACCTCTTGGTGACGTCGGACAGACCGCTTGATCCTGGTCT 919

GAV St-7 GTCCGACGTCACCAAGAGGTCTGTTGACGACTTCATCAGC 920

GAV st-8 AGTGCAATAGGCGTCTCGGTGCTGATGAAGTCGTCAACAG 921

GAV St-9 ACCGAGACGCCTATTGCACTGAACAATU I C I 1 I GCAATG 922

GAV st- 10 ACGGCATCCATCAGGACCAACATTGCAAAGAAGATTGTTC 923

GAV st-11 TTGGTCCTGATGGATGCCGTGCATTCGGCACATCAGCTGG 924

GAV St- 12 TGGGAGATGCAATCACCGCACCAGCTGATGTGCCGAATGC 925

GAV st-13 TGCGGTGATTGCATCTCCCAGCACACAAGACCCGGACTAC 926

GAV st-14 TCTCGCGTCCACATGTAATAGTAGTCCGGGTCTTGTGTGC 927

GAV st-15 TATTACATGTGGACGCGAGATAGCGCTCTTGTCTTCAAGA 928 SEQ ID

Primer DNA sequence

NO:

GAV St- 16 GGTGAAGCGGTCGATGAGGTTCTTGAAGACAAGAGCGCTA 929

GAV st- 17 ACCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT 930

GAV St- 18 ACTGCTCGATGCGGCGCTGCAGGCCCGCATCGTACGTTTC 931

GAV st- 19 GCAGCGCCGCATCGAGCAGTACATTACTGCCCAGGTCACT 932

GAV st-20 GGGTTAGAGAGGCCCTGGAGAGTGACCTGGGCAGTAATGT 933

GAV st-21 CTCCAGGGCCTCTCTAACCCCTCGGGCTCCCTCGCGGACG 934

GAV st-22 GGGCTCGCCGAGACCAGAGCCGTCCGCGAGGGAGCCCGAG 935

GAV st-23 GCTCTGGTCTCGGCGAGCCCAAGTTTGAGTTGACCCTGAA 936

GAV St-24 CCCAGTTGCCGGTGAAAGGCTTCAGGGTCAACTCAAACTT 937

GAV st-25 GCCTTTCACCGGCAACTGGGGTCGACCGCAGCGGGATGGC 938

GAV St-26 GCAATGGCTCGCAGAGCTGGGCCATCCCGCTGCGGTCGAC 939

GAV st-27 CCAGCTCTGCGAGCCATTGCCTTGATTGGATACTCAAAGT 940

GAV St- 28 ATAGTTGTTGTTGATGAGCCAC 1 1 1 GAGTATCCAATCAAG 941

GAV st- 29 GGCTCATCAACAACAACTATCAGTCGACTGTGTCCAACGT 942

GAV st-30 TGCGCACAATAGGCCAGATGACGTTGGACACAGTCGACTG 943

GAV st-31 CATCTGGCCTATTGTGCGCAACGACCTCAACTATGTTGCC 944

GAV St-32 CCGGTTTGGTTCCAGTACTGGGCAACATAGTTGAGGTCGT 945

GAV st-33 CAGTACTGGAACCAAACCGGCTTTGACCTCTGGGAAGAAG 946

GAV St-34 AAAGAATGAGCTCCCATTGACTTCTTCCCAGAGGTCAAAG 947

GAV st-35 TCAATGGGAGCTCA 1 I C I 1 1 ACTGTTGCCAACCAGCACCG 948

GAV St-36 TGGCGCCCTCGACAAGTGCTCGGTGCTGGTTGGCAACAGT 949

GAV St-37 AGCACTTGTCGAGGGCGCCACTCTTGCTGCCACTCTTGGC 950

GAV st-38 GAATAAGCGCTTCCCGACTGGCCAAGAGTGGCAGCAAGAG 951

GAV st- 39 CAGTCGGGAAGCGCTTATTCATCTGTTGCTCCCCAGGTTT 952

GAV St-40 GAATCGTTGGAGAAAGCACAAAACCTGGGGAGCAACAGAT 953

GAV st-41 TGTGCTTTCTCCAACGATTCTGGGTGTCGTCTGGTGGATA 954

GAV st-42 TGTTGATGTTGGAGTCGACGTATCCACCAGACGACACCCA 955

GAV st-43 CGTCGACTCCAACATCAACACCAACGAGGGCAGGACTGGC 956

GAV St-44 AGGACGGAGTTGACATCCTTGCCAGTCCTGCCCTCGTTGG 957

GAV St-45 AAGGATGTCAACTCCGTCCTGACTTCCATCCACACCTTCG 958 SEQ ID

Primer DNA sequence

NO:

GAV st-46 GTCACAGCCAAGGTTGGGATCGAAGGTGTGGATGGAAGTC 959

GAV st-47 ATCCCAACCTTGGCTGTGACGCAGGCACCTTCCAGCCATG 960

GAV st-48 TGGAGAGCGCTTTGTCACTGCATGGCTGGAAGGTGCCTGC 961

GAV st-49 CAGTGACAAAGCGCTCTCCAACCTCAAGGTTGTTGTCGAC 962

GAV st-50 CCGTAGATGGAGCGGAAGGAGTCGACAACAACCTTGAGGT 963

GAV st-51 TCCTTCCGCTCCATCTACGGCGTGAACAAGGGCATTCCTG 964

GAV st- 52 AATGGCGACGGCAGCACCGGCAGGAATGCCCTTGTTCACG 965

GAV st-53 CCGGTGCTGCCGTCGCCATTGGCCGGTATGCAGAGGATGT 966

GAV st- 54 AAGGGTTGCCGTTGTAGTACACATCCTCTGCATACCGGCC 967

GAV st- 55 GTACTACAACGGCAACCCTTGGTATCTTGCTACATTTGCT 968

GAV st-56 TCGTACAGCTGCTCGGCAGCAGCAAATGTAGCAAGATACC 969

GAV st-57 GCTGCCGAGCAGCTGTACGATGCCATCTACGTCTGGAAGA 970

GAV st-58 CACCGTGATGGAGCCCGTCTTCTTCCAGACGTAGATGGCA 971

GAV st-59 AGACGGGCTCCATCACGGTGACCGCCACCTCCCTGGCCTT 972

GAV St-60 CAGGAACAAGCTCCTGGAAGAAGGCCAGGGAGGTGGCGGT 973

GAV st-61 CTTCCAGGAGCTTGTTCCTGGCGTGACGGCCGGGACCTAC 974

GAV st-62 AAGGTCGAAGAGCTGCTGGAGTAGGTCCCGGCCGTCACGC 975

GAV St-63 TCCAGCAGCTCTTCGACCTTTACCAACATCATCAACGCCG 976

GAV St- 64 GCCATCGGCGTATGTCGAGACGGCGTTGATGATGTTGGTA 977

GAV st-65 TCTCGACATACGCCGATGGCTTCCTCAGCGAGGCTGCCAA 978

GAV st-66 AACCGTCGGCGGGGACGTACTTGGCAGCCTCGCTGAGGAA 979

GAV st-67 GTACGTCCCCGCCGACGGTTCGCTGGCCGAGCAGTTTGAC 980

GAV st-68 AGCGGAGTGCCGCTGTTGCGGTCAAACTGCTCGGCCAGCG 981

GAV st-69 CGCAACAGCGGCACTCCGCTGTCTGCGCTTCACCTGACGT 982

GAV st-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAAGCGCAGAC 983

GAV st-71 GGTCGTACGCCTCGTTCTTGACAGCCACGGCCCGTCGGGC 984

GAV st-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGTGGCTGT 985

GAV st-73 TGGCATCGTGCCCCCCTCGTGGGCCAACAGCAGCGCTAGC 986

GAV st-74 GAGCACGTCGAGGGGATCGTGCTAGCGCTGCTGTTGGCCC 987

GAV St-75 ACGATCCCCTCGACGTGCTCCGGCGCGTCCGTGGTCGGAT 988 SEQ ID

Primer DNA sequence

NO:

GAV st-76 GGCGGTGGGACGCGAGTAGGATCCGACCACGGACGCGCCG 989

GAV st-77 CCTACTCGCGTCCCACCGCCACGTCATTCCCTCCGTCGCA 990

GAV st-78 GCACGCCAGGCTTGGGCGTCTGCGACGGAGGGAATGACGT 991

GAV st-79 GACGCCCAAGCCTGGCGTGCCTTCCGGTACTCCCTACACG 992

GAV st-80 GGGGTCGCGCAGGGCAGGGGCGTGTAGGGAGTACCGGAAG 993

GAV st-81 CCCCTGCCCTGCGCGACCCCAACCTCCGTGGCCGTCACCT 994

GAV st-82 TGTCGACACGAGCTCGTGGAAGGTGACGGCCACGGAGGTT 995

GAV st-83 TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 996

GAV st-84 CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG 997

GAV st-85 GGTGGCGGGCAACGCCGCGGCCCTGGGCAACTGGAGCACG 998

GAV st-86 TCCAGAGCCACGGCGGCGCTCGTGCTCCAGTTGCCCAGGG 999

GAV st-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATGCCGATA 1000

GAV st- 88 CCCAATCCACAGGGGGTGGTTATCGGCATAGTTGACGGCG 1001

GAV st- 89 ACCACCCCCTGTGGATTGGGACGGTCAACCTCGAGGCTGG 1002

GAV st-90 ACTTGTACTCCACGACGTCTCCAGCCTCGAGGTTGACCGT 1003

GAV st-91 AGACGTCGTGGAGTACAAGTACATCAATGTGGGCCAAGAT 1004

GAV st-92 CTCTCCC AG GTCACG G AG CCATCTTG G CCCAC ATTG ATGT 1005

GAV st-93 GGCTCCGTGACCTGGGAGAGTGATCCCAACCACACTTACA 1006

GAV st-94 ACAAGCCACCGCAGGAACCGTGTAAGTGTGGTTGGGATCA 1007

GAV st-95 CGGTTCCTGCGGTGGCTTGTGTGACGCAGGTTGTCAAGGA 1008

GAV st-96 TTTACGACTGCCAGGTGTCCTCCTTGACAACCTGCGTCAC 1009

GAV st-97 GGACACCTGGCAGTCGTAAACCCAGCTTTCTTGTACAAAG 1010

GAV st-98 CTCTGGGGACCACTTTGTACAAGAAAGCTGGG 1011

RB1-16 GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA 1012

RB1-17 TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT 1013

RB1-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC 1014

RB1-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT 1015

RB2-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 1016

RB2-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 1017

RB2-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC 1018 SEQ ID

Primer DNA sequence

NO:

RB2-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT 1019

RB3-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGCTCCGTCGGGC 1020

RB3-72 ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGCGGCTGT 1021

RB1-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC 1022

RB1-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT 1023

RB4-82 TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT 1024

RB4-83 TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 1025

RB4-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC 1026

RB4-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT 1027

RB5-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC 1028

RB5-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT 1029

RB5-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA 1030

RB5-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG 1031

RB6-16 GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA 1032

RB6-17 TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT 1033

RB6-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC 1034

RB6-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT 1035

RB6-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA 1036

RB6-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG 1037

RB7-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 1038

RB7-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 1039

RB7-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC 1040

RB7-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT 1041

RB7-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA 1042

RB7-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG 1043

RB8-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGCTCCGTCGGGC 1044

RB8-72 ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGCGGCTGT 1045

RB8-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA 1046

RB8-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG 1047

RB9-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGCTCCGTCGGGC 1048 SEQ ID

Primer DNA sequence

NO:

RB9-72 ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGCGGCTGT 1049

RB9-82 TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT 1050

RB9-83 TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 1051

RB10-16 GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA 1052

RB10-17 TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT 1053

RB10-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGCTCCGTCGGGC 1054

RB10-72 ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGCGGCTGT 1055

RB10-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA 1056

RB10-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG 1057

RBI 1-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 1058

RB11-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 1059

RB11-71 GGTCGTACGCCTCGTTCTTGACAGCCGCGCTCCGTCGGGC 1060

RB11-72 ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGCGGCTGT 1061

RBI 1-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA 1062

RBI 1-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG 1063

RB12-16 GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA 1064

RB12-17 TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT 1065

RB13-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 1066

RB13-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 1067

RB14-71 GGTCGTACGCCTCGTTCTTGACAGCCACGCTCCGTCGGGC 1068

RB14-72 ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGTGGCTGT 1069

RB15-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA 1070

RB15-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG 1071

RB16-16 GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA 1072

RB16-17 TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT 1073

RB16-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA 1074

RB16-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG 1075

RB17-82 TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT 1076

RB17-83 TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA 1077

RB17-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA 1078 SEQ ID

Primer DNA sequence

NO:

RB17-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG 1079

RB18-71 GGTCGTACGCCTCGTTCTTGACAGCCATGGCCCGTCGGGC 1080

RB18-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCATGGCTGT 1081

RB18-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA 1082

RB18-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG 1083

RB19-16 GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA 1084

RB19-17 TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT 1085

RB19-71 GGTCGTACGCCTCGTTCTTGACAGCCATGGCCCGTCGGGC 1086

RB19-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCATGGCTGT 1087

RB19-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA 1088

RB19-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG 1089

RB20-16 GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA 1090

RB20-17 TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT 1091

RB20-69 CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT 1092

RB20-70 CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC 1093

RB20-71 GGTCGTACGCCTCGTTCTTGACAGCCATGGCCCGTCGGGC 1094

RB20-72 ACGAGGGGGGCACGATGCCAGCCCGACGGGCCATGGCTGT 1095

RB20-87 AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA 1096

RB20-88 CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG 1097

Variants were purified from large-scale fermentation, i.e., 100 ml or 500 ml fermentation, and Pis of thermal stability (Ts) and specific activities were determined . Specifically, specific activities were determined using different substrates, including DP2, DP3, DP4, DP5, DP6, DP7, cornstarch (CS), and liquefact (Liq). Pis are presented in Table 8. "N/D" in Table 8 stands for "not done." Table 8: Pis of representative combinatorial variants

a

H

—\

z

—\

m

Example 9: Homology between TrGA and AaGA

The crystal structure of the TrGA identified in Example 11 in WO2009/067218 (Danisco US Inc., Genencor Division) page 89-93 incorporated herein by reference was superposed on the previously identified crystal structure of the Aspergillus awamori GA (AaGA). The AaGA crystal structure was obtained from the protein database (PDB) and the form of AaGA that was crystallized was the form containing only a catalytic domain (PDB entry number: 1GLM). The structure of the TrGA with all three regions intact was determined to 1.8 Angstrom resolution herein (see Table 20 in WO2009/067218 (Danisco US Inc., Genencor Division) page 94-216 incorporated herein by reference and Example 11 in in WO2009/067218 (Danisco US Inc., Genencor Division) page 89-93 incorporated herein by reference). Using the coordinates (see Table 20 in WO2009/067218 (Danisco US Inc., Genencor Division) page 94-216 incorporated herein by reference), the structure was aligned with the coordinates of the catalytic domain from Aspergillus awamori strain X100 that was determined previously (Aleshin et al., J. Mol. Biol. 238: 575-591 (1994)). As seen in Figures 6-7, the structure of the catalytic domain overlapped very closely and allowed the identification of equivalent residues based on the structural superposition.

Based on this analysis, sites were identified that could be mutated in TrGA and result in increased thermostability and/or specific activity. There sites include 108, 124, 175, and 316 at the active site. Also identified were specific pairwise variants Y47W/Y315F and

Y47F/Y315W. Other sites identified were 143, D44, P45, D46, R122, R125, V181, E242, Y310, D313, V314, N317, R408, and N409. Because of the high structural homology, it is expected that beneficial variants found at sites in the TrGA would have similar consequence in Aspergillus awamori and other homologous glucoamylases.

The TrGA linker, residues 454-490 is defined as the segment spanning the region between two disulfide bridges, one between residues 222 and 453 and one between residues 491 and 587. Nine of the residues in the linker are prolines. From the crystal structure, the linker extends from the back of the molecule in a wide arc followed by an abrupt turn after the lysine 477 residue on the surface near the substrate binding surface. The linker extends as a random coil that is anchored by interactions of the side chains of Tyr 452, Pro 465, Phe 470, Gin 474, Pro 475, Lys 477, Val 480 and Tyr 486 to regions on the surface of the catalytic domain.

The starch binding domain is composed of a beta-sandwich of two twisted beta sheets, tethered at one end by a disulfide bridge between Cys 491 and Cys 587 and at the other end, having a series of loops that comprise a binding site for starch connected by long loops. The structure of the TrGA SBD is quite similar to the averaged structure of the AnGA SBD determined by NMR (Sorimachi et al., Structure 5 : 647-661( 1997)) and the SBD of beta amylase from Bacillus cereus (Mikami, B. et al., Biochemistry 38 : 7050-61( 1999)). Figure 9 shows an alignment of the AnGA and TrGA crystal structures including the SBD. When aligned with one or both of these SBD's, one loop stands out as being highly variable, corresponding to residues 537-543 (in A. niger the loop is 554-560 and in B. cereus the loop is 462-465). In the NMR structure of beta-cyclodextrin, a starch analog complexed to the SBD of AnGA (Sorimachi et al. ( 1997) supra), the loop shifts substantially upon binding to cyclodextrin. Thus, this loop is designated the "flexible loop." This flexible loop forms part of the "binding site 2" (see Figure 9 for this binding site in TrGA). A second binding site was also identified in AnGA (binding site 1), a primary site that shares similarities with other carbohydrate binding proteins. Overall, conservation of residues and even side

conformations in the binding site 1 of these SBDs is very high. The figures demonstrate the interactions in these binding sites between the SBD and the catalytic domain that serve to bind to the starch. Taken together, there appears to be a common pattern for the interactions between the linker and SBD with the catalytic domain. The interaction is in the form of an anchoring side chain that interacts with the surface area of the neighboring domain. In general, the anchor residue is found on the linker segment. In the case of interactions between the CD and SBD, the anchor residues can be contributed from either domain as in the case of residues He 43 and Phe 29 that come from the CD or residue 592, which comes from the SBD.

Example 10: Model of acarbose binding to TrGA

The crystal structure of the TrGA complexed with the inhibitor acarbose has been

determined. Crystals of the complex were obtained by soaking pre-grown native TrGA crystals in acarbose. After soaking for 3 days the crystals were mounted in a seal glass capillary tube and x-ray diffraction was collected with a Rigaku Raxis IV++ image plate detector to a resolution of 2.0 A. The coordinates were fitted to a difference electron density map. The model was refined to an R-factor of 0.154 with an R-free of 0.201 for a total of 41276 reflection representing all data collected between 27 and 2.0 A resolution. The model of the resulting refined structure is shown in Figure 9. Based on the knowledge that the presence of the SBD has an impact on hydrolysis of insoluble starch, it followed that there should be an interaction of the SBD with larger starch molecules. Thus, the structure of the TrGA was compared with known structures of ( 1) an acarbose bound CD of AaGA and (2) an SBD from A. niger complexed with beta-cyclodextrin. This showed that the beta-cyclodextrin bound at binding site 2 was close to the substrate location as indicated by the location of acarbose bound to the A. awamori CD. Thus, the coordinates of acarbose from the structure model of the AaGA (pdb entylGAI, Aleshin, et al. 1994 supra) were aligned into the TrGA active site. Further, the AnGA SBD structure bound to cyclodextrin (pdb entry 1ACO: Sorimachi, et al 1997 supra) was aligned. From this, a model was made for acarbose binding to TrGA (see Figure 9). The model showed that the SBD would localize the TrGA CD near disrupted starch, and also prevent the enzyme from diffusing away from the substrate while releasing the product from the active site after hydrolysis. The SBD of TrGA would bind to starch along site 1, and favor localization where a disrupted fragment could bind to site 2 within a loose end that points into the catalytic site (the active side for the catalytic domain). This model shows how the proposed function of the enzyme is contributed by the structure of the SBD and linker. The amino acid side chains involved in the specific interaction between the CD, the linker and the SBD are specific for Trichoderma reesei GA, however, in other glucoamylases, complementary sequence changes would enable similar overall interactions and domain juxtaposition.

Based on this model, sites were identified for which substitutions could be made in the TrGA SBD to result in increased stability and/or specific activity. Thus, two loops that are part of binding site 1 are likely candidates for alterations to increase or decrease binding to the larger starch molecule. These are loop 1 (aa 560-570) and loop 2 (aa 523-527). Because the two Trp (tryptophan) residues at amino acids 525 and 572 are likely involved directly in starch binding, they would not be as conducive to change. However, the underlying residues, including 516-518 would be conducive, as would the underlying residues 558-562. The loop from residues 570-578 is also a good candidate for alterations. Residues 534-541 are part of the binding site 2 that interacts with the catalytic site on the CD. Thus, these might be a good candidate for alterations that may increase or decrease specific activity.

Because of the high structural homology of the TrGA SBD, it is expected that beneficial variants found at sites in Trichoderma reesei GA would have similar consequences in

Aspergillus awamori and other homologous glucoamylases. Thus, the structure of the TrGA SBD provides a basis for engineering this and related enzymes for altered properties as compared to a parent glucoamylase. These altered properties may be advantageous for processes in the generation of fuels based on starch feed stocks. Example 11

Enzymes used:

Purified variant of the Trichoderma reesei glucoamulase (TrGA) with mutations D44R and A539R. The variant is expressed in Trichoderma reesei and is hereafter called BRW 1.

Glucoamylase product from fermentation of Aspergillus niger, sold under the name DIAZYME® X4. Pullulanase product from Bacillus deramnificans expressed in Bacillus licheniformis, sold under the name DIAZYME® PIO. Acid a-amylase product from Aspergillus kawachi expressed in Trichoderma reesei, sold under the name GC626.

Results: Table 9 below shows the RDF values obtained with different combinations of glucoamylase, pullulanase and alpha-amylase using the above described "Malt-adjunct brew analysis"- method. The glucoamylase, pullulanase and alpha-amylase activity were measured as described above. Three replicates were made for each dose. The average RDF and standard deviation are listed. For glucoamylases the amount of glucoamylase protein added/kg of grist is listed. Additionally the corresponding activity in GAU/kg of grist is listed. For alpha-amylase and pullulanase the number of enzyme units added/kg of grist is listed together with the corresponding amount of enzyme product (GC626 and DIAZYME® PIO respectively) added/kg of grist. *DIAZYME® X4 also contains some alpha-amylase activity. The number of units of alpha-amylase added when dosing this product is also listed in the table.

It is seen from Table 9, dose 2 and 3 that BRWl performs better in terms of RDF obtained than TrGA. This correlates well with the fact that the BRWl variant has a lower level of reversion activity. When dosed at 1022 mg glucoamylase protein/kg of grist BRWl performs on level with DIAZYME® X4 (compare dose 1 and 3). Note that the glucoamylase in

DIAZYME® X4 (Aspergillus niger glucoamylase) and the BRWl glucoamylase have similar levels of reversion activity. The alpha-amylase activity present in DIAZYME® X4 probably means that the RDF value obtained is slightly higher than what pure Aspergillus niger glucoamylase would give. This only emphasizes that the BRWl molecule performs as well if not better than Aspergillus niger glucoamylase.

When the dose of BRWl is doubled from 1022 to 2044 mg/kg of grist, the RDF value increases from 83.2 to 84.8 (compare doses 3 and 4). RDF values can also be increased by adding auxiliary enzymes. When BRWl is combined with alpha-amylase at 28172 SSU/kg of grist and pullulanase at 1961 PU/kg of grist the RDF value increases from 83.2 to 84.1 (compare doses 3 and 5). At high dose of BRWl (2044 mg/kg of grist) there is also a benefit of adding auxiliary enzymes, but not as pronounced as with the low dose of BRWl (compare differences in RDF between dose 3 and 5 and dose 4 and 6).

SEQUENCES

Following are sequences, which are herein incorporated by reference in their entirety.

SEQ ID NO: 1: Trichoderma reesei glucoamylase, full length; with signal peptide

<210> 1

<211> 632

<212> PRT

<213> Trichoderma reesei

<400> 1

Met His Val Leu Ser Thr Ala Val Leu Leu Gly Ser Val Ala Val Gin

1 5 10 15

Lys Val Leu Gly Arg Pro Gly Ser Ser Gly Leu Ser Asp Val Thr Lys

20 25 30

Arg Ser Val Asp Asp Phe He Ser Thr Glu Thr Pro He Ala Leu Asn

35 40 45

Asn Leu Leu Cys Asn Val Gly Pro Asp Gly Cys Arg Ala Phe Gly Thr

50 55 60

Ser Ala Gly Ala Val He Ala Ser Pro Ser Thr He Asp Pro Asp Tyr

65 70 75 80

Tyr Tyr Met Trp Thr Arg Asp Ser Ala Leu Val Phe Lys Asn Leu He

85 90 95

Asp Arg Phe Thr Glu Thr Tyr Asp Ala Gly Leu Gin Arg Arg He Glu

100 105 110

Gin Tyr He Thr Ala Gin Val Thr Leu Gin Gly Leu Ser Asn Pro Ser

115 120 125

Gly Ser Leu Ala Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu Leu

130 135 140

Thr Leu Lys Pro Phe Thr Gly Asn Trp Gly Arg Pro Gin Arg Asp Gly

145 150 155 160

Pro Ala Leu Arg Ala He Ala Leu He Gly Tyr Ser Lys Trp Leu He

165 170 175

Asn Asn Asn Tyr Gin Ser Thr Val Ser Asn Val He Trp Pro He Val

180 185 190

Arg Asn Asp Leu Asn Tyr Val Ala Gin Tyr Trp Asn Gin Thr Gly Phe

195 200 205

Asp Leu Trp Glu Glu Val Asn Gly Ser Ser Phe Phe Thr Val Ala Asn

210 215 220

Gin His Arg Ala Leu Val Glu Gly Ala Thr Leu Ala Ala Thr Leu Gly 225 230 235 240

Gin Ser Gly Ser Ala Tyr Ser Ser Val Ala Pro Gin Val Leu Cys Phe

245 250 255

Leu Gin Arg Phe Trp Val Ser Ser Gly Gly Tyr Val Asp Ser Asn He

260 265 270

Asn Thr Asn Glu Gly Arg Thr Gly Lys Asp Val Asn Ser Val Leu Thr

275 280 285

Ser He His Thr Phe Asp Pro Asn Leu Gly Cys Asp Ala Gly Thr Phe

290 295 300

Gin Pro Cys Ser Asp Lys Ala Leu Ser Asn Leu Lys Val Val Val Asp

305 310 315 320

Ser Phe Arg Ser He Tyr Gly Val Asn Lys Gly He Pro Ala Gly Ala

325 330 335

Ala Val Ala He Gly Arg Tyr Ala Glu Asp Val Tyr Tyr Asn Gly Asn

340 345 350

Pro Trp Tyr Leu Ala Thr Phe Ala Ala Ala Glu Gin Leu Tyr Asp Ala

355 360 365

He Tyr Val Trp Lys Lys Thr Gly Ser He Thr Val Thr Ala Thr Ser

370 375 380

Leu Ala Phe Phe Gin Glu Leu Val Pro Gly Val Thr Ala Gly Thr Tyr

385 390 395 400

Ser Ser Ser Ser Ser Thr Phe Thr Asn He He Asn Ala Val Ser Thr

405 410 415

Tyr Ala Asp Gly Phe Leu Ser Glu Ala Ala Lys Tyr Val Pro Ala Asp

420 425 430

Gly Ser Leu Ala Glu Gin Phe Asp Arg Asn Ser Gly Thr Pro Leu Ser

435 440 445

Ala Leu His Leu Thr Trp Ser Tyr Ala Ser Phe Leu Thr Ala Thr Ala

450 455 460

Arg Arg Ala Gly He Val Pro Pro Ser Trp Ala Asn Ser Ser Ala Ser

465 470 475 480

Thr He Pro Ser Thr Cys Ser Gly Ala Ser Val Val Gly Ser Tyr Ser

485 490 495

Arg Pro Thr Ala Thr Ser Phe Pro Pro Ser Gin Thr Pro Lys Pro Gly

500 505 510

Val Pro Ser Gly Thr Pro Tyr Thr Pro Leu Pro Cys Ala Thr Pro Thr

515 520 525

Ser Val Ala Val Thr Phe His Glu Leu Val Ser Thr Gin Phe Gly Gin

530 535 540

Thr Val Lys Val Ala Gly Asn Ala Ala Ala Leu Gly Asn Trp Ser Thr

545 550 555 560

Ser Ala Ala Val Ala Leu Asp Ala Val Asn Tyr Ala Asp Asn His Pro

565 570 575

Leu Trp He Gly Thr Val Asn Leu Glu Ala Gly Asp Val Val Glu Tyr

580 585 590

Lys Tyr He Asn Val Gly Gin Asp Gly Ser Val Thr Trp Glu Ser Asp

595 600 605

Pro Asn His Thr Tyr Thr Val Pro Ala Val Ala Cys Val Thr Gin Val

610 615 620

Val Lys Glu Asp Thr Trp Gin Ser

625 630

SEQ ID NO: 2: Trichoderma reesei glucoamylase, mature protein; without signal peptide

<210> 2

<211> 599

<212> PRT

<213> Trichoderma reesei

<400> 2

Ser Val Asp Asp Phe He Ser Thr Glu Thr Pro He Ala Leu Asn Asn 1 5 10 15

Leu Leu Cys Asn Val Gly Pro Asp Gly Cys Arg Ala Phe Gly Thr Ser

20 25 30

Ala Gly Ala Val He Ala Ser Pro Ser Thr He Asp Pro Asp Tyr Tyr

35 40 45

Tyr Met Trp Thr Arg Asp Ser Ala Leu Val Phe Lys Asn Leu He Asp

50 55 60

Arg Phe Thr Glu Thr Tyr Asp Ala Gly Leu Gin Arg Arg He Glu Gin

65 70 75 80

Tyr He Thr Ala Gin Val Thr Leu Gin Gly Leu Ser Asn Pro Ser Gly

85 90 95

Ser Leu Ala Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu Leu Thr

100 105 110

Leu Lys Pro Phe Thr Gly Asn Trp Gly Arg Pro Gin Arg Asp Gly Pro

115 120 125

Ala Leu Arg Ala He Ala Leu He Gly Tyr Ser Lys Trp Leu He Asn

130 135 140

Asn Asn Tyr Gin Ser Thr Val Ser Asn Val He Trp Pro He Val Arg

145 150 155 160

Asn Asp Leu Asn Tyr Val Ala Gin Tyr Trp Asn Gin Thr Gly Phe Asp

165 170 175

Leu Trp Glu Glu Val Asn Gly Ser Ser Phe Phe Thr Val Ala Asn Gin

180 185 190

His Arg Ala Leu Val Glu Gly Ala Thr Leu Ala Ala Thr Leu Gly Gin

195 200 205

Ser Gly Ser Ala Tyr Ser Ser Val Ala Pro Gin Val Leu Cys Phe Leu

210 215 220

Gin Arg Phe Trp Val Ser Ser Gly Gly Tyr Val Asp Ser Asn He Asn

225 230 235 240

Thr Asn Glu Gly Arg Thr Gly Lys Asp Val Asn Ser Val Leu Thr Ser

245 250 255

He His Thr Phe Asp Pro Asn Leu Gly Cys Asp Ala Gly Thr Phe Gin

260 265 270

Pro Cys Ser Asp Lys Ala Leu Ser Asn Leu Lys Val Val Val Asp Ser

275 280 285

Phe Arg Ser He Tyr Gly Val Asn Lys Gly He Pro Ala Gly Ala Ala

290 295 300

Val Ala He Gly Arg Tyr Ala Glu Asp Val Tyr Tyr Asn Gly Asn Pro

305 310 315 320

Trp Tyr Leu Ala Thr Phe Ala Ala Ala Glu Gin Leu Tyr Asp Ala He

325 330 335

Tyr Val Trp Lys Lys Thr Gly Ser He Thr Val Thr Ala Thr Ser Leu

340 345 350

Ala Phe Phe Gin Glu Leu Val Pro Gly Val Thr Ala Gly Thr Tyr Ser

355 360 365

Ser Ser Ser Ser Thr Phe Thr Asn He He Asn Ala Val Ser Thr Tyr

370 375 380

Ala Asp Gly Phe Leu Ser Glu Ala Ala Lys Tyr Val Pro Ala Asp Gly

385 390 395 400

Ser Leu Ala Glu Gin Phe Asp Arg Asn Ser Gly Thr Pro Leu Ser Ala

405 410 415

Leu His Leu Thr Trp Ser Tyr Ala Ser Phe Leu Thr Ala Thr Ala Arg

420 425 430

Arg Ala Gly He Val Pro Pro Ser Trp Ala Asn Ser Ser Ala Ser Thr

435 440 445

He Pro Ser Thr Cys Ser Gly Ala Ser Val Val Gly Ser Tyr Ser Arg

450 455 460

Pro Thr Ala Thr Ser Phe Pro Pro Ser Gin Thr Pro Lys Pro Gly Val

465 470 475 480

Pro Ser Gly Thr Pro Tyr Thr Pro Leu Pro Cys Ala Thr Pro Thr Ser

485 490 495

Val Ala Val Thr Phe His Glu Leu Val Ser Thr Gin Phe Gly Gin Thr 500 505 510

Val Lys Val Ala Gly Asn Ala Ala Ala Leu Gly Asn Trp Ser Thr Ser

515 520 525

Ala Ala Val Ala Leu Asp Ala Val Asn Tyr Ala Asp Asn His Pro Leu

530 535 540

Trp lie Gly Thr Val Asn Leu Glu Ala Gly Asp Val Val Glu Tyr Lys

545 550 555 560

Tyr lie Asn Val Gly Gin Asp Gly Ser Val Thr Trp Glu Ser Asp Pro

565 570 575

Asn His Thr Tyr Thr Val Pro Ala Val Ala Cys Val Thr Gin Val Val

580 585 590

Lys Glu Asp Thr Trp Gin Ser

595

SEQ ID NO: 3: Trichoderma reesei glucoamylase catalytic domain, 1-453 of matu TrGA, CD

<210> 3

<211> 453

<212> PRT

<213> Trichoderma reesei

<400> 3

Ser Val Asp Asp Phe lie Ser Thr Glu Thr Pro lie Ala Leu Asn Asn

1 5 10 15

Leu Leu Cys Asn Val Gly Pro Asp Gly Cys Arg Ala Phe Gly Thr Ser

20 25 30

Ala Gly Ala Val lie Ala Ser Pro Ser Thr lie Asp Pro Asp Tyr Tyr

35 40 45

Tyr Met Trp Thr Arg Asp Ser Ala Leu Val Phe Lys Asn Leu lie Asp

50 55 60

Arg Phe Thr Glu Thr Tyr Asp Ala Gly Leu Gin Arg Arg lie Glu Gin

65 70 75 80

Tyr lie Thr Ala Gin Val Thr Leu Gin Gly Leu Ser Asn Pro Ser Gly

85 90 95

Ser Leu Ala Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu Leu Thr

100 105 110

Leu Lys Pro Phe Thr Gly Asn Trp Gly Arg Pro Gin Arg Asp Gly Pro

115 120 125

Ala Leu Arg Ala lie Ala Leu lie Gly Tyr Ser Lys Trp Leu lie Asn

130 135 140

Asn Asn Tyr Gin Ser Thr Val Ser Asn Val lie Trp Pro lie Val Arg

145 150 155 160

Asn Asp Leu Asn Tyr Val Ala Gin Tyr Trp Asn Gin Thr Gly Phe Asp

165 170 175

Leu Trp Glu Glu Val Asn Gly Ser Ser Phe Phe Thr Val Ala Asn Gin

180 185 190

His Arg Ala Leu Val Glu Gly Ala Thr Leu Ala Ala Thr Leu Gly Gin

195 200 205

Ser Gly Ser Ala Tyr Ser Ser Val Ala Pro Gin Val Leu Cys Phe Leu

210 215 220

Gin Arg Phe Trp Val Ser Ser Gly Gly Tyr Val Asp Ser Asn lie Asn

225 230 235 240

Thr Asn Glu Gly Arg Thr Gly Lys Asp Val Asn Ser Val Leu Thr Ser

245 250 255 lie His Thr Phe Asp Pro Asn Leu Gly Cys Asp Ala Gly Thr Phe Gin

260 265 270

Pro Cys Ser Asp Lys Ala Leu Ser Asn Leu Lys Val Val Val Asp Ser

275 280 285

Phe Arg Ser lie Tyr Gly Val Asn Lys Gly lie Pro Ala Gly Ala Ala

290 295 300

Val Ala lie Gly Arg Tyr Ala Glu Asp Val Tyr Tyr Asn Gly Asn Pro 305 310 315 320

Trp Tyr Leu Ala Thr Phe Ala Ala Ala Glu Gin Leu Tyr Asp Ala He

325 330 335

Tyr Val Trp Lys Lys Thr Gly Ser He Thr Val Thr Ala Thr Ser Leu

340 345 350

Ala Phe Phe Gin Glu Leu Val Pro Gly Val Thr Ala Gly Thr Tyr Ser

355 360 365

Ser Ser Ser Ser Thr Phe Thr Asn He He Asn Ala Val Ser Thr Tyr

370 375 380

Ala Asp Gly Phe Leu Ser Glu Ala Ala Lys Tyr Val Pro Ala Asp Gly

385 390 395 400

Ser Leu Ala Glu Gin Phe Asp Arg Asn Ser Gly Thr Pro Leu Ser Ala

405 410 415

Leu His Leu Thr Trp Ser Tyr Ala Ser Phe Leu Thr Ala Thr Ala Arg

420 425 430

Arg Ala Gly He Val Pro Pro Ser Trp Ala Asn Ser Ser Ala Ser Thr

435 440 445

lie Pro Ser Thr Cys

450

SEQ ID NO: 4: Trichoderma reesei glucoamylase cDNA

<210> 4

<211> 1899

<212> DNA

<213> Trichodi

<400> 4

atgcacgtcc tgtcgactgc ggtgctgctc ggctccgttg ccgttcaaaa ggtcctggga 60 agaccaggat caagcggtct gtccgacgtc accaagaggt ctgttgacga cttcatcagc 120 accgagacgc ctattgcact gaacaatctt ctttgcaatg ttggtcctga tggatgccgt 180 gcattcggca catcagctgg tgcggtgatt gcatctccca gcacaattga cccggactac 240 tattacatgt ggacgcgaga tagcgctctt gtcttcaaga acctcatcga ccgcttcacc 300 gaaacgtacg atgcgggcct gcagcgccgc atcgagcagt acattactgc ccaggtcact 360 ctccagggcc tctctaaccc ctcgggctcc ctcgcggacg gctctggtct cggcgagccc 420 aagtttgagt tgaccctgaa gcctttcacc ggcaactggg gtcgaccgca gcgggatggc 480 ccagctctgc gagccattgc cttgattgga tactcaaagt ggctcatcaa caacaactat 540 cagtcgactg tgtccaacgt catctggcct attgtgcgca acgacctcaa ctatgttgcc 600 cagtactgga accaaaccgg ctttgacctc tgggaagaag tcaatgggag ctcattcttt 660 actgttgcca accagcaccg agcacttgtc gagggcgcca ctcttgctgc cactcttggc 720 cagtcgggaa gcgcttattc atctgttgct ccccaggttt tgtgctttct ccaacgattc 780 tgggtgtcgt ctggtggata cgtcgactcc aacatcaaca ccaacgaggg caggactggc 840 aaggatgtca actccgtcct gacttccatc cacaccttcg atcccaacct tggctgtgac 900 gcaggcacct tccagccatg cagtgacaaa gcgctctcca acctcaaggt tgttgtcgac 960 tccttccgct ccatctacgg cgtgaacaag ggcattcctg ccggtgctgc cgtcgccatt 1020 ggccggtatg cagaggatgt gtactacaac ggcaaccctt ggtatcttgc tacatttgct 1080 gctgccgagc agctgtacga tgccatctac gtctggaaga agacgggctc catcacggtg 1140 accgccacct ccctggcctt cttccaggag cttgttcctg gcgtgacggc cgggacctac 1200 tccagcagct cttcgacctt taccaacatc atcaacgccg tctcgacata cgccgatggc 1260 ttcctcagcg aggctgccaa gtacgtcccc gccgacggtt cgctggccga gcagtttgac 1320 cgcaacagcg gcactccgct gtctgcgctt cacctgacgt ggtcgtacgc ctcgttcttg 1380 acagccacgg cccgtcgggc tggcatcgtg cccccctcgt gggccaacag cagcgctagc 1440 acgatcccct cgacgtgctc cggcgcgtcc gtggtcggat cctactcgcg tcccaccgcc 1500 acgtcattcc ctccgtcgca gacgcccaag cctggcgtgc cttccggtac tccctacacg 1560 cccctgccct gcgcgacccc aacctccgtg gccgtcacct tccacgagct cgtgtcgaca 1620 cagtttggcc agacggtcaa ggtggcgggc aacgccgcgg ccctgggcaa ctggagcacg 1680 agcgccgccg tggctctgga cgccgtcaac tatgccgata accaccccct gtggattggg 1740 acggtcaacc tcgaggctgg agacgtcgtg gagtacaagt acatcaatgt gggccaagat 1800 ggctccgtga cctgggagag tgatcccaac cacacttaca cggttcctgc ggtggcttgt 1860 gtgacgcagg ttgtcaagga ggacacctgg cagtcgtaa 1899

SEQ ID NO: 5: Aspergillus awamori GA (AaGA) ; CD <210> 5

<211> 448

<212> PRT

<213> Aspergillus awamori

<400> 5

Ala Thr Leu Asp Ser Trp Leu Ser Asn Glu Ala Thr Val Ala Arg Thr

1 5 10 15

Ala lie Leu Asn Asn He Gly Ala Asp Gly Ala Trp Val Ser Gly Ala

20 25 30

Asp Ser Gly He Val Val Ala Ser Pro Ser Thr Asp Asn Pro Asp Tyr

35 40 45

Phe Tyr Thr Trp Thr Arg Asp Ser Gly Leu Val He Lys Thr Leu Val

50 55 60

Asp Leu Phe Arg Asn Gly Asp Thr Asp Leu Leu Ser Thr He Glu Asn

65 70 75 80

Tyr He Ser Ser Gin Ala He Val Gin Gly He Ser Asn Pro Ser Gly

85 90 95

Asp Leu Ser Ser Gly Gly Leu Gly Glu Pro Lys Phe Asn Val Asp Glu

100 105 110

Thr Ala Tyr Thr Gly Ser T p Gly Arg Pro Gin Arg Asp Gly Pro Ala

115 120 125

Leu Arg Ala Thr Ala Met He Gly Phe Arg Gin Trp Leu Leu Asp Asn

130 135 140

Gly Tyr Thr Ser Ala Ala Thr Glu He Val Trp Pro Leu Val Arg Asn

145 150 155 160

Asp Leu Ser Tyr Val Ala Gin Tyr Trp Asn Gin Thr Gly Tyr Asp Leu

165 170 175

Trp Glu Glu Val Asn Gly Ser Ser Phe Phe Thr He Ala Val Gin His

180 185 190

Arg Ala Leu Val Glu Gly Ser Ala Phe Ala Thr Ala Val Gly Ser Ser

195 200 205

Cys Ser Trp Cys Asp Ser Gin Ala Pro Gin He Leu Cys Tyr Leu Gin

210 215 220

Ser Phe Trp Thr Gly Glu Tyr He Leu Ala Asn Phe Asp Ser Ser Arg

225 230 235 240

Ser Gly Lys Asp Thr Asn Thr Leu Leu Gly Ser He His Thr Phe Asp

245 250 255

Pro Glu Ala Gly Cys Asp Asp Ser Thr Phe Gin Pro Cys Ser Pro Arg

260 265 270

Ala Leu Ala Asn His Lys Glu Val Val Asp Ser Phe Arg Ser He Tyr

275 280 285

Thr Leu Asn Asp Gly Leu Ser Asp Ser Glu Ala Val Ala Val Gly Arg

290 295 300

Tyr Pro Lys Asp Ser Tyr Tyr Asn Gly Asn Pro Trp Phe Leu Cys Thr

305 310 315 320

Leu Ala Ala Ala Glu Gin Leu Tyr Asp Ala Leu Tyr Gin Trp Asp Lys

325 330 335

Gin Gly Ser Leu Glu He Thr Asp Val Ser Leu Asp Phe Phe Gin Ala

340 345 350

Leu Tyr Ser Asp Ala Ala Thr Gly Thr Tyr Ser Ser Ser Ser Ser Thr

355 360 365

Tyr Ser Ser He Val Asp Ala Val Lys Thr Phe Ala Asp Gly Phe Val

370 375 380

Ser He Val Glu Thr His Ala Ala Ser Asn Gly Ser Leu Ser Glu Gin

385 390 395 400

Tyr Asp Lys Ser Asp Gly Asp Glu Leu Ser Ala Arg Asp Leu Thr Trp

405 410 415

Ser Tyr Ala Ala Leu Leu Thr Ala Asn Asn Arg Arg Asn Ser Val Met

420 425 430

Pro Pro Ser rp Gly Glu Thr Ser Ala Ser Ser Val Pro Gly Thr Cys 435 440

ID NO: 6: Aspergillus niger (AnGA) ,

)> 6

L> 449

>> PRT

> Aspergillus niger

<400> 6

Ala Thr Leu Asp Ser Trp Leu Ser Asn Glu Ala Thr Val Ala Arg Thr

1 5 10 15

Ala He Leu Asn Asn He Gly Ala Asp Gly Ala Trp Val Ser Gly Ala

20 25 30

Asp Ser Gly He Val Val Ala Ser Pro Ser Thr Asp Asn Pro Asp Tyr

35 40 45

Phe Tyr Thr Trp Thr Arg Asp Ser Gly Leu Val Leu Lys Thr Leu Val

50 55 60

Asp Leu Phe Arg Asn Gly Asp Thr Ser Leu Leu Ser Thr He Glu Asn

65 70 75 80

Tyr He Ser Ala Gin Ala He Val Gin Gly He Ser Asn Pro Ser Gly

85 90 95

Asp Leu Ser Ser Gly Ala Gly Leu Gly Glu Pro Lys Phe Asn Val Asp

100 105 110

Glu Thr Ala Tyr Thr Gly Ser Trp Gly Arg Pro Gin Arg Asp Gly Pro

115 120 125

Ala Leu Arg Ala Thr Ala Met He Gly Phe Gly Gin Trp Leu Leu Asp

130 135 140

Asn Gly Tyr Thr Ser Thr Ala Thr Asp He Val Trp Pro Leu Val Arg

145 150 155 160

Asn Asp Leu Ser Tyr Val Ala Gin Tyr Trp Asn Gin Thr Gly Tyr Asp

165 170 175

Leu Trp Glu Glu Val Asn Gly Ser Ser Phe Phe Thr He Ala Val Gin

180 185 190

His Arg Ala Leu Val Glu Gly Ser Ala Phe Ala Thr Ala Val Gly Ser

195 200 205

Ser Cys Ser Trp Cys Asp Ser Gin Ala Pro Glu He Leu Cys Tyr Leu

210 215 220

Gin Ser Phe Trp Thr Gly Ser Phe He Leu Ala Asn Phe Asp Ser Ser

225 230 235 240

Arg Ser Gly Lys Asp Ala Asn Thr Leu Leu Gly Ser He His Thr Phe

245 250 255

Asp Pro Glu Ala Ala Cys Asp Asp Ser Thr Phe Gin Pro Cys Ser Pro

260 265 270

Arg Ala Leu Ala Asn His Lys Glu Val Val Asp Ser Phe Arg Ser He

275 280 285

Tyr Thr Leu Asn Asp Gly Leu Ser Asp Ser Glu Ala Val Ala Val Gly

290 295 300

Arg Tyr Pro Glu Asp Thr Tyr Tyr Asn Gly Asn Pro Trp Phe Leu Cys

305 310 315 320

Thr Leu Ala Ala Ala Glu Gin Leu Tyr Asp Ala Leu Tyr Gin Trp Asp

325 330 335

Lys Gin Gly Ser Leu Glu Val Thr Asp Val Ser Leu Asp Phe Phe Lys

340 345 350

Ala Leu Tyr Ser Asp Ala Ala Thr Gly Thr Tyr Ser Ser Ser Ser Ser

355 360 365

Thr Tyr Ser Ser He Val Asp Ala Val Lys Thr Phe Ala Asp Gly Phe

370 375 380

Val Ser He Val Glu Thr His Ala Ala Ser Asn Gly Ser Met Ser Glu

385 390 395 400

Gin Tyr Asp Lys Ser Asp Gly Glu Gin Leu Ser Ala Arg Asp Leu Thr

405 410 415 Trp Ser Tyr Ala Ala Leu Leu Thr Ala Asn Asn Arg Arg Asn Ser Val

420 425 430

Val Pro Ala Ser Trp Gly Glu Thr Ser Ala Ser Ser Val Pro Gly Thr

435 440 445

Cys

SEQ ID NO: 7: Aspergillus oryzae (AoGA) , CD

<210> 7

<211> 450

<212> PRT

<213> Aspergillus oryzae

<400> 7

Gin Ser Asp Leu Asn Ala Phe He Glu Ala Gin Thr Pro He Ala Lys

1 5 10 15

Gin Gly Tyr Leu Asn Asn He Gly Ala Asp Gly Lys Leu Val Glu Gly

20 25 30

Ala Ala Ala Gly He Val Tyr Ala Ser Pro Ser Lys Ser Asn Pro Asp

35 40 45

Tyr Phe Tyr Thr Trp Thr Arg Asp Ala Gly Leu Thr Met Glu Glu Tyr

50 55 60

lie Glu Gin Phe He Gly Gly Asp Ala Thr Leu Glu Ser Thr He Gin

65 70 75 80

Asn Tyr Val Asp Ser Gin Ala Asn Glu Gin Ala Val Ser Asn Pro Ser

85 90 95

Gly Gly Leu Ser Asp Gly Ser Gly Leu Ala Glu Pro Lys Phe Tyr Tyr

100 105 110

Asn He Ser Gin Phe Thr Asp Ser Trp Gly Arg Pro Gin Arg Asp Gly

115 120 125

Pro Ala Leu Arg Ala Ser Ala Leu He Ala Tyr Gly Asn Ser Leu He

130 135 140

Ser Ser Asp Lys Gin Ser Val Val Lys Ala Asn He Trp Pro He Tyr

145 150 155 160

Gin Asn Asp Leu Ser Tyr Val Gly Gin Tyr Trp Asn Gin Thr Gly Phe

165 170 175

Asp Leu Trp Glu Glu Val Gin Gly Ser Ser Phe Phe Thr Val Ala Val

180 185 190

Gin His Lys Ala Leu Val Glu Gly Asp Ala Phe Ala Lys Ala Leu Gly

195 200 205

Glu Glu Cys Gin Ala Cys Ser Val Ala Pro Gin He Leu Cys His Leu

210 215 220

Gin Asp Phe Trp Asn Gly Ser Ala Val Leu Ser Asn Leu Pro Thr Asn

225 230 235 240

Gly Arg Ser Gly Leu Asp Thr Asn Ser Leu Leu Gly Ser He His Thr

245 250 255

Phe Asp Pro Ala Ala Ala Cys Asp Asp Thr Thr Phe Gin Pro Cys Ser

260 265 270

Ser Arg Ala Leu Ser Asn His Lys Leu Val Val Asp Ser Phe Arg Ser

275 280 285

Val Tyr Gly He Asn Asn Gly Arg Gly Ala Gly Lys Ala Ala Ala Val

290 295 300

Gly Pro Tyr Ala Glu Asp Thr Tyr Gin Gly Gly Asn Pro Trp Tyr Leu

305 310 315 320

Thr Thr Leu Val Ala Ala Glu Leu Leu Tyr Asp Ala Leu Tyr Gin Trp

325 330 335

Asp Lys Gin Gly Gin Val Asn Val Thr Glu Thr Ser Leu Pro Phe Phe

340 345 350

Lys Asp Leu Ser Ser Asn Val Thr Thr Gly Ser Tyr Ala Lys Ser Ser

355 360 365

Ser Ala Tyr Glu Ser Leu Thr Ser Ala Val Lys Thr Tyr Ala Asp Gly 370 375 380

Phe lie Ser Val Val Gin Glu Tyr Thr Pro Asp Gly Gly Ala Leu Ala 385 390 395 400

Glu Gin Tyr Ser Arg Asp Gin Gly Thr Pro Val Ser Ala Ser Asp Leu

405 410 415

Thr Trp Ser Tyr Ala Ala Phe Leu Ser Ala Val Gly Arg Arg Asn Gly

420 425 430

Thr Val Pro Ala Ser Trp Gly Ser Ser Thr Ala Asn Ala Val Pro Ser

435 440 445

Gin Cys

450

SEQ ID NO: 8: Hu icola grisea glucoamylase (HgGA) ; CD

<210> 8

<211> 441

<212> PRT

<213> Humicola grisea

<400> 8

Ala Ala Val Asp Thr Phe lie Asn Thr Glu Lys Pro lie Ala Trp Asn

1 5 10 15

Lys Leu Leu Ala Asn lie Gly Pro Asn Gly Lys Ala Ala Pro Gly Ala

20 25 30

Ala Ala Gly Val Val lie Ala Ser Pro Ser Arg Thr Asp Pro Pro Tyr

35 40 45

Phe Phe Thr Trp Thr Pro Asp Ala Ala Leu Val Leu Thr Gly lie lie

50 55 60

Glu Ser Leu Gly His Asn Tyr Asn Thr Thr Leu Gin Gin Val Ser Asn 65 70 75 80

Pro Ser Gly Thr Phe Ala Asp Gly Ser Gly Leu Gly Glu Ala Lys Phe

85 90 95

Asn Val Asp Leu Thr Ala Phe Thr Gly Glu Trp Gly Arg Pro Gin Arg

100 105 110

Asp Gly Pro Pro Leu Arg Ala lie Ala Leu lie Gin Tyr Ala Lys Trp

115 120 125

Leu lie Ala Asn Gly Tyr Lys Ser Thr Ala Lys Ser Val Val Trp Pro

130 135 140

Val Val Lys Asn Asp Leu Ala Tyr Thr Ala Gin Tyr Trp Asn Glu Thr 145 150 155 160

Gly Phe Asp Leu Trp Glu Glu Val Pro Gly Ser Ser Phe Phe Thr lie

165 170 175

Ala Ser Ser His Arg Ala Leu Thr Glu Gly Ala Tyr Leu Ala Ala Gin

180 185 190

Leu Asp Thr Glu Cys Pro Pro Cys Thr Thr Val Ala Pro Gin Val Leu

195 200 205

Cys Phe Gin Gin Ala Phe Trp Asn Ser Lys Gly Asn Tyr Val Val Ser

210 215 220

Thr Ser Thr Ala Gly Glu Tyr Arg Ser Gly Lys Asp Ala Asn Ser lie 225 230 235 240

Leu Ala Ser lie His Asn Phe Asp Pro Glu Ala Gly Cys Asp Asn Leu

245 250 255

Thr Phe Gin Pro Cys Ser Glu Arg Ala Leu Ala Asn His Lys Ala Tyr

260 265 270

Val Asp Ser Phe Arg Asn Leu Tyr Ala lie Asn Lys Gly lie Ala Gin

275 280 285

Gly Lys Ala Val Ala Val Gly Arg Tyr Ser Glu Asp Val Tyr Tyr Asn

290 295 300

Gly Asn Pro Trp Tyr Leu Ala Asn Phe Ala Ala Ala Glu Gin Leu Tyr 305 310 315 320

Asp Ala He Tyr Val Trp Asn Lys Gin Gly Ser He Thr Val Thr Ser

325 330 335 Val Ser Leu Pro Phe Phe Arg Asp Leu Val Ser Ser Val Ser Thr Gly 340 345 350

Thr Tyr Ser Lys Ser Ser Ser Thr Phe Thr Asn He Val Asn Ala Val

355 360 365

Lys Ala Tyr Ala Asp Gly Phe lie Glu Val Ala Ala Lys Tyr Thr Pro 370 375 380

Ser Asn Gly Ala Leu Ala Glu Gin Tyr Asp Arg Asn Thr Gly Lys Pro

385 390 395 400

Asp Ser Ala Ala Asp Leu Thr Trp Ser Tyr Ser Ala Phe Leu Ser Ala

405 410 415

He Asp Arg Arg Ala Gly Leu Val Pro Pro Ser Trp Arg Ala Ser Val

420 425 430

Ala Lys Ser Gin Leu Pro Ser Thr Cys

435 440

SEQ ID NO: 9: Hypocrea vinosa glucoamylase (HvGA) ; CD

<210> 9

<211> 452

<212> PRT

<213> Hypocrea vinosa

<400> 9

Ser Val Asp Asp Phe He Asn Thr Gin Thr Pro He Ala Leu Asn Asn

1 5 10 15

Leu Leu Cys Asn Val Gly Pro Asp Gly Cys Arg Ala Phe Gly Thr Ser

20 25 30

Ala Gly Ala Val He Ala Ser Pro Ser Thr Thr Asp Pro Asp Tyr Tyr

35 40 45

Tyr Met Trp Thr Arg Asp Ser Ala Leu Val Phe Lys Asn He Val Asp

50 55 60

Arg Phe Thr Gin Gin Tyr Asp Ala Gly Leu Gin Arg Arg He Glu Gin

65 70 75 80

Tyr He Ser Ala Gin Val Thr Leu Gin Gly He Ser Asn Pro Ser Gly

85 90 95

Ser Leu Ser Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu Leu Thr

100 105 110

Leu Ser Gin Phe Thr Gly Asn Trp Gly Arg Pro Gin Arg Asp Gly Pro

115 120 125

Ala Leu Arg Ala He Ala Leu He Gly Tyr Ser Lys Trp Leu He Asn

130 135 140

Asn Asn Tyr Gin Ser Thr Val Ser Asn He He Trp Pro He Val Arg

145 150 155 160

Asn Asp Leu Asn Tyr Val Ala Gin Tyr Trp Asn Gin Thr Gly Phe Asp

165 170 175

Leu Trp Glu Glu Val Asn Gly Ser Ser Phe Phe Thr Val Ala Asn Gin

180 185 190

His Arg Ala Leu Val Glu Gly Ala Thr Leu Ala Ala Thr Leu Gly Gin

195 200 205

Ser Gly Ser Thr Tyr Ser Ser Val Ala Pro Gin He Leu Cys Phe Leu

210 215 220

Gin Arg Phe Trp Val Ser Gly Gly Tyr He Asp Ser Asn He Asn Thr

225 230 235 240

Asn Glu Gly Arg Thr Gly Lys Asp Ala Asn Ser Leu Leu Ala Ser He

245 250 255

His Thr Phe Asp Pro Ser Leu Gly Cys Asp Ala Ser Thr Phe Gin Pro

260 265 270

Cys Ser Asp Lys Ala Leu Ser Asn Leu Lys Val Val Val Asp Ser Phe

275 280 285

Arg Ser He Tyr Gly Val Asn Lys Gly He Pro Ala Gly Ser Ala Val 290 295 300

Ala lie Gly Arg Tyr Pro Glu Asp Val Tyr Phe Asn Gly Asn Pro Trp 305 310 315 320

Tyr Leu Ala Thr Phe Ala Ala Ala Glu Gin Leu Tyr Asp Ser Val Tyr

325 330 335

Val Trp Lys Lys Thr Gly Ser lie Thr Val Thr Ser Thr Ser Ser Ala

340 345 350

Phe Phe Gin Glu Leu Val Pro Gly Val Ala Ala Gly Thr Tyr Ser Ser

355 360 365

Ser Gin Ser Thr Phe Thr Ser lie lie Asn Ala lie Ser Thr Tyr Ala

370 375 380

Asp Gly Phe Leu Ser Glu Ala Ala Lys Tyr Val Pro Ala Asp Gly Ser 385 390 395 400

Leu Ala Glu Gin Phe Asp Arg Asn Thr Gly Thr Pro Leu Ser Ala Val

405 410 415

His Leu Thr Trp Ser Tyr Ala Ser Phe Leu Thr Ala Ala Ala Arg Arg

420 425 430

Ala Gly Val Val Pro Pro Ser Trp Ala Ser Ser Gly Ala Asn Thr Val

435 440 445

Pro Ser Ser Cys

450

SEQ ID NO: 10: TrGA, linker region

<210> 10

<211> 37

<212> PRT

<213> Trichoderma reesei

<400> 10

Ser Gly Ala Ser Val Val Gly Ser Tyr Ser Arg Pro Thr Ala Thr Ser

1 5 10 15

Phe Pro Pro Ser Gin Thr Pro Lys Pro Gly Val Pro Ser Gly Thr Pro

20 25 30

Tyr Thr Pro Leu Pro

35

SEQ ID NO: 11: TrGA, SBD

<210> 11

<211> 109

<212> PRT

<213> Trichoderma reesei

<400> 11

Cys Ala Thr Pro Thr Ser Val Ala Val Thr Phe His Glu Leu Val Ser

1 5 10 15

Thr Gin Phe Gly Gin Thr Val Lys Val Ala Gly Asn Ala Ala Ala Leu

20 25 30

Gly Asn Trp Ser Thr Ser Ala Ala Val Ala Leu Asp Ala Val Asn Tyr

35 40 45

Ala Asp Asn His Pro Leu Trp lie Gly Thr Val Asn Leu Glu Ala Gly

50 55 60

Asp Val Val Glu Tyr Lys Tyr lie Asn Val Gly Gin Asp Gly Ser Val 65 70 75 80

Thr Trp Glu Ser Asp Pro Asn His Thr Tyr Thr Val Pro Ala Val Ala

85 90 95

Cys Val Thr Gin Val Val Lys Glu Asp Thr Trp Gin Ser

100 105

SEQ ID NO: 12 SVDDFI: start of the TrGA mature protein <210> 12

<211> 6

<212> PRT

<213> Trichoderma reesei

<400> 12

Ser Val Asp Asp Phe He

1 5

SEQ ID NO: 384 Talaromyces GA mature protein

<210> 384

<211> 588

<212> PRT

<213> Talaromyces sp.

<400> 384

Gly Ser Leu Asp Ser Phe Leu Ala Thr Glu Thr Pro He Ala Leu Gin

1 5 10 15

Gly Val Leu Asn Asn He Gly Pro Asn Gly Ala Asp Val Ala Gly Ala

20 25 30

Ser Ala Gly He Val Val Ala Ser Pro Ser Arg Ser Asp Pro Asp Tyr

35 40 45

Phe Tyr Ser Trp Thr Arg Asp Ala Ala Leu Thr Ala Lys Tyr Leu Val

50 55 60

Asp Ala Phe He Ala Gly Asn Lys Asp Leu Glu Gin Thr He Gin Glu

65 70 75 80

Tyr He Ser Ala Gin Ala Gin Val Gin Thr He Ser Asn Pro Ser Gly

85 90 95

Asp Leu Ser Thr Gly Gly Leu Gly Glu Pro Lys Phe Asn Val Asn Glu

100 105 110

Thr Ala Phe Thr Gly Pro Trp Gly Arg Pro Gin Arg Asp Gly Pro Ala

115 120 125

Leu Arg Ala Thr Ala Leu He Ala Tyr Ala Asn Tyr Leu He Asp Asn

130 135 140

Gly Gin Ala Ser Thr Ala Asp Glu He He Trp Pro He Val Gin Asn

145 150 155 160

Asp Leu Ser Tyr Val Thr Gin Tyr T p Asn Ser Ser Thr Phe Asp Leu

165 170 175

Trp Glu Glu Val Glu Gly Ser Ser Phe Phe Thr Thr Ala Val Gin His

180 185 190

Arg Ala Leu Val Glu Gly Asn Ala Leu Ala Thr Arg Leu Asn His Thr

195 200 205

Cys Pro Asn Cys Val Ser Gin Ala Pro Gin Val Leu Cys Phe Leu Gin

210 215 220

Ser Tyr Trp Thr Gly Ser Tyr Val Leu Ala Asn Phe Gly Gly Ser Gly

225 230 235 240

Arg Ser Gly Lys Asp Val Asn Ser He Leu Gly Ser He His Thr Phe

245 250 255

Asp Pro Ala Gly Gly Cys Asp Asp Ser Thr Phe Gin Pro Cys Ser Ala

260 265 270

Arg Ala Leu Ala Asn His Lys Val Val Thr Asp Ser Phe Arg Ser Val

275 280 285

Tyr Ala Val Asn Ser Gly He Ala Glu Gly Ser Ala Val Ala Val Gly

290 295 300

Arg Tyr Pro Glu Asp Val Tyr Gin Gly Gly Asn Pro Trp Tyr Leu Ala

305 310 315 320

Thr Ala Ala Ala Ala Glu Gin Leu Tyr Asp Ala He Tyr Gin Trp Asn

325 330 335

Lys He Gly Ser He Ser He Thr Asp Val Ser Leu Ala Phe Phe Gin

340 345 350

Asp He Tyr Pro Ser Ala Ala Val Gly Thr Tyr Asn Ser Gly Ser Ser 355 360 365

Thr Phe Asn Asp He He Ser Ala Val Gin Thr Tyr Ala Asp Gly Tyr 370 375 380

Leu Ser He He Glu Lys Tyr Thr Pro Ser Asp Gly Ser Leu Thr Glu 385 390 395 400

Gin Phe Ser Arg Ser Asp Gly Thr Pro Leu Ser Ala Ser Gly Leu Thr

405 410 415

Trp Ser Tyr Ala Ser Leu Leu Thr Ala Ala Ala Arg Arg Gin Ser He

420 425 430

Val Pro Ala Ser Trp Gly Glu Ser Ser Ala Ser Ser Val Pro Ala Val

435 440 445

Cys Ser Ala Thr Ser Ala Thr Gly Pro Tyr Ser Thr Ala Thr Asn Thr 450 455 460

Ala Trp Pro Ser Ser Gly Ser Gly Pro Ser Thr Thr Thr Ser Val Pro 465 470 475 480

Cys Thr Thr Pro Thr Ser Val Ala Val Thr Phe Asp Glu He Val Ser

485 490 495

Thr Thr Tyr Gly Glu Thr He Tyr Leu Ala Gly Ser He Pro Glu Leu

500 505 510

Gly Asn Trp Ser Pro Ser Ser Ala He Pro Leu Arg Ala Asp Ala Tyr

515 520 525

Thr Ser Ser Asn Pro Leu Trp Tyr Val Thr Leu Asn Leu Pro Ala Gly 530 535 540

Thr Ser Phe Glu Tyr Lys Phe Phe Lys Lys Glu Thr Asp Gly Thr He 545 550 555 560

Val Trp Glu Asp Asp Pro Asn Arg Ser Tyr Thr Val Pro Ala Tyr Cys

565 570 575 Gly Gin Thr Thr Ala He Leu Asp Asp Ser Trp Gin

580 585

SEQ ID NO: 385 Humicola grisea GA SBD

<210> 385

<211> 112

<212> PRT

<213> Humicola grisea

<400> 385

Cys Ala Asp Ala Ser Glu Val Tyr Val Thr Phe Asn Glu Arg Val Ser

1 5 10 15

Thr Ala Trp Gly Glu Thr He Lys Val Val Gly Asn Val Pro Ala Leu

20 25 30

Gly Asn Trp Asp Thr Ser Lys Ala Val Thr Leu Ser Ala Ser Gly Tyr

35 40 45

Lys Ser Asn Asp Pro Leu Trp Ser He Thr Val Pro He Lys Ala Thr 50 55 60

Gly Ser Ala Val Gin Tyr Lys Tyr He Lys Val Gly Thr Asn Gly Lys

65 70 75 80

He Thr Trp Glu Ser Asp Pro Asn Arg Ser He Thr Leu Gin Thr Ala

85 90 95

Ser Ser Ala Gly Lys Cys Ala Ala Gin Thr Val Asn Asp Ser Trp Arg

100 105 110

SEQ ID NO: 386 Thermomyces lanuginosus GA SBD

<210> 386

<211> 109

<212> PRT

<213> Thermomyces lanuginosus <400> 386

Cys Thr Pro Pro Ser Glu Val Thr Leu Thr Phe Asn Ala Leu Val Asp 1 5 10 15 Thr Ala Phe Gly Gin Asn lie Tyr Leu Val Gly Ser lie Pro Glu Leu

20 25 30

Gly Ser Trp Asp Pro Ala Asn Ala Leu Leu Met Ser Ala Lys Ser Trp

35 40 45

Thr Ser Gly Asn Pro Val Trp Thr Leu Ser lie Ser Leu Pro Ala Gly

50 55 60

Thr Ser Phe Glu Tyr Lys Phe lie Arg Lys Asp Asp Gly Ser Ser Asp 65 70 75 80 Val Val Trp Glu Ser Asp Pro Asn Arg Ser Tyr Asn Val Pro Lys Asp

85 90 95

Cys Gly Ala Asn Thr Ala Thr Val Asn Ser Trp Trp Arg

100 105

SEQ ID NO: 387 Talaromyces emersonii GA SBD

<210> 387

<211> 108

<212> PRT

<213> Talaromyces emersonii

<400> 387

Cys Thr Thr Pro Thr Ser Val Ala Val Thr Phe Asp Glu He Val Ser

1 5 10 15

Thr Ser Tyr Gly Glu Thr He Tyr Leu Ala Gly Ser He Pro Glu Leu

20 25 30

Gly Asn Trp Ser Thr Ala Ser Ala He Pro Leu Arg Ala Asp Ala Tyr

35 40 45

Thr Asn Ser Asn Pro Leu Trp Tyr Val Thr Val Asn Leu Pro Pro Gly

50 55 60

Thr Ser Phe Glu Tyr Lys Phe Phe Lys Asn Gin Thr Asp Gly Thr He 65 70 75 80

Val Trp Glu Asp Asp Pro Asn Arg Ser Tyr Thr Val Pro Ala Tyr Cys

85 90 95

Gly Gin Thr Thr Ala He Leu Asp Asp Ser Trp Gin

100 105

SEQ ID NO: 388 Aspergillus niger GA SBD

<210> 388

<211> 108

<212> PRT

<213> Aspergillus niger

<400> 388

Cys Thr Thr Pro Thr Ala Val Ala Val Thr Phe Asp Leu Thr Ala Thr

1 5 10 15

Thr Thr Tyr Gly Glu Asn He Tyr Leu Val Gly Ser He Ser Gin Leu

20 25 30

Gly Asp Trp Glu Thr Ser Asp Gly He Ala Leu Ser Ala Asp Lys Tyr

35 40 45

Thr Ser Ser Asp Pro Leu Trp Tyr Val Thr Val Thr Leu Pro Ala Gly

50 55 60

Glu Ser Phe Glu Tyr Lys Phe He Arg He Glu Ser Asp Asp Ser Val 65 70 75 80

Glu Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val Pro Gin Ala Cys

85 90 95 Gly Thr Ser Thr Ala Thr Val Thr Asp Thr Trp Arg

100 105

SEQ ID NO: 389 Aspergillus awamori GA SBD

<210> 389

<211> 108

<212> PRT

<213> Aspergillus awamori

<400> 389

Cys Thr Thr Pro Thr Ala Val Ala Val Thr Phe Asp Leu Thr Ala Thr

1 5 10 15

Thr Thr Tyr Gly Glu Asn He Tyr Leu Val Gly Ser He Ser Gin Leu

20 25 30

Gly Asp Trp Asp Thr Ser Asp Gly He Ala Leu Ser Ala Asp Lys Tyr

35 40 45

Thr Ser Ser Asn Pro Leu Trp Tyr Val Thr Val Thr Leu Pro Ala Gly

50 55 60

Glu Ser Phe Glu Tyr Lys Phe He Arg He Glu Ser Asp Asp Ser Val

65 70 75 80

Glu Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val Pro Gin Ala Cys

85 90 95

Gly Glu Ser Thr Ala Thr Val Thr Asp Thr Trp Arg

100 105

SEQ ID NO: 390 Thielavia terrestris GA SBD

<210> 390

<211> 108

<212> PRT

<213> Thielavia terrestris

<400> 390

Cys Ser Thr Pro Thr Ala Val Ala Val Thr Phe Asn Glu Arg Val Thr

1 5 10 15

Thr Gin Trp Gly Gin Thr He Lys Val Val Gly Asp Ala Ala Ala Leu

20 25 30

Gly Gly Trp Asp Thr Ser Lys Ala Val Pro Leu Ser Ala Ala Gly Tyr

35 40 45

Thr Ala Ser Asp Pro Leu Trp Ser Gly Thr Val Asp Leu Pro Ala Gly

50 55 60

Leu Ala Val Gin Tyr Lys Tyr He Asn Val Ala Ala Asp Gly Gly Val

65 70 75 80

Thr Trp Glu Ala Asp Pro Asn His Ser Phe Thr Val Pro Ala Ala Cys

85 90 95

Gly Thr Thr Ala Val Thr Arg Asp Asp Thr Trp Gin

100 105

SEQ ID NO: 1098 Trichoderma reesei glucoamylase variant

SVDDFISTETPIALNNLLCNVGPDGCRAFGTSAGAVIASPSTIRPDYYYMWTRDSALVFK NLIDRFTETYDAGLQRRIE QYITAQVTLQGLSNPSGSLADGSGLGEPKFELTLKPFTGNWGRPQRDGPALRAIALIGYS KWLINNNYQSTVSNVIWPI VRNDLNYVAQYWNQTGFDLWEEVNGSSFFTVANQHRALVEGATLAATLGQSGSAYSSVAP QVLCFLQRFWVSSGGYVDS NINTNEGRTGKDVNSVLTSIHTFDPNLGCDAGTFQPCSDKALSNLKVVVDSFRSIYGVN GIPAGAAVAIGRYAEDVYY NGNPWYLATFAAAEQLYDAIYVWKKTGSITVTATSLAFFQELVPGVTAGTYSSSSSTFTN IINAVSTYADGFLSEAAKY VPADGSLAEQFDRNSGTPLSALHLTWSYASFLTATARRAGIVPPSWANSSASTIPSTCSG ASVVGSYSRPTATSFPPSQ TPKPGVPSGTPYTPLPCATPTSVAVTFHELVSTQFGQTVKVAGNAAALGNWSTSAAVALD AVNYRDNHPLWIGTVNLEA GDVVEYKYINVGQDGSVTWESDPNHTYTVPAVACVTQVVKEDTWQS

SEQ ID NO: 1099 Trichoderma reesei glucoamylase variant

SVDDFISTETPIALNNLLCNVGPDGCRAFGTSAGAVIASPSTIRPDYYYMWTRDSALVFK ILIDRFTETYDAGLQRRIE QYITAQVTLQGLSNPSGSLADGSGLGEPKFELTLKPFTGNWGRPQRDGPALRAIALIGYS EWLINNNYQSTVSNVIWPI VRNDLNYVAQYWNQTGFDLWEEVNGSSFFTVANQHRALVEGATLAATLGQSGSAYSSVAP QVLCFLQRFWVSSGGYVDS NINTNEGRTGKDVNSVLTSIHTFDPNLGCDAGTFQPCSDKALSNL VVVDSFRSIYGVNKGI PAGAAVAIGRYAEDVYY NGNPWYLATFAAAEQLYDAIYVWKKTGSITVTATSLAFFQELVPGVTAGTYSSSSSTFTN IINAVSTYADGFLSEAAKY VPADGSLAEQFDRNSGTPLSALHLTWSYASFLTATARRAGIVPPSWANSSASTIPSTCSG ASVVGSYSRPTATSFPPSQ TPKPGVPSGTPYTPLPCATPTSVAVTFHELVSTQFGQTVKVAGNAAALGNWSTSAAVALD AVNYRDNHPLWIGTVNLEA GDVVEYKYINVGQDGSVTWESDPNHTYTVPAVACVTQVVKEDTWQS

The foregoing applications, and all documents cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

Various modifications and variations of the described methods and system of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific representative embodiments, it should be understood that the subject matters as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the following claims.