A METHOD OF PREPARING FOOD PRODUCTS USING TS-23 ALPHA-AMYLASE

FIELD OF THE INVENTION

A TS-23 alpha-amylase are useful in a method for preparing feed or food products such as dough and baked products made thereof, and in a composition. The TS-23 alpha-amylase may suitably be used in combination with a further amylase such as an anti-staling amylase.

BACKGROUND OF THE INVENTION

Improved amylases can ameliorate problems inherent in certain processes, such as baking. Crystallisation of amylopectin takes place in starch granules days after baking, which leads to increased firmness of bread and causes bread staling. When bread stales, bread loses crumb softness and crumb moisture. As a result, crumbs become less elastic, and bread develops a leathery crust.

Enzymatic hydrolysis (by amylases, for example) of amylopectin side chains can reduce crystallization and increase anti-staling. Crystallization depends upon the length of amylopectin side chains: the longer the side chains, the greater the crystallization. Most starch granules are composed of a mixture of two polymers: amylopectin and amylose, of which about 75% is amylopectin. Amylopectin is a very large, branched molecule consisting of chains of α-D-glucopyranosyl units joined by (1-4) linkages, where the chains are attached by α-D-(l-6) linkages to form branches. Amylose is a linear chain of (1-4) linked α-D-glucopyranosyl units having few α-D-(l-6) branches.

Baking of farinaceous bread products such as white bread, bread made from bolted rye flour and wheat flour and rolls is accomplished by baking the bread dough at oven temperatures in the range of from 180 to 250 ⁰ C for about 15 to 60 minutes. During the baking process a steep temperature gradient (200 → 120 ⁰ C) prevails over the outer dough layers where the crust of the baked product is developed. However, due to steam, the temperature in the crumb is only about 100 ⁰ C at the end of the baking process. Above temperatures of about

85°C, enzyme inactivation can take place and the enzyme will have no anti- staling properties. Only thermostable amylases, thus, are able to modify starch efficiently during baking.

Excessive endoamylase activity can negatively affect the quality of the final bread product by producing a sticky or gummy crumb due to the accumulation of branched dextrins. A high degree of exo-amylase activity is preferred, because it accomplishes the desired modification of starch that leads to retardation of staling, with fewer of the negative effects associated with endoamylase activity. Reduction of endoamylase activity can lead to greater exospecifity, which can reduce branched dextrins and produce a higher quality bread.

Alpha-Amylases (alpha-l,4-glucan-4-glucanohydrolases, E. C. 3.2.1.1) constitute a group of enzymes, which hydrolyze starch, glycogen, and related polysaccharides by cleaving internal α-l,4-glucosidic bonds at random. This enzyme class has a number of important commercial applications in, for example, starch liquefaction, textile desizing, starch modification in the paper and pulp industry, sweetener (e.g., sugar) manufacture, baking and for brewing. These enzymes can also be used to remove starchy stains during dishwashing and laundry washing, α-amylases are isolated from a wide variety of bacterial, fungal, plant and animal sources. Industrially, many important α-amylases are those isolated from Bacilli.

Pseudomonas saccharophila expresses a maltotetraose-forming maltotetraohydrolase (EC 3.2.1.60; G4-forming amylase; G4-amylase; "Amy3A"; or "PS4" herein). The nucleotide sequence of the P. saccharophila gene encoding PS4 has been determined. Zhou et al., "Nucleotide sequence of the maltotetraohydrolase gene from Pseudomonas saccharophila," FEBS Lett. 255: 37-41 (1989); GenBank Ace. No. X16732. PS4 is expressed as a precursor protein with an N-terminal 21-residue signal peptide. The mature form of PS4, as set forth in SEQ ID NO: 1, contains 530 amino acid residues with a catalytic domain at the N-terminus and a starch binding domain at the C-terminus. PS4 displays both endo- and exo-α-amylase activity. Endo-α-amylase activity is

useful for decreasing the viscosity of gelatinized starch, and exo-α-amylase activity is useful for breaking down maltodextrins to smaller saccharides.

One characterized α-amylase is that of an alkaliphilic Bacillus sp. strain TS-23 which produces at least five kinds of enzymes exhibiting starch hydrolyzing activity. (Lin et al., 1998, Production and properties of a raw-starch-degrading amylase from the thermophilic and alkaliphilic Bacillus sp. TS-23, Biotechnol. Appl. Biochem. 28:61-68).

There is a need in the industry for the identification and optimization of compositions and methods using amylases, for various uses, including baking. These second generation amylases will offer improved manufacturing and/or performance characteristics over the industry standard enzymes.

BRIEF DISCLOSURE OF THE INVENTION

The object of the present invention is to provide methods suitable for preparing a food product and compositions comprising amylases such as a variant amylase, which variant has alpha-amylase activity and exhibits an alteration in at least one of the following properties relative to the parent alpha-amylase: substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH activity profile, pH stability profile, stability towards oxidation, Ca ²⁺ dependency, specific activity, stability under, e.g., high temperature and/or low pH conditions, in particular at low calcium concentrations.

SUMMARY OF THE INVENTION

Provided herein are compositions, including food additives, food products, bakery products, improver compositions, feed products including animal feeds, etc comprising an AmyTS-23 alpha amylase and optionally a further amylase such as an anti-staling amylase, preferably such a further anti-staling amylase which have exoamylase activity, as well as methods of making and using such amylase polypeptides and the compositions in the preparation of food products.

Thus, in a first aspect the present invention relates to a method of preparing a food product, comprising incorporating into the food product an AmyTS-23 alpha-amylase.

In a second aspect the present invention relates to a composition comprising an AmyTS-23 alpha-amylase and optionally a further amylase such as an anti- staling amylase.

In a third aspect the present invention relates to a dough which comprises an AmyTS-23 alpha-amylase and optionally a further amylase such as an anti- staling amylase.

In a further aspect the present invention relates to a pre-mix for dough comprising flour, an AmyTS-23 alpha-amylase and optionally a further amylase such as an anti-staling amylase.

In a further aspect the present invention relates to an enzyme preparation which comprises an AmyTS-23 alpha-amylase and optionally a further amylase such as an anti-staling amylase.

In a further aspect the present invention relates to the use of an AmyTS-23 alpha-amylase and optionally a further amylase such as an anti-staling amylase as a food product additive, such as a feed product additive.

In a further aspect the present invention relates to a method for treating a starch comprising contacting the starch with an AmyTS-23 alpha-amylase and optionally a further amylase such as an anti-staling amylase and allowing the generation from the starch of one or more linear products.

In a further aspect the present invention relates to the use of an AmyTS-23 alpha-amylase and optionally a further amylase such as an anti-staling amylase in preparing a food product, such as a feed product.

In a further aspect the present invention relates to a method of preparing a food or feed product comprising admixing an AmyTS-23 alpha-amylase and optionally

a further amylase such as an anti-staling amylase with a food ingredient, such as a feed ingredient.

In a further aspect the present invention relates to a method for making a bakery product comprising : (a) providing a starch medium; (b) adding to the starch medium an AmyTS-23 alpha-amylase and optionally a further amylase such as an anti-staling amylase; and (c) applying heat to the starch medium during or after step (b) to produce a bakery product.

In a further aspect the present invention relates to an improver composition for a dough, in which the improver composition comprises an AmyTS-23 alpha- amylase, optionally in combination with a further amylase such as an anti- staling amylase, and at least one further dough ingredient or dough additive.

In a further aspect the present invention relates to a composition comprising a flour and an AmyTS-23 alpha-amylase, optionally in combination with a further amylase such as an anti-staling amylase.

In a further aspect the present invention relates to the use of an AmyTS-23 alpha-amylase, optionally in combination with a further amylase such as an anti- staling amylase, in a dough product to retard or reduce staling, preferably detrimental retrogradation, of the dough product.

In a further aspect the present invention relates to the use of an AmyTS-23 alpha-amylase, optionally in combination with a further amylase such as an anti- staling amylase, in a dough product to improve any one or more of firmness, resilience, cohesiveness, crumbliness or foldability of the dough product.

In a further aspect the present invention relates to a combination of an AmyTS- 23 alpha-amylase and optionally a further amylase such as an anti-staling amylase, together with any one or more of the following :

(a) maltogenic alpha-amylase also called glucan 1,4-α-maltohydrolase (EC 3.2.1.133) from Bacillus stearothermophilus, or a variant, homologue, or mutants thereof which have maltogenic alpha-amylase activity;

(b) a bakery xylanase (EC 3.2.1.8) from e.g. Bacillus sp., Aspergillus sp., Thermomyces sp. or Trichoderma sp.;

(c) α-amylase (EC 3.2.1.1) from Bacillus amyloliqufaciens or a variant, homologue, or mutants thereof which have alpha-amylase activity; and

(d) a lipase such as glycolipase from Fusarium heterosporum.

In a further aspect the present invention relates to a combination of an AmyTS- 23 alpha-amylase together with any one or more of the following :

(a) maltogenic alpha-amylase also called glucan 1,4-α-maltohydrolase (EC 3.2.1.133) from Bacillus stearothermophilus, or a variant, homologue, or mutants thereof which have maltogenic alpha-amylase activity;

(b) a bakery xylanase (EC 3.2.1.8) from e.g. Bacillus sp., Aspergillus sp., Thermomyces sp. or Trichoderma sp.; (c) α-amylase (EC 3.2.1.1) from Bacillus amyloliqufaciens or a variant, homologue, or mutants thereof which have alpha-amylase activity; and (d) a lipase such as glycolipase from Fusarium heterosporum.

The AmyTS-23 alpha amylase optionally used in combination with a further amylase such as an anti-staling amylase may comprise one or more improved handling properties, preferably improved baking properties. Thus, the AmyTS-23 alpha amylase optionally used in combination with a further amylase such as an anti-staling amylase are such that the food products so treated have one or more of (preferably all of) a lower firmness, a higher resilience, a higher cohesiveness, a lower crumbliness or a higher foldability. Such improved handling or baking properties exhibited by the AmyTS-23 alpha amylase optionally used in combination with a further amylase such as an anti-staling amylase are described in further detail below.

Further is provided for the treatment of food products, particularly doughs and bakery products with such polypeptides, and preferably such that the food products exhibit the desired qualities set out above.

Further is provided for other uses of such compositions such as in sweeteners, syrups, etc.

Described herein are further compositions and use of variants of a parent AmyTS-23 alpha-amylase, which variant has alpha-amylase activity and exhibits an alteration in at least one of the following properties relative to said parent AmyTS-23 alpha-amylase:

Substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH activity profile, pH stability profile, stability towards oxidation, Ca ²⁺ dependency, reduced and increased pi and improved wash performance, specific activity, stability under, e.g., high temperature and/or low pH conditions, in particular at low calcium concentrations. The AmyTS-23 α-amylases including parent and variants described herein are suitable for starch conversion and ethanol production, and/or sweetener production.

Provided for herein are, inter alia, a parent AmyTS-23 α-amylase and novel α- amylolytic variants (mutants) of a parent AmyTS-23 α-amylase, in particular variants exhibiting altered properties for use in food products.

AmyTS-23 α-amylases may also be applied in feed products to improve starch digestion and thereby feed conversion. Due to pelleting of feed materials with enzymes included, the AmyTS-23 α-amylase preferably should be highly thermostable for this application.

Alterations in properties which may be achieved in AmyTS-23 α-amylase variants are alterations in, e.g., substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile [such as increased stability at low (e.g. pH<6, in particular pH<5) or high (e.g. pH>9) pH values], stability towards oxidation, Ca ²⁺ dependency, specific activity, and other properties of interest. For instance, the alteration may result in a variant which, as compared to the parent AmyTS-23 α-amylase, has a reduced Ca ²⁺ dependency and/or an altered pH/activity profile and/or thermostability.

In one embodiment, there is provided herein the use in the preparation of food products of a variant of a parent AmyTS-23 alpha-amylase, wherein the variant has an amino acid sequence which has at least 80%, 81,%, 82%, 83%, 84%,

85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the parent AmyTS-23 alpha-amylase and comprises at least two of the following : (a) truncation of the C-terminus, (b) substitution of amino acid 201, using SEQ ID NO: 1 for numbering, or (c) deletion of residues R180 and S181 and wherein the variant has alpha-amylase activity.

In a specific embodiment the parent amyTS23 alpha-amylase is SEQ ID No: 1.

In yet a further aspect, a baking composition is contemplated using a Bacillus sp. no. TS-23 α-amylase or variant thereof such as in a solution or in a gel. Also contemplated is a method of baking using such a baking composition.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the amino acid sequence of the parent AmyTS-23 amylase (full- length molecule (AmyTS-23fl), mature chain (SEQ ID NO: I)).

Figure 2 shows the amino acid sequence of the AmyTS-23t: truncated polypeptide (mature, SEQ ID NO: 2). Bold and underlined text indicates amino acid residues R180, S181 and M201.

Figure 3 shows the DNA sequence of the optimized AmyTS-23 gene (SEQ ID NO: 3).

Figure 4 shows the DNA sequence of the optimized AmyTS-23t gene (SEQ ID NO: 4).

Figure 5 shows an expression cassette for AmyTS-23 and AmyTS-23t.

Figure 6 shows a graph of the cohessiveness effect of TS-23 and a variant PS4 as well as TS-23 combined with a variant PS4 in a US toast.

Figure 7 shows a graph of the firmness effect of TS-23 and a variant PS4 as well as TS-23 combined with a variant PS4 in a US toast.

Figure 8 shows a graph of the firmness effect of TS-23t RSdel in a US toast.

Figure 9 shows a texture analysis curve, i.e. a measure of firmness, resilience and cohesiveness effect of TS-23 as described in example 7.

DETAILED DISCLOSURE OF THE INVENTION

The following relates to compounds, compositions, methods of making said compounds, and methods of using said compounds and compositions, wherein the compounds are a Bacillus sp. no. TS-23 α-amylase or variants thereof.

The α-amylase of Bacillus sp. no. TS-23 has a pH optimum of 9 and is stable over a broad pH range {i.e., pH 4.7 to 10.8). The polypeptide had a temperature optimum of 45°C. The enzyme has activity at lower temperatures, e.g., 15-20 ⁰ C.

In some aspects, the present invention relies on routine techniques and methods used in the field of genetic engineering and molecular biology. The following resources include descriptions of general methodology useful in accordance with the invention : Sambrook et al., MOLECULAR CLONING : A LABORATORY MANUAL (2nd Ed., 1989); Kreigler, GENE TRANSFER AND EXPRESSION; A LABORATORY MANUAL (1990) and Ausubel et al., Eds. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (1994). These general references provide definitions and methods known to those in the art. However, it is not intended that the present invention be limited to any particular methods, protocols, and reagents described, as these may vary.

Unless defined otherwise herein, 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 invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994) and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with general dictionaries of many of the terms used in this invention.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

The invention will now be described in detail by way of reference only using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

Numeric ranges are inclusive of the numbers defining the range.

Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole.

1. Abbreviations and Definitions

In accordance with this detailed description, the following abbreviations and definitions apply. It must be noted that as used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes and reference to "the formulation" includes reference to one or more formulations and equivalents thereof known to those skilled in the art, and so forth.

All patents and publications referred to herein, including all sequences disclosed within such patents and publications are expressly incorporated by reference.

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 invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994) and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with general dictionaries of many of the terms used in this invention. The following terms are provided below.

1.1 Definitions

Suitably, the term "food product" as used herein may mean a food product in a form which is ready for consumption. Alternatively or in addition, however, the term food product as used herein may mean one or more food materials which are used in the preparation of a food product. By way of example only, the term food product encompasses both baked goods produced from dough as well as the dough used in the preparation of said baked goods.

Suitably, the term "food product" as used herein means a substance which is suitable for human and/or animal consumption.

As used herein the term "starch" refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (C ₆ Hi ₀ O ₅ ) _X , wherein X can be any number. In particular, the term refers to any plant-based material including but not limited to grains, grasses, tubers and roots and more specifically wheat, barley, corn, rye, rice, sorghum, brans, cassava, millet, potato, sweet potato, and tapioca.

As used herein, an "Amylase" is an enzyme capable of catalyzing the degradation of starch. Amylases are hydrolases that cleave the α-D-(l→4) O- glycosidic linkages in starch. Generally, α-amylases (EC 3.2.1.1; α-D-(l→4)- glucan glucanohydrolase) are defined as endo-acting enzymes cleaving α-D- (1→4) O-glycosidic linkages within the starch molecule in a random fashion. In contrast, the exo-acting amylolytic enzymes, such as β-amylases (EC 3.2.1.2; α- D-(l→4)-glucan maltohydrolase) and some product-specific amylases like maltogenic α-amylase (EC 3.2.1.133) cleave the starch molecule from the non-

reducing end of the substrate. β-Amylases, α-glucosidases (EC 3.2.1.20; α-D- glucoside glucohydrolase), glucoamylases (EC 3.2.1.3; α-D-(l→4)-glucan glucohydrolase), and product-specific amylases can produce malto- oligosaccharides of a specific length from starch. As used herein, amylase include any/all amylases such as glucoamylases, α-amylases, β-amylases and wild-type α-amylases of bacteria such as Bacillus sp., such as B. licheniformis and B. subtil is.

As used herein, "Bacillus sp. strain TS-23 α-amylase", "AmyTS-23 alpha- amylase" and similar phrases, refer to an α-amylase derived from Bacillus sp. strain TS-23. The gene encoding the α-amylase can be the wild-type gene or a codon optimized polynucleotide that encodes the α-amylase. The mature α- amylase of Bacillus sp. strain TS-23 is (amino to carboxy orientation) (SEQ ID NO: 1, Figure 1) :

ntapinetmm qyfewdlpnd gtlwtkvkne aanlsslgit alwlppaykg 50 tsqsdvgygv ydlydlgefn qkgtirtkyg tktqyiqaiq aakaagmqvy 100 advvfnhkag adgtefvdav evdpsnrnqe tsgtyqiqaw tkfdfpgrgn 150 tyssfkwrwy hfdgtdwdes rklnriykfr stgkawdwev dtengnydyl 200 mfadldmdhp evvtelknwg twyvnttnid gfrldavkhi kysffpdwlt 250 yvrnqtgknl favgefwsyd vnklhnyitk tngsmslfda plhnnfytas 300 kssgyfdmry llnntlmkdq pslavtlvdn hdtqpgqslq swvepwfkpl 350 ayafiltrqe gypcvfygdy ygipkynipg lkskidplli arrdyaygtq 400 rdyidhqdii gwtregidtk pnsglaalit dgpggskwmy vgkkhagkvf 450 ydltgnrsdt vtinadgwge fkvnggsvsi wvaktsnvtf tvnnatttsg 500 qnvyvvanip elgnwntana ikmnpssypt wkatialpqg kaiefkfikk 550 dqagnviwes tsnrtytvpf sstgsytasw nvp 583

As used herein, the phrases "parent Bacillus sp. strain TS-23 alpha-amylase", "parent AmyTS-23 alpha-amylase", "wild-type Bacillus sp. strain TS-23 α- amylase" and similar terms, refers to the polypeptide of Bacillus sp. strain TS- 23. The term may be abbreviated "wild-type enzyme", "parent enzyme" and "parent polypeptide" or the like, for convenience. The parent Bacillus sp. strain TS-23 α-amylase may include mutations in the signal sequence of the parent

polypeptide, or elsewhere in the α-amylase parent polypeptide. In addition, the parent Bacillus sp. strain TS-23 α-amylase may be in the form of a fusion protein containing a heterologous α-amylase signal sequence, such as B. licheniformis (LAT), which is well know in the art.

A "parent nucleic acid/polynucleotide," "wild-type nucleic acid/polynucleotide," or "reference nucleic acid/polynucleotide," refers to a nucleic acid sequence encoding a parent polypeptide, and a nucleic acid complementary thereto.

As used herein, the term "AmyTS-23 alpha-amylase variant" and similar phrases, refer to variants/mutants of the wild-type Bacillus sp. strain TS-23 α- amylase, which includes an amino acid sequence substitution, addition, or deletion with respect to the parent (wild-type; reference) amino acid sequence of Bacillus sp. strain TS-23 amylase. The term "variant" is used interchangeably with the term "mutant". The variant Bacillus sp. strain TS-23 α-amylase may include mutations in the signal sequence with respect to parent signal sequence. In addition, the variant Bacillus sp. strain TS-23 α-amylase can be in the form of a fusion protein containing a heterologous α-amylase signal sequence, such as from B. licheniformis (LAT).

Variant nucleic acids refers to a nucleic acid sequence that encode a vaiant polypeptide with additional substitutions, transversions, insertions, and deletions to the Bacillus sp. strain TS-23 α-amylase. Variants can include sequences that are complementary to sequences that are capable of hybridizing to the nucleotide sequences presented herein. For example, a variant nucleic acid sequence is complementary to sequences capable of hybridizing under stringent conditions (e.g., 50 ⁰ C and 0.2X SSC {IX SSC = 0.15 M NaCI, 0.015 M Na ₃ citrate, pH 7.0}) to the nucleotide sequences presented herein. The term variant nucleic acid sequence encompasses sequences that are complementary to sequences that are capable of hybridizing under high stringent conditions (e.g., 65°C and 0.1X SSC {IX SSC = 0.15 M NaCI, 0.015 M Na ₃ citrate, pH 7.0}) to the nucleotide sequences presented herein.

The term "recombinant" when used in reference to a cell, nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found 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. The terms "recovered", "isolated", and "separated" as used herein refer to a compound, protein, cell, nucleic acid or amino acid that is removed from at least one component with which it is naturally associated and found in nature.

As used herein , the term "purified" refers to material (e.g., an isolated polypeptide or polynucleotide) that is in a relatively pure state, e.g., at least about 90% pure, or at least about 95% pure, or at least about 98% pure, or even at least about 99% pure.

The term "thermostable" and "thermostability" refers to the ability of the enzyme to retain activity after exposure to an elevated temperature. The thermostability of an enzyme, such as an α-amylase enzymes, is measured by its half-life given in minutes, hours, or days, during which half the enzyme activity is lost under defined conditions. The half-life value may be calculated by measuring the residual α-amylase activity.

As used herein the term "anti-staling amylase" or an "amylase with anti-staling activity" refers to an amylase that is effective in retarding the staling (crumb firming) of products such as baked products. Included within this definition is amylases that reduces staling after baking and leads to a reduction in firmness increase and a reduction in resilience decrease from day 1 to day 7 after baking relative to a control without enzyme. An anti-staling amylase may be assayed by doing a baking trial and using texture analysis to determine firmness and resilience development over time. Texture analysis is described e.g. in examples example 8 and 9.

As used herein the term "pH range" refers to the ability of the enzyme to exhibit catalytic activity from acidic to basic conditions spanning 5 or more pH units.

As used herein, the terms "pH stable" and "pH stability" refers to the ability of the enzyme to retain activity over a wide range of pHs for a predetermined period of time (e.g., 15 min., 30 min., 1 hour).

As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein" and are used interchangeably herein. In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme". The conventional one-letter or three-letter code for amino acid residues are used herein.

The term "nucleic acid" encompasses DNA, RNA, single stranded or double stranded and chemical modifications thereof. The terms "nucleic acid" and "polynucleotide" may be used interchangeably herein.

As used herein, "nucleotide sequence" or "nucleic acid sequence" refers to an oligonucleotide sequence or polynucleotide sequence encoding a Bacillus sp. strain TS-23 α-amylase polypeptide or variant thereof, and fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic, synthetic, or recombinant origin, and may be double-stranded or single-stranded whether representing the sense or anti-sense strand. As used herein, the term nucleotide sequence includes genomic DNA, cDNA, synthetic DNA, and RNA. For example, the DNA can be a cDNA sequence coding for a Bacillus sp. strain TS-23 α-amylase or variant thereof. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present invention encompasses use of nucleotide sequences which encode a particular amino acid sequence.

Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

By "homologue" shall mean an entity having a certain degree of identity with the subject amino acid sequences and the subject nucleotide sequences. A homologous sequence is taken to include an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identical to the subject sequence. Typically, homologues will comprise the same active sites as the subject amino acid sequence.

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 well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies. The α-amylase or variant thereof nucleic acid may exist as single- or double-stranded DNA or RNA, an RNA/DNA heteroduplex or an RNA/DNA copolymer.

As used herein, the term "synthetic" shall refer to that which is produced by in vitro chemical or enzymatic synthesis, rather than by an organism. It includes, but is not limited to, nucleic acids encoding Bacillus sp. strain TS-23 α-amylase or variants thereof made with optimal codon usage for host organisms, such as the methylotrophic yeasts (e.g., Pichia, Hansenula, etc) or filamentous fungi (e.g., Trichoderma (e.g., T. reesei), etc) or other expression hosts (e.g., Bacillus, Streptomyces,etc).

As used herein, the terms "transformed", "stably transformed" and "transgenic" used with reference to a cell means the cell has a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or as an episomal plasmid that is maintained through multiple generations.

The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell wherein the nucleic acid sequence may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA),

converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

As used herein, the term "transformed cell" shall include cells that have been genetically altered by use of recombinant DNA techniques. Transformation typically occurs by insertion of one or more nucleotide sequences into a cell. The inserted nucleotide sequence may be a heterologous nucleotide sequence {i.e. is a sequence that is not natural to the cell that is to be transformed, such as a DNA sequence encoding a fusion protein or a non-native sequence).

"Host strain" or "host cell" means a suitable host for an expression vector or DNA construct comprising a polynucleotide encoding a parent AmyTS-23 α- amylases or a variant alpha-amylase enzyme according to the present disclosure. Specifically, host strains are preferably bacterial cells. In a preferred embodiment of the invention, "host cell" means both the cells and protoplasts created from the cells of a microbial strain and particularly a Bacillus sp.

The terms "selective marker" or "selectable marker" refers to a gene capable of expression in a host that allows for ease of selection of those hosts containing an introduced nucleic acid or vector. Examples of selectable markers include but are not limited to antimicrobials (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage on the host cell.

The term "culturing" refers to growing a population of microbial cells under suitable conditions in a liquid or solid medium. In one embodiment, culturing refers to fermentative bioconversion of a starch substrate containing granular starch to an end-product (typically in a vessel or reactor). Fermentation is the enzymatic and anaerobic breakdown of organic substances by microorganisms to produce simpler organic compounds. While fermentation occurs under anaerobic conditions it is not intended that the term be solely limited to strict anaerobic conditions, as fermentation also occurs in the presence of oxygen.

A "gene" refers to a DNA segment that is involved in producing a polypeptide and includes coding regions as well as regions preceding and following the

coding regions as well as intervening sequences (introns) between individual coding segments (exons).

A "vector" refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.

An "expression vector" as used herein means a DNA construct comprising a DNA sequence encoding a polypeptide of interest which is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

A "promoter" is a regulatory sequence that is involved in binding RNA polymerase to initiate transcription of a gene. The promoter may be an inducible promoter or a constitutive promoter. An exemplary promoter used according to the invention may be the Bacillus licheniformis alpha-amylase (AmyL) promotor.

The term "operably linked" refers to juxtaposition wherein the elements are in an arrangement allowing them to be functionally related. Thus, as used herein, "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

"Under transcriptional control" is a term well understood in the art that indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operably linked to an element which contributes to the initiation of, or promotes transcription.

"Under translational control" is a term well understood in the art that indicates a regulatory process that occurs after mRNA has been formed.

A "signal sequence" means a sequence of amino acids bound to the N-terminal portion of a protein, which facilitates the secretion of the mature form of the protein outside the cell. The definition of a signal sequence is a functional one. The mature form of the extracellular protein lacks the signal sequence which is cleaved off during the secretion process.

The term "heterologous" with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell. In some embodiments, the protein is a commercially important industrial protein. It is intended that the term encompass proteins that are encoded by naturally occurring genes, mutated genes, and/or synthetic genes.

The term "endogenous" with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.

As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

As used herein, "biologically active" shall refer to a sequence having a similar structural function (but not necessarily to the same degree), and/or similar regulatory function (but not necessarily to the same degree) and/or similar biochemical function (but not necessarily to the same degree) of the naturally occurring sequence.

As used herein the term "saccharification" refers to enzymatic conversion of starch to glucose.

The term "gelatinization" means solubilization of a starch molecule by cooking to form a viscous suspension.

The term "liquefaction" refers to the stage in starch conversion in which gelatinized starch is hydrolyzed to give low molecular weight soluble dextrins.

The term "degree of polymerization (DP)" refers to the number (n) of anhydroglucopyranose units in a given saccharide. Examples of DPI are the monosaccharides, such as glucose and fructose. Examples of DP2 are the disaccharides, such as maltose and sucrose. A DP>3 denotes polymers with a degree of polymerization of greater than 3.

The terms "end-product" or "desired end-product" refer to any carbon-source derived molecule product which is enzymatically converted from the starch substrate.

As used herein the term "dry solids content (ds)" refers to the total solids of a slurry in % on a dry weight basis. The term "slurry" refers to an aqueous mixture containing insoluble solids.

The term "residual starch" refers to the remaining starch (soluble or insoluble) left in a composition after fermentation or enzymatic hydrolysis of a starch containing substrate.

As used herein "a recycling step" refers to the recycling of mash components, which may include residual starch, enzymes and/or microorganisms to ferment substrates comprising starch.

The term "mash" refers to a mixture of a fermentable carbon source (carbohydrate) in water used to produce a fermented product, such as an alcohol. In some embodiments, the term "beer" and "mash" are used interchangeability.

The term "stillage" means a mixture of non-fermented solids and water, which is the residue after removal of alcohol from a fermented mash.

The terms "distillers dried grain (DDG)" and "distillers dried grain with solubles (DDGS)" refer to a useful by-product of grain fermentation.

As used herein "ethanologenic microorganism" refers to a microorganism with the ability to convert a sugar or oligosaccharide to ethanol. The ethanologenic

microorganisms are ethanologenic by virtue of their ability to express one or more enzymes that individually or together convert sugar to ethanol.

As used herein the term "ethanol producer" or ethanol producing microorganism" refers to any organism or cell that is capable of producing ethanol from a hexose or pentose. Generally, ethanol-producing cells contain an alcohol dehydrogenase and a pyruvate decarboxylase. Examples of ethanol producing microorganisms include fungal microorganisms such as yeast. A preferred yeast includes strains of Sacchromyces, particularly, S. cerevisiae.

As used herein when describing proteins and genes that encode them, the term for the gene is italicized, (e.g., the gene that encodes amyL (β. licheniformis AA) may be denoted as amyL). The term for the protein is generally not italicized and the first letter is generally capitalized, (e.g., the protein encoded by the amyL gene may be denoted as AmyL or amyL). Similarly, the amylase gene and protein from Bacillus sp. strain TS-23 provided for herein are amyTS-23 and AmyTS-23, respectively.

With respect to amylase enzymes and their substrates, the term "contacting" refers to the placing of the respective enzyme(s) in sufficiently close proximity to the respective substrate to enable the enzyme(s) to convert the substrate to the end-product. Those skilled in the art will recognize that mixing solutions of the enzyme with the respective substrates can effect contacting.

The term "derived from" encompasses the terms "originated from", "obtained" or "obtainable from", and "isolated from".

The term "enzymatic conversion" in general refers to the modification of a substrate (e.g., starch) by enzyme action (e.g., amylase). The term as used herein also refers to the modification of a starch substrate by the action of an enzyme.

As used herein the term "specific activity" means an enzyme unit defined as the number of moles of substrate converted to product by an enzyme preparation

per unit time under specific conditions. Specific activity is expressed as units (U)/mg of protein.

The term "yield" refers to the amount of end-product or desired end-products produced using the methods of the present invention. In some preferred embodiments, the yield is greater than that produced using methods known in the art. In some embodiments, the term refers to the volume of the end product and in other embodiment the term refers to the concentration of the end product.

"ATCC" refers to American Type Culture Collection located at Manassas, Va. 20108 (ATCC).

"NRRL" refers to the Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research (and previously known as USDA Northern Regional Research Laboratory), Peoria, III.

As used herein the term "comprising" and its cognates are used in their inclusive sense; that is, equivalent to the term "including" and its corresponding cognates.

1.2 Abbreviations

The following abbreviations apply unless indicated otherwise:

AE alcohol ethoxylate AEO alcohol ethoxylate

AEOS alcohol ethoxysulfate

AES alcohol ethoxysulfate

AFAU acid fungal α-amylase units

AGU glucoamylase activity unit AOS α-olefinsulfonate

AS alcohol sulfate

BAA Bacillus amyloliquefaciens α-amylase

BLA Bacillus licheniformis (or LAT)

BSA bovine serum albumin

CDNA complementary DNA

CMC carboxymethylcellulose

DNA deoxyribonucleic acid

DP3 degree of polymerization with three subunits

DPn degree of polymerization with n subunits

DTMPA diethyltriaminepentaacetic acid

EC enzyme commission for enzyme classification

EDTA ethylenediaminetetraacetic acid

EO ethylene oxide

F&HC fabric and household care

FAU fungal amylase unit

GA glucoamylase gpg grains per gallon

HFCS high fructose corn syrup

HFSS high fructose starch based syrup

IPTG isopropyl β-D-1-thiogalactopyranoside

LAS linear alkylbenezenesulfonate

LOM Launder-O-meter

LU Liquiphon unit

MW molecular weight

MWU modified Wohlgemuth unit

NOBS nonanoyloxybenzenesulfonate

NTA nitrilotriacetic acid

PCR polymerase chain reaction

PEG polyethyleneglycol

PVA poly(vinyl alcohol)

PVP poly(vinyl pyrrol idone)

RNA ribonucleic acid

SAS secondary alkane sulfonates

TAED tetraacetylethylenediamine

TCA trichloroacetic acid

TSB tryptic soy broth

UFC ultrafiltration concentrate w/v weight/volume w/w weight/weight

Wt wild-type

1.3 Nomenclature

In the present description and claims, the conventional one-letter and three- letter codes for amino acid residues are used. For ease of reference, AmyTS-23 α-amylases including parent and alpha-amylase variants used according to the present invention are described by use of the following nomenclature:

Original amino acid(s) : position(s) : substituted amino acid(s)

According to this nomenclature, for instance the substitution of serine by an alanine in position 242 is shown as:

Ser242Ala or S242A

a deletion of alanine in position 30 is shown as:

Ala30* or A30* or δA30

and insertion of an additional amino acid residue, such as lysine, is shown as:

Ala30Alal_ys or A30AK

A deletion of a consecutive stretch of amino acid residues, such as amino acid residues 30-33, is indicated as (30-33)* or δ(A30-N33) or δ30-33. A deletion of two consecutive amino acids, such as amino acid residues R180-S181, is indicated as δRS or δ180-181.

Where a specific alpha-amylase contains a "deletion" in comparison with other alpha-amylases and an insertion is made in such a position this is indicated as:

*36Asp or *36D

for insertion of an aspartic acid in position 36.

Multiple mutations are separated by plus signs, i.e. :

Ala30Asp+Glu34Ser or A30N + E34S

representing mutations in positions 30 and 34 substituting alanine and glutamic acid for asparagine and serine, respectively.

When one or more alternative amino acid residues may be inserted in a given position it is indicated as

A30N,E or

A30N or A30E

Furthermore, when a position suitable for modification is identified herein without any specific modification being suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position. Thus, for instance, when a modification of an alanine in position 30 is mentioned, but not specified, it is to be understood that the alanine may be deleted or substituted for any other amino acid, i.e., any one of:

R, N, D, A, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.

Further, "A30X" means any one of the following substitutions:

A30R, A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A30I, A30L, A30K, A30M, A30F, A30P, A30S, A30T, A30W, A30Y, or A30 V;

or in short: A30R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

If the parent enzyme--used for the numbering—already has the amino acid residue in question suggested for substitution in that position the following nomenclature is used :

"X30N" or "X30N,V"

in the case where for instance one of N or V is present in the wildtype. Thus, it means that other corresponding parent enzymes are substituted to an "Asn" or "VaI" in position 30.

1.4 Characteristics of Amino Acid Residues

Charged amino acids:

Asp, GIu, Arg, Lys, His

Negatively charged amino acids (with the most negative residue first) :

Asp, GIu

Positively charged amino acids (with the most positive residue first):

Arg, Lys, His

Neutral amino acids:

GIy, Ala, VaI, Leu, lie, Phe, Tyr, Trp, Met, Cys, Asn, GIn, Ser, Thr, Pro

Hydrophobic amino acid residues (with the most hydrophobic residue listed last) :

GIy, Ala, VaI, Pro, Met, Leu, lie, Tyr, Phe, Trp,

Hydrophilic amino acids (with the most hydrophilic residue listed last) :

Thr, Ser, Cys, GIn, Asn

1.5 Homology (Identity)

A polynucleotide or a polypeptide having a certain percent (e.g. 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of sequence identity with another sequence means that, when aligned, that percentage of bases or amino acid residues are the same in comparing the two sequences. This alignment and the percent homology or identity can be determined using any suitable software program known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. (eds) 1987, Supplement 30, section 7.7.18). Preferred programs include the Vector NTI Advance™ 9.0 (Invitrogen Corp. Carlsbad, CA), GCG Pileup program, FASTA (Pearson et al. (1988) Proc. Natl, Acad. Sci USA 85: 2444-2448), and BLAST (BLAST Manual, Altschul et al., Natl Cent. Biotechnol. Inf., Natl Lib. Med. (NCIB NLM NIH), Bethesda, Md., and Altschul et al., (1997) NAR 25: 3389-3402). Another preferred alignment program is ALIGN Plus (Scientific and Educational Software, PA), preferably using default parameters. Another sequence software program that finds use is the TFASTA Data Searching Program available in the Sequence Software Package Version 6.0 (Genetics Computer Group, University of Wisconsin, Madison, WI).

The homology may be determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (described above). Thus, Gap GCG v8 may be used with the default scoring matrix for identity and the following default parameters: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, respectively for nucleic acidic sequence comparison, and GAP creation penalty of 3.0 and GAP extension penalty of 0.1, respectively, for protein sequence comparison. GAP uses the method of Needleman and Wunsch, (1970), J. MoI. Biol. 48:443-453, to make alignments and to calculate the identity.

A structural alignment between AmyTS-23 (SEQ ID NO: 1) and, e.g., another alpha-amylase may be used to identify equivalent/corresponding positions in other alpha-amylases having a high degree of homology, e.g., 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with AmyTS-23. One method of obtaining said structural alignment is to use the Pile Up programme from the GCG package using default values of gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1. Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al., (1987), FEBS LETTERS 224, pp. 149-155) and reverse threading (Huber, T; Torda, AE, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).

The "percent (%) nucleic acid sequence identity" or "percent (%) amino acid sequence identity" may be measured 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., PS4). The sequence identity can be measured over the entire length of the starting sequence.

1.6 Hybridisation

The oligonucleotide probe used in the characterization of AmyTS-23, above, may suitably be prepared on the basis of the full or partial nucleotide or amino acid sequence of the alpha-amylase in question.

Suitable conditions for testing hybridization involve pre-soaking in 5X SSC and prehybridizing for 1 hour at 4O ⁰ C in a solution of 20% formamide, 5X Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 mg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 100 mM ATP for 18 hours at 4O ⁰ C, followed by three times washing of the filter in 2X SSC, 0.2% SDS at 4O ⁰ C for 30 minutes (low stringency), preferred at 5O ⁰ C (medium stringency), more preferably at 65 ⁰ C (high stringency), even more preferably at 75 ⁰ C (very high stringency). More details about the

hybridization method can be found in Sambrook et al., Molecular Cloning : A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.

In the present context, "derived from" is intended not only to indicate an alpha- amylase produced or producible by a strain of the organism in question, but also an alpha-amylase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence. Finally, the term is intended to indicate an alpha-amylase, which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the alpha-amylase in question. The term is also intended to indicate that the parent alpha-amylase may be a variant of a naturally occurring alpha-amylase, i.e., a variant, which is the result of a modification (insertion, substitution, deletion) of one or more amino acid residues of the naturally occurring alpha-amylase.

One skilled in the art will recognize that sequences encompassed by the use according to the present invention are also defined by the ability to hybridize under stringent hybridization conditions with the exemplified amyTS-23 sequence (e.g., SEQ ID NO:4 shown in Figure 4). A nucleic acid is hybridizable to another nucleic acid sequence when a single stranded form of the nucleic acid can anneal to the other nucleic acid under appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known in the art (See, e.g., Sambrook (1989) supra, particularly chapters 9 and 11). In some embodiments, stringent conditions correspond to a Tm of 65°C and 0. I xSSC, 0.1% SDS.

1.7 Parent Alpha -Amylases

According to the present disclosure any AmyTS-23 alpha-amylase, as defined above, may be used as the parent (i.e., backbone) alpha-amylase. In a preferred embodiment the parent alpha-amylase is derived from Bacillus sp. strain TS-23, e.g., one of those referred to above, such as the TS-23 alpha- amylase having the amino acid sequence shown in SEQ ID NO: 1 (see Figure 1).

1.8 Altered Properties

The following section describes the relationship between mutations, which are present in a variant amylase described herein, and desirable alterations in properties (relative to those of a parent TS-23 alpha-amylase), which may result therefrom.

As mentioned above the invention relates to the use of as well as compositions comprising an alpha-amylase derivable from Bacillus sp strain TS-23 and mutants thereof with altered properties.

Parent TS-23 alpha-amylases specifically contemplated in connection with the specifically contemplated altered properties are the above mentioned parent TS- 23 alpha-amylase and hybrids or chimeric amylases that include at least a portion of a TS-23 alpha-amylase.

The Bacillus sp strain TS-23 alpha-amylase (SEQ ID NO: 1) is used as the starting point, but corresponding positions in other Bacillus alpha-amylases having a high degree of homology should be understood as disclosed and specifically contemplated too. This is particularly true of other naturally-occuring Bacillus α-amylases that include only minor sequence different in comparison to Bacillus sp strain TS-23 α-amylase, not including the substitutions, deletions, or insertions, that are the subject of the present disclosure.

In an aspect the invention relates to the use of a variant with altered properties as mentioned above.

In the first aspect a variant of a parent Bacillus sp. strain TS-23 alpha-amylase, comprising at least two of the following alterations:

(a) truncation of the C-terminus,

(b) substitution of amino acid 201 (i.e., M201), using SEQ ID NO: 1 for numbering, or

(c) deletion of at least two residues selected from the group consisting of R180, S181, T182 and G183, using SEQ ID NO: 1 for numbering, and wherein the variant has alpha-amylase activity.

1.8.1 Stability

In the context of the variants described herein, mutations (including amino acid substitutions and deletion) of importance with respect to achieving altered stability (i.e., higher or lower), in particular improved stability, at especially high temperatures (i.e., 70-120 ⁰ C) and/or extreme pH (i.e. low or high pH, i.e, pH 4- 6 or pH 8-11, respectively), in particular at free (i.e., unbound, therefore in solution) calcium concentrations below 60 ppm, include any of the mutations listed in the "Altered Properties" section. The stability may be determined as described in the "Methods" section below.

1.8.2 Ca ²⁺ Stability

Altered Ca ²⁺ stability means the stability of the enzyme under Ca ²⁺ depletion has been improved, i.e., higher or lower stability. In the context of the presently described variants, mutations (including amino acid substitutions and deletions) of importance with respect to achieving altered Ca ²⁺ stability, in particular improved Ca ²⁺ stability, i.e., higher or lower stability, at especially high pH (i.e., pH 8-10.5) include any of the mutations listed in the in "Altered Properties" section.

1.8.3 Specific Activity

In a further aspect, important mutations (including amino acid substitutions and deletions) with respect to obtaining variants exhibiting altered specific activity, in particular increased or decreased specific activity, especially at temperatures from 10-60 ⁰ C, preferably 20-50 ⁰ C, especially 30-40 ⁰ C, include any of the mutations listed in the in "Altered properties" section. The specific activity may be determined as described in the "Methods" section below.

1.8.4 Oxida tion Stability

The described variants may have altered oxidation stability, in particular higher oxidation stability, in comparison to the parent alpha-amylase. Increased oxidation stability is advantageous in, e.g., detergent compositions and decreased oxidation stability may be advantageous in composition for starch liquefaction. Oxidation stability may be determined as described in the "Methods" section below.

1.8.5 Altered pH Profile

Important positions and mutations with respect to obtaining variants with altered pH profile, in particular improved activity at especially high pH (i.e., pH 8-10.5) or low pH (i.e., pH 4-6) include mutations of amino residues located close to the active site residues.

Preferred specific mutations/substitutions are the ones listed above in the section "Altered Properties" for the positions in question. Suitable assays are described in the "Methods" section below.

2. Methods of Preparing α-amylase Variants

Thus, one aspect provides for use of the Bacillus sp. strain TS-23 α-amylase sequence in creating recombinant forms that include other previously determined amino acid substitutions, deletions, transversions, insertions, and combinations thereof to produce variants of the Bacillus sp. strain TS-23 α- amylase. These variants can have additional production enhancement, increased pH stability, increased temperature stability, reduced requirements for Ca ²⁺ , increased specific activity, increased solubility, increased storage stability, or combinations thereof. Methods of recombinantly generating the variants could be performed using the provided sequences and vectors, or using other modalities known in the art.

Several methods for introducing mutations into genes are known in the art. After a brief discussion of the cloning of α-amylase-encoding DNA sequences, methods for generating mutations at specific sites within the α-amylase-encoding sequence will be discussed.

2.1 Cloning a DNA Sequence Encoding an α-amylase

The DNA sequence encoding a parent α-amylase may be isolated from any cell or microorganism producing the α-amylase in question, using various methods well known in the art. First, a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the α-amylase to be studied. Then, if the amino acid sequence of the α-amylase is known, homologous, labelled oligonucleotide probes may be synthesized and used to identify α-amylase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labelled oligonucleotide probe containing sequences homologous to a known α-amylase gene could be used as a probe to identify α-amylase-encoding clones, using hybridization and washing conditions of lower stringency.

Yet another method for identifying α-amylase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming α-amylase-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for α-amylase, thereby allowing clones expressing the α-amylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S. L. Beaucage and M. H. Caruthers (1981) or the method described by Matthes et al. (1984). In the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et al. (1988).

2.2 Site-directed Mutagenesis

Once an α-amylase-encoding DNA sequence has been isolated, and desirable sites for mutation identified, mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis. In a specific method, a single-stranded gap of DNA, bridging the α-amylase-encoding sequence, is created in a vector carrying the α- amylase gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase. A specific example of this method is described in Morinaga et al. (1984). U.S. Pat. No. 4,760,025 discloses the introduction of oligonucleotides encoding multiple mutations by performing minor alterations of the cassette. However, an even greater variety of mutations can be introduced at any one time by the Morinaga method, because a multitude of oligonucleotides, of various lengths, can be introduced.

Another method of introducing mutations into α-amylase-encoding DNA sequences is described in Nelson and Long (1989). It involves the 3-step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions. From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.

Alternative methods for providing variants used according to the invention include gene shuffling, e.g., as described in WO 95/22625 (from Affymax Technologies N. V.) or in WO 96/00343 (from Novo Nordisk A/S), or other corresponding techniques resulting in a hybrid enzyme comprising the mutation(s), e.g., substitution(s) and/or deletion(s), in question.

2.3 Expression of Alpha- Amylase Variants

According to the invention, a DNA sequence encoding the variant produced by methods described above, or by any alternative methods known in the art, can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding an alpha-amylase variant used according to the present invention may be any vector, which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome.

Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA sequence encoding an alpha-amylase variant used according to the present invention, especially in a bacterial host, are the promoter of the lac operon of E. coli, the

Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis alpha-amylase gene (amyL), the promoters of the Geobacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens alpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.

The expression vector used to produce an AmyTS-23 alpha-amylase may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the alpha-amylase variant used according to the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl and pIJ702.

The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g., as described in WO 91/17243.

While intracellular expression may be advantageous in some respects, e.g., when using certain bacteria as host cells, it is generally preferred that the expression is extracellular. In general, the Bacillus alpha-amylases mentioned herein comprise a preregion permitting secretion of the expressed protease into

the culture medium. If desirable, this preregion may be replaced by a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions.

The procedures used to ligate the DNA construct encoding an alpha-amylase variant, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., Molecular Cloning : A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).

The cell used to produce an AmyTS-23 alpha-amylase, either comprising a DNA construct or an expression vector as defined above, is advantageously used as a host cell in the recombinant production of an alpha-amylase variant of the used according to the invention. The cell may be transformed with the DNA construct encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

The cell used in producing an AmyTS-23 alpha-amylase may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g., a bacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are Gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyces lividans or Streptomyces murinus, or gram- negative bacteria such as E. coli. The transformation of the bacteria may, for

instance, be effected by protoplast transformation or by using competent cells in a manner known per se.

The yeast organism may favorably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. The filamentous fungus may advantageously belong to a species of Aspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.

Methods of producing an alpha-amylase, such as a variant, which method comprises cultivating a host cell as described above under conditions conducive to the production of the alpha-amylase, such as a variant and recovering the alpha-amylase, such as a variant from the cells and/or culture medium are provided herein.

The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the alpha-amylase, such as a variant used according to the present invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).

The alpha-amylase, such as an alpha-amylase variant secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

3. Industrial Applications

The alpha-amylase, such as alpha-amylase variants presented herein possess valuable properties allowing for a variety of industrial applications. In particular, the enzymes, such as enzyme variants are applicable as a component in baking applications.

One or more of the variants with altered properties may be used for starch processes, in particular starch conversion, especially liquefaction of starch (see, e.g., U.S. Pat. No. 3,912,590, EP patent application nos. 252 730 and 63 909, WO 99/19467, and WO 96/28567 all references hereby incorporated by reference). Also contemplated are compositions of the invention for starch conversion purposes, which may beside the alpha-amylase, such as a variant also comprise a glucoamylase, pullulanase, and other alpha-amylases.

Further, one or more of the alpha-amylase, such as variants are also particularly useful in the production of sweeteners and ethanol (see, e.g., U.S. Pat. No. 5,231,017 hereby incorporated by reference), such as fuel, drinking and industrial ethanol, from starch or whole grains.

The alpha-amylase, such as variants as described herein may also be useful for beer making or brewing.

3.1 Starch Conversion

Conventional starch-conversion processes, such as liquefaction and saccharification processes are described, e.g., in U.S. Pat. No. 3,912,590 and EP patent publications Nos. 252,730 and 63,909, hereby incorporated by reference.

In an embodiment the starch conversion process degrading starch to lower molecular weight carbohydrate components such as sugars or fat replacers includes a debranching step.

3.2 Starch to Sugar Conversion

In the case of converting starch into a sugar the starch is depolymerized. A such depolymerization process consists of a Pre-treatment step and two or three consecutive process steps, viz a liquefaction process, a saccharification process and dependent on the desired end product optionally an isomerization process.

3.3 Pre-Treatment of Native Starch

Native starch consists of microscopic granules, which are insoluble in water at room temperature. When an aqueous starch slurry is heated, the granules swell and eventually burst, dispersing the starch molecules into the solution. During this "gelatinization" process there is a dramatic increase in viscosity. As the solids level is 30-40% in a typical industrial process, the starch has to be thinned or "liquefied" so that it can be handled. This reduction in viscosity is today mostly obtained by enzymatic degradation.

3.4 Liquefaction

During the liquefaction step, the long chained starch is degraded into branched and linear shorter units (maltodextrins) by an alpha-amylase. The liquefaction process is carried out at 105-110 ⁰ C for 5 to 10 minutes followed by 1-2 hours at 95 ⁰ C. The pH lies between 5.5 and 6.2. In order to ensure optimal enzyme stability under these conditions, 1 mM of calcium is added (40 ppm free calcium ions). After this treatment the liquefied starch will have a "dextrose equivalent" (DE) of 10-15.

3.5 Saccharification

After the liquefaction process the maltodextrins are converted into dextrose by addition of a glucoamylase (e.g., OPTIDEX® L-400) and a debranching enzyme, such as an isoamylase (U.S. Pat. No. 4,335,208) or a pullulanase. Before this step the pH is reduced to a value below 4.5, maintaining the high temperature

(above 95 ⁰ C) to inactivate the liquefying alpha-amylase to reduce the formation of short oligosaccharide called "panose precursors" which cannot be hydrolyzed properly by the debranching enzyme.

The temperature is lowered to 6O ⁰ C, and glucoamylase and debranching enzyme are added. The saccharification process proceeds for 24-72 hours.

Normally, when denaturing the α-amylase after the liquefaction step about 0.2- 0.5% of the saccharification product is the branched trisaccharide Glcpαl-6Glc pαl-4Glc (panose) which cannot be degraded by a pullulanase. If active amylase from the liquefaction step is present during saccharification (i.e., no denaturing) this level can be as high as 1-2%, which is highly undesirable as it lowers the saccharification yield significantly.

3.6 Isomerization

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, preferably 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 immmobilized glucose isomerase (such as Gensweet® IGI-HF).

3.7 Ethanol Production

In general alcohol production (ethanol) from whole grain can be separated into 4 main steps:

Milling Liquefaction Saccharification Fermentation

3.7.1 Milling

The grain is milled in order to open up the structure and allow for further processing. Two processes used are wet or dry milling. In dry milling the whole kernel is milled and used in the remaining part of the process. Wet milling gives a very good separation of germ and meal (starch granules and protein) and is with a few exceptions applied at locations where there is a parallel production of syrups.

3.7.2 Liquefaction

In the liquefaction process the starch granules are solubilized by hydrolysis to maltodextrins mostly of a DP higher than 4. The hydrolysis may be carried out by acid treatment or enzymatically by alpha-amylase. Acid hydrolysis is used on a limited basis. The raw material can be milled whole grain or a side stream from starch processing.

Enzymatic liquefaction is typically carried out as a three-step hot slurry process. The slurry is heated to between 60-95 ⁰ C, preferably 80-85 ⁰ C, and the enzyme(s) is (are) added. Then the slurry is jet-cooked at between 95-14O ⁰ C, preferably 105-125 ⁰ C, cooled to 60-95 ⁰ C and more enzyme(s) is (are) added to obtain the final hydrolysis. The liquefaction process is carried out at pH 4.5-6.5, typically at a pH between 5 and 6. Milled and liquefied grain is also known as mash.

3.7.3 Saccharification

To produce low molecular sugars DPi _-3 that can be metabolized by yeast, the maltodextrin from the liquefaction must be further hydrolyzed. The hydrolysis is typically done enzymatically by glucoamylases, alternatively alpha-glucosidases or acid alpha-amylases can be used. A full saccharification step may last up to 72 hours, however, it is common only to do a pre-saccharification of typically 40-90 minutes and then complete saccharification during fermentation (SSF).

Saccharification is typically carried out at temperatures from 30-65 ⁰ C, typically around 6O ⁰ C, and at pH 4.5.

3.7.4 Fermentation

Yeast typically from Saccharomyces spp. is added to the mash and the fermentation is ongoing for 24-96 hours, such as typically 35-60 hours. The temperature is between 26-34 ⁰ C, typically at about 32 ⁰ C, and the pH is from pH 3-6, preferably around pH 4-5.

Note that the most widely used process is a simultaneous saccharification and fermentation (SSF) process where there is no holding stage for the saccharification, meaning that yeast and enzyme is added together. When doing SSF it is common to introduce a pre-saccharification step at a temperature above 5O ⁰ C, just prior to the fermentation.

3.8 Distillation

Following the fermentation the mash is distilled to extract the ethanol.

The ethanol obtained according to the process of the invention may be used as, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol.

3.9 By-Products

Left over from the fermentation is the grain, which is typically used for animal feed either in liquid form or dried.

Further details on how to carry out liquefaction, saccharification, fermentation, distillation, and recovery of ethanol are well known to the skilled person.

According to the process of the invention the saccharification and fermentation may be carried out simultaneously or separately.

3.10 Beer Making

The alpha-amylase, such as variant alpha-amylases provided for herein may also be very useful in a beer-making process; the alpha-amylases will typically be added during the mashing process.

In some embodiments the compositions or preparations according to the invention, further comprises a lipase.

Upases: Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include but are not limited to lipases from Humicola (synonym Thermomyces), e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g., from P. a lea Ii genes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeή (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from B. subtilis (Dartois et al. (1993), Biochemica et

Biophysica Acta, 1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422). Additional exemplary lipase variants contemplated for use in the formulations include those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.

Commercially available lipase enzymes include LIPOLASE™ and LIPOLASE ULTRA™ (Novozymes A/S).

Amylases: One or more additional amylases (in addition to the parent AmyTS-23 alpha-amylase and/or variants thereof described herein) may also be included. Such amylases may be from a fungus, bacterium or plant. In one aspect it is a alpha-amylase (EC 3.2.1.1). In a further aspect it is a fungal alpha-amylase from Aspergillus or Trichoderma, e.g. from Aspergillus oryzae. In yet a further aspect it is a alpha-amylase from Bacillus, e.g. B. amyloliquefaciens or B.licheniformis. In a further aspect it is a plant beta- amylase (EC 3.2.1.2), e.g. beta-amylase from barley malt or soy bean. In a further aspect it is an exo-

amylase used for anti-staling, e.g. a non-maltogenic exo-amylase (EC 3.2.1.60) developed from Pseudomonas saccharophila maltotetraohydrolase (such as e.g. SEQ ID NO:7, or alternatively SEQ ID NO:7 without the M in position 1) or a maltogenic amylase (EC 3.2.1.133) developed from Bacillus stearothermophilus (such as e.g. SEQ ID No: 10).

Amylases include, for example, alpha-amylases obtained from Bacillus, e.g., a special strain of B. licheniformis, described in more detail in GB 1,296,839. Examples of useful alpha-amylases are the variants described in WO 94/18314, WO 96/39528, WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

Commercially available alpha-amylases are DURAMYL™, LIQUEZYME™ TERMAMYL™, NATALASE™, STAINZYME™ PLUS, STAINZYME™ ULTRA, FUNGAMYL™ and BAN™ (Novozymes A/S), RAPIDASE™ and PURASTAR™ (from Genencor).

In some embodiments the compositions or preparations according to the invention, further comprises a cellulase.

Cellulases: Cellulases may be added to the compositions. Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include but are not limited to cellulases from the genera Bacillus, Pseudomonas, Trichoderma, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No.

5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259. Exemplary Trichoderma reesei cellulases are disclosed in U.S. Pat. No. 4,689,297, U.S. Pat. No. 5,814,501, U.S. Pat. No. 5,324,649, WO 92/06221 and WO 92/06165. Exemplary Bacillus cellulases are disclosed in U.S. Pat. No. 6,562,612.

Commercially available cellulases include CELLUZYME®, and CAREZYME® (Novozymes A/S), CLAZINASE®, and PURADAX HA® (Genencor International Inc.), and KAC-500(B)® (Kao Corporation).

4. Compositions and Use

One or more of the alpha-amylase, such as variant enzymes described herein may also be used in methods for using an alpha-amylase, such as a variant in baking, beer making, ethanol production, and starch conversion processes as described above.

The composition may comprise a Bacillus sp. strain TS-23 α-amylase or variants thereof as the major enzymatic component, e.g., a mono-component composition. Alternatively, the composition may comprise multiple enzymatic activities, such as an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, α-galactosidase, β- galactosidase, glucoamylase, α-glucosidase, β-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase, as well as other enzymes discussed below. The additional enzyme(s) may be producible by means of a microorganism belonging to the genera Aspergillus, Trichoderma, Humicola

(e.g., H. insolens), and Fusarium. Exemplary members of the Aspergillus genus include Aspergillus aculeatus, Aspergillus awamori, Aspergillus niger, or Aspergillus oryzae. Exemplary members of the genus Fusarium include Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundinis, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides , and Fusarium venenatum.

As discussed above, in one aspect of the invention, a further amylase such as an anti-staling amylase is used in combination with the AmyTS-23 alpha-amylase such as for incorporating into a food product.

In a further aspect, the AmyTS-23 alpha-amylase used according to the invention has anti-staling amylase activity.

In a further aspect, the further amylase is a non-maltogenic exo-amylase.

In a further aspect, the further amylase is a EC 3.2.1.60 amylase.

In a further aspect, wherein the further amylase is a maltogenic exo-amylase.

In a further aspect, wherein the further amylase is a EC 3.2.1.133 amylase.

In a further aspect, the further amylase is an exo-amylase.

In one aspect of the invention, the further amylase is selected from the group consisting of a Bacillus stearothermophilus having SEQ ID No 10 or a variant, homologue, or mutant thereof and a Pseudomonas saccharophila (PS4) variant polypeptide.

In one aspect, the further amylase is a maltogenic alpha-amylase. A "maltogenic alpha-amylase" (glucan 1,4-alpha-maltohydrolase, E. C. 3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose in the alpha-configuration. A maltogenic alpha-amylase from Bacillus (EP 120 693) is commercially available under the trade name Novamyl (Novozymes, Denmark) and is widely used in the baking industry as an anti-staling agent due to its ability to reduce retrogradation of starch. Novamyl is described in detail in International Patent Publication WO 91/04669. The maltogenic alpha-amylase Novamyl shares several characteristics with cyclodextrin glucanotransferases (CGTases), including sequence homology (Henrissat B, Bairoch A; Biochem. J., 316, 695- 696 (1996)) and formation of transglycosylation products (Christophersen, C, et al., 1997, Starch, vol. 50, No. 1, 39-45). The Novamyl may in particular comprise Novamyl 1500 MG.

Other documents describing Novamyl and its uses include Christophersen, C, Pedersen, S., and Christensen, T., (1993) Method for production of maltose an a limit dextrin, the limit dextrin, and use of the limit dextrin. Denmark, and WO 95/10627. It is further described in U.S. Pat. No. 4,598,048 and U.S. Pat. No. 4,604,355. Each of these documents is hereby incorporated by reference, and any of the Novamyl polypeptides described therein may be used in combinations with any of the AmyTS-23 alpha amylase polypeptides described here.

Variants, homologues, and mutants of Novamyl may be used for the combinations. For example, any of the Novamyl variants disclosed in US Patent Number 6,162,628, the entire disclosure of which is hereby incorporated by reference, may be used in combination with the AmyTS-23 alpha amylase polypeptide described here. In particular, any of the polypeptides described in that document, specifically variants of SEQ ID NO: 1 of US 6,162,628 at any one or more positions corresponding to Q13, 116, D17, N26, N28, P29, A30, S32, Y33, G34, L35, K40, M45, P73, V74, D76 N77, D79, N86, R95, N99, 1100, H103, Q119, N120, N131, S141, T142, A148, N152, A163, H169, N171, G172, 1174, N176, N187, F188, A192, Q201, N203, H220, N234, G236, Q247, K249, D261, N266, L268, R272, N275, N276, V279, N280, V281, D285, N287, F297, Q299, N305, K316, N320, L321, N327, A341, N342, A348, Q365, N371, N375, M378, G397, A381, F389, N401, A403, K425, N436, S442, N454, N468, N474, S479, A483, A486, V487, S493, T494, S495, A496, S497, A498, Q500, N507, 1510, N513, K520, Q526, A555, A564, S573, N575, Q581, S583, F586, K589, N595, G618, N621, Q624, A629, F636, K645, N664 and/or T681 may be used.

In one aspect of the invention, the further amylase is a Pseudomonas saccharophila (PS4) variant polypeptide.

In a further aspect of the invention, the further amylase has an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID No:8.

SEQ ID No:8 is the mature protein sequence of G4-amylase from Pseudomonas saccharophila (PS4) with the starch binding domain removed.

A "PS4 variant" or "PS4 variants" refers to either polypeptides or nucleic acids. The term "variant" may be used interchangeably with the term "mutant." Variants include insertions, substitutions, transversions, truncations, and/or inversions at one or more locations in the amino acid or nucleotide sequence, respectively. The phrases "PS4 variant polypeptide" and "PS4 variant enzyme" mean a PS4 protein that has an amino acid sequence that has been modified compared to the amino acid sequence of e.g. a wild-type PS4, a mature protein sequence of G4-amylase from Pseudomonas saccharophila (PS4) with the starch binding domain removed having SEQ ID No :8, SEQ ID No: 7, or alternatively SEQ ID No: 7 without the M in position 1. In one aspect, the variant polypeptides include a polypeptide having a certain percent, e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, of sequence identity with the parent PS4 enzyme. As used herein, "parent PS4 enzymes," "parent PS4 sequence," "parent PS4 polypeptide," "wild-type PS4," and "parent polypeptides" mean enzymes and polypeptides from which the variant PS4 polypeptides are based, e.g., the PS4 of SEQ ID NO: 8. In a further aspect, the variant polypeptides include a polypeptide having a certain percent, e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, of sequence identity with SEQ ID No: 8. In yet a further aspect, the variant polypeptides include a polypeptide having a certain percent, e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, of sequence identity with SEQ ID No: 7. To describe the various PS4 variants that are contemplated to be encompassed by the present disclosure, the following nomenclature will be adopted for ease of reference. Where the substitution includes a number and a letter, e.g., 141P, then this refers to {position according to the numbering system/substituted amino acid}. Accordingly, for example, the substitution of an amino acid to proline in position 141 is designated as 141P. Where the substitution includes a letter, a number, and a letter, e.g., A141P, then this refers to {original amino acid/position according to the numbering system/substituted amino acid}.

Accordingly, for example, the substitution of alanine with proline in position 141 is designated as A141P. 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. MoI. 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)).

"Thermostable" means the enzyme retains activity after exposure to elevated temperatures. The thermostability of an enzyme is measured by its half-life (ti _/2 ), where half of the enzyme activity is lost by the half-life. The half-life value is calculated under defined conditions by measuring the residual amylase activity. To determine the half-life of the enzyme, the sample is heated to the test temperature for 1-10 min, and activity is measured using a standard assay e.g. for PS4 activity, such as the Betamyl® assay (Megazyme, Ireland).

An isolated and/or purified polypeptide comprising a PS4 or variant thereof is provided. In one embodiment, the PS4 is a mature form of the polypeptide (SEQ ID NO: 9), wherein the 21 amino acid leader sequence is cleaved, so that the N-terminus of the polypeptide begins at the aspartic acid (D) residue.

Variants of PS4 include a PS4 in which the C-terminal starch binding domain is removed. A representative amino acid sequence of a mature PS4 variant in which the starch binding domain is removed is the one having an amino acid sequence of residues 1 to 429 of SEQ ID NO: 9, or SEQ ID NO: 8 with residues 419-429 of SEQ ID NO:9 fused at the C-terminus. Other PS4 variants include variants wherein between one and about 25 amino acid residues have been added or deleted with respect to wild-type PS4 or the PS4 having an amino acid sequence of residues 1 to 429 of SEQ ID NO: 9, or SEQ ID NO: 8 with residues 419-429 of SEQ ID NO:9 fused at the C-terminus. In one aspect, the PS4 variant has the amino acid sequence of residues 1 to 429 of SEQ ID NO: 9, wherein any number between one and about 25 amino acids have been substituted.

In one aspect of the invention, the further amylase comprises the amino acid sequence of SEQ ID No: 7, or alternatively SEQ ID No: 7 without the M in position 1. In yet a further aspect of the invention, the further amylase has the amino acid sequence of SEQ ID No: 7. In yet a further aspect of the invention,

the further amylase has the amino acid sequence of SEQ ID No: 7 without the M in position 1. In a further aspect, the further amylase is a G4-amylase contained in Grindamyl™ PowerFresh products. In a further aspect, the further amylase is as described in WO2007148224.

In a further aspect, the further amylase compared to SEQ ID No: 8 comprises a substitution at one or more residues selected from the group consisting of residue 33, 34, 70, 121, 134, 141, 146, 157, 161, 178, 179, 223, 229, 272, 303, 307, 309 and 334 wherein said residue refer to the amino acid residue at a position corresponding to the identical position of SEQ ID NO:8.

In a further aspect, the further amylase compared to SEQ ID No:8 comprises a substitution at one or more residues selected from the group consisting of residue 121, 134, 141, 146, 157, 161, 178, 179, 223, 229, 307, 309 and 334 wherein said residue refer to the amino acid residue at a position corresponding to the identical position of SEQ ID NO:8. In yet a further aspect, the one or more substitutions are selected from the group consisting of 33Y, 34N, 7OD, 7OK, 121F, 121Y, 121D, 134R, 141P, 146G, 157L, 157M, 161A, 178F, 179T, 223A, 223E, 223S, 229P, 272Q, 303E, 307K, 307R, 309P and 334P wherein said amino acid residues refer to the amino acid residue at a position corresponding to the identical position of SEQ ID NO:8. In yet a further aspect, the one or more substitutions are selected from the group consisting 121F, 134R, 141P,

146G, 157L, 161A, 178F, 179T, 223A, 223E, 223S, 229P, 307K, 309P and 334P, wherein said amino acid residues refer to the amino acid residue at a position corresponding to the identical position of SEQ ID NO:8.

In yet a further aspect, the further amylase has a higher thermostability compared to wild-type PS4 having SEQ ID NO:9 when tested under the same conditions.

In yet a further aspect, the further amylase has a half life (tl/2), which is increased by 15% or more, preferably by 50% or more, most preferably by 100% or more, relative to wild-type PS4 having SEQ ID NO:9 when measured under same conditions, preferably at a temperature of 60 degrees C.

Examples of PS4 polypeptides or variants thereof is e.g. described in any one of international patent applications WO2004111217, WO2005003339, WO2005007867, WO2005007818, WO2006003461, WO2007007053, and/or WO2007148224, which patent applications are hereby incorporated by reference in their entirety.

4.1 Starch Processing Compositions and Use

In another aspect, compositions with a disclosed Bacillus sp. strain TS-23 α- amylase or variants thereof, can be utilized for starch liquefaction or saccharification.

One aspect contemplates compositions and uses of compositions to produce sweeteners from starch. A "traditional" process for conversion of starch to fructose syrups normally consists of three consecutive enzymatic processes, viz. a liquefaction process followed by a saccharification process, and an isomerization process. During the liquefaction process, starch is degraded to dextrins by a Bacillus sp. strain TS-23 α-amylase or variants thereof, at pH values between about 5.5 and about 6.2 and at temperatures of about 95°C to about 160 ⁰ C for a period of approximately 2 hours. In order to ensure optimal enzyme stability under these conditions, 1 mM of calcium is added (40 ppm free calcium ions). Starch processing is useful for producing alcohol (e.g., cereal liquefaction for fuel and potable alcohol, alcohol brewing), starch liquefaction for sweetener production, cane sugar processing, and other food related starch processing goals. Other conditions can be used for different Bacillus sp. strain TS-23 α-amylases or variants thereof.

After the liquefaction process, the dextrins are converted into dextrose by addition of a glucoamylase (e.g. AMG™) and a debranching enzyme, such as an isoamylase or a pullulanase (e.g., Promozyme®). Before this step, the pH is reduced to a value below about 4.5, maintaining the high temperature (above 95°C), and the liquefying Bacillus sp. strain TS-23 α-amylase or variant thereof, activity is denatured. The temperature is lowered to 60 ⁰ C, and a glucoamylase

and a debranching enzyme can be added. The saccharification process proceeds typically for about 24 to about 72 hours.

After the saccharification process, the pH is increased to a value in the range of about 6.0 to about 8.0, e.g., 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®).

At least one enzymatic improvement of this process can be performed. Reduction of the calcium dependency of the liquefying Bacillus sp. strain TS-23 α-amylase or variant thereof. Addition of free calcium is required to ensure adequately high stability of the Bacillus sp. strain TS-23 α-amylase or variant thereof, but free calcium strongly inhibits the activity of the glucose isomerase and needs to be removed, by means of an expensive unit operation, to an extent that reduces the level of free calcium to below 3-5 ppm. Cost savings can be obtained if such an operation could be avoided, and the liquefaction process could be performed without addition of free calcium ions.

For example, a less calcium-dependent enzyme, which is stable and highly active at low concentrations of free calcium (<40 ppm) can be utilized in the composition and procedures. Such a Bacillus sp. strain TS-23 α-amylase or variant thereof should have a pH optimum at a pH in the range of about 4.5 to about 6.5, or in the range of about 4.5 to about 5.5.

A Bacillus sp. strain TS-23 α-amylase or variant thereof can be used in laboratory and in industrial settings to hydrolyze starch or any maltodextrine- comprising compound for a variety of purposes. These Bacillus sp. strain TS-23 α-amylases or variants thereof can be used alone to provide specific hydrolysis or can be combined with other amylases to provide a "cocktail" with a broad spectrum of activity. Exemplary uses include the removal or partial or complete hydrolysis of starch or any maltodextrine-comprising compound from biological, food, animal feed, pharmaceutical, or industrial samples.

Another aspect contemplates compositions and methods of using the compositions in a fermentation process, wherein a starch substrate is liquefied

and/or saccharified in the presence of the Bacillus sp. strain TS-23 α-amylase or variant thereof to produce glucose and/or maltose suitable for conversion into a fermentation product by a fermenting organism, such as a yeast. Such fermentation processes include a process for producing ethanol for fuel or drinking ethanol (potable alcohol), a process for producing a beverage, a process for producing desired organic compounds (e.g., such as citric acid, itaconic acid, lactic acid, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta lactone, or sodium erythorbate), ketones, amino acids (such as glutamic acid, sodium monoglutaminate), but also more complex compounds (e.g., antibiotics, such as penicillin, tetracyclin), enzymes, vitamins (e.g., riboflavin, vitamin Bi ₂ , β-carotene), and hormones, which are difficult to produce synthetically.

The starch to be processed may be a highly refined starch quality, such as at least 90%, at least 95%, at least 97%, or at least 99.5% pure. Alternatively, the starch can be a more crude starch containing material comprising milled whole grain including non-starch fractions such as germ residues and fibers. The raw material, such as whole grain, is milled in order to open up the structure and allowing for further processing. Two milling processes can be used : wet and dry milling. Also, corn grits such as milled corn grits may be applied.

Dry milled grain will, in addition to starch, comprise significant amounts of non- starch carbohydrate compounds. When such a heterogeneous material is processed by jet cooking Bacillus sp. strain TS-23 often only a partial gelatinization of the starch is achieved. As the Bacillus sp. strain TS-23 α- amylase or variant thereof has a high activity towards ungelatinized starch, the enzyme(s) may be advantageously applied in a process comprising liquefaction and/or saccharification jet cooked dry milled starch.

Furthermore, due to the superior hydrolysis activity of the Bacillus sp. strain TS- 23 α-amylases or variants thereof, the need for glucoamylase during the saccharification step is greatly reduced. This allows saccharification to be performed at very low levels of glucoamylase activity. Glucoamylase activity is

either absent, or if present, then present in an amount of no more than or even less than 0.5 AGU/g DS, or no more than or even less than 0.4 AGU/g DS, or no more than or even less than about 0.3 AGU/g DS, or less than 0.1 AGU, such as no more than or even less than about 0.05 AGU/g DS of starch substrate. "DS" is the unit of enzyme added per gram of dry solid substrate. Expressed in mg enzyme protein, the enzyme having glucoamylase activity is either absent or present in an in an amount of no more than or even less than about 0.5 mg EP/g DS, or no more than or even less than about 0.4 mg EP/g DS, or no more than or even less than about 0.3 mg EP/g DS, or no more than or even less than about 0.1 mg EP/g DS (e.g., no more than or even less than about 0.05 mg EP/g DS or no more than or even less than 0.02 mg EP/g DS of starch substrate). The glucoamylase may be derived from a strain within Aspergillus sp., Talaromyces sp., Pachykytospora sp., or Trametes sp., with exemplary examples being Aspergillus niger, Talaromyces emersonii, Trametes cingulata, or Pachykytospora papyracea.

The process may comprise a) contacting a starch substrate with a Bacillus sp. strain TS-23 α-amylase or variant thereof comprising a catalytic module having α-amylase activity and a carbohydrate-binding module, e.g., the polypeptide of the first aspect; b) incubating said starch substrate with said enzyme for a time and at a temperature sufficient to achieve conversion of at least 90%, or at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% w/w of said starch substrate into fermentable sugars; c) fermenting to produce a fermentation product; and d) optionally recovering the fermentation product. During the process steps b) and/or c), an enzyme having glucoamylase activity is either absent or present in an amount from

0.001 to 2.0 AGU/g DS, from 0.01 to 1.5 AGU/g DS, from 0.05 to 1.0 AGU/g DS, from 0.01 to 0.5 AGU/g DS. The enzyme having glucoamylase activity can either absent or present in an amount of no more than or even less than 0.5 AGU/g DS, or no more than or even less than 0.4 AGU/g DS, or no more than or even less than 0.3 AGU/g DS, or no more than or even less than 0.1 AGU/g DS (e.g., no more than or even less than 0.05 AGU/g DS of starch substrate). Expressed in mg enzyme protein, the enzyme having glucoamylase activity is either absent or present in an in an amount of no more than or even less than

0.5 mg EP/g DS, or no more than or even less than 0.4 mg EP/g DS, or no more than or even less than 0.3 mg EP/g DS, or no more than or even less than 0.1 mg EP/g DS (e.g., no more than or even less than 0.05 mg EP/g DS or no more than or even less than 0.02 mg EP/g DS of starch substrate). In the process steps a), b), c), and/or d) may be performed separately or simultaneously.

In another aspect the process may comprise: a) contacting a starch substrate with a yeast cell transformed to express a Bacillus sp. strain TS-23 α-amylase or variant thereof comprising a catalytic module having α-amylase activity and a carbohydrate-binding module; b) incubating said starch substrate with said yeast for a time and at a temperature sufficient to achieve conversion of at least 90% w/w of said starch substrate into fermentable sugars; c) fermenting to produce ethanol; d) optionally recovering ethanol. The steps a), b), and c) may performed separately or simultaneously.

In yet another aspect, the process comprising hydrolysis of a slurry of gelatinized or granular starch, in particular hydrolysis of granular starch into a soluble starch hydrolysate at a temperature below the initial gelatinization temperature of said granular starch. In addition to being contacted with a polypeptide comprising a catalytic module having α-amylase activity and a carbohydrate-binding module. The starch can be contacted with any one or more of the following a fungal α-amylase (EC 3.2.1.1) and one or more of the following : a β-amylase (EC 3.2.1.2), and a glucoamylase (EC 3.2.1.3). In a further aspect, another amylolytic enzyme or a debranching enzyme, such as an isoamylase (EC 3.2.1.68), or a pullulanases (EC 3.2.1.41) may be added to the Bacillus sp. strain TS-23 α-amylase or variant thereof.

In an embodiment, the process is conducted at a temperature below the initial gelatinization temperature. Such processes are oftentimes conducted at least at 30 ⁰ C, at least 31°C, at least 32°C, at least 33°C, at least 34°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 39°C, at least 40 ⁰ C, at least 41°C, at least 42°C, at least 43°C, at least 44°C, at least 45°C, at least 46°C, at least 47°C, at least 48°C, at least 49°C, at least 50 ⁰ C, at least 51°C, at least

52°C, at least 53°C, at least 54°C, at least 55°C, at least 56°C, at least 57°C, at

least 58°C, at least 59°C, or at least 60 ⁰ C. The pH at which the process is conducted may in be in the range of about 3.0 to about 7.0, or from about 3.5 to about 6.0, or from about 4.0 to about 5.0. One aspect contemplates a process comprising fermentation, e.g. with a yeast to produce ethanol, e.g., at a temperature around 32°C, such as from 30 ⁰ C to 35°C.

In another aspect, the process comprises simultaneous saccharification and fermentation, e.g., with a yeast to produce ethanol, or another suitable fermentation organism to produce a desired organic compound, such as at a temperature from 30 ⁰ C to 35°C, e.g., at around 32°C.

In the above fermentation processes, the ethanol content reaches at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15% such as at least about 16% ethanol.

The starch slurry to be used in any of the above aspects may have about 20% to about 55% dry solids granular starch, about 25% to about 40% dry solids granular starch, or from about 30% to about 35% dry solids granular starch.

After being contacted with a Bacillus sp. strain TS-23 α-amylase or variant thereof, the enzyme converts the soluble starch into a soluble starch hydrolysate of the granular starch in the amount of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

98%, or at least 99%.

In another embodiment, a Bacillus sp. strain TS-23 α-amylase or variant thereof comprises a catalytic module having α-amylase activity and a carbohydrate- binding module, e.g., the polypeptide of the first aspect, is used in a process for liquefaction, saccharification of a gelatinized starch, e.g., but not limited to gelatinization by jet cooking. The process may comprise fermentation to produce a fermentation product, e.g., ethanol. Such a process for producing ethanol from starch-containing material by fermentation comprises: (i) liquefying said starch-containing material with a polypeptide comprising a

catalytic module having α-amylase activity and a carbohydrate-binding module, e.g., the polypeptide of the first aspect; (ii) saccharifying the liquefied mash obtained; and (iii) fermenting the material obtained in step (ii) in the presence of a fermenting organism. Optionally the process further comprises recovery of the ethanol. The saccharification and fermentation processes may be carried out as a simultaneous saccharification and fermentation process (SSF process). During the fermentation, the ethanol content reaches at least about 7%, at least about 8%, at least about 9%, at least about 10% such as at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least 15% such as at least 16% ethanol.

The starch to be processed in the processes of the above aspects may in particular be obtained from tubers, roots, stems, legumes, cereals or whole grain. More specifically, the granular starch may be obtained from corns, cobs, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, bean, banana, or potatoes. Also contemplated are both waxy and non-waxy types of corn and barley.

The composition described above may be used for liquefying and/or saccharifying a gelatinized or a granular starch, and a partly gelatinized starch. A partly gelatinized starch is a starch that to some extent is gelatinized, i.e., wherein part of the starch has irreversibly swelled and gelatinized and part of the starch is still present in a granular state.

The composition described above may comprise an acid α-amylase variant present in an amount of 0.01 to 10.0 AFAU/g DS, or 0.1 to 5.0 AFAU/g DS, or 0.5 to 3.0 AFAU/AGU, or 0.3 to 2.0 AFAU/g DS. The composition may be applied in any of the starch processes described above.

As used herein, the term "liquefaction" or "liquefy" means a process by which starch is converted to shorter chain and less viscous dextrins. Generally, this process involves gelatinization of starch simultaneously with or followed by the addition of a Bacillus sp. strain TS-23 α-amylase or variant thereof. Additional liquefaction inducing enzymes may also be added.

As used herein, the term "primary liquefaction" refers to a step of liquefaction when the slurry's temperature is raised to or near its gelatinization temperature. Subsequent to the raising of the temperature, the slurry is sent through a heat exchanger or jet to temperatures from 200-300 ⁰ F, e.g., 220-235 ⁰ F. Subsequent to application to a heat exchange or jet temperature, the slurry is held for a period of 3-10 minutes at that temperature. This step of holding the slurry at 200-300 ⁰ F is primary liquefaction.

As used herein, the term "secondary liquefaction" refers the liquefaction step subsequent to primary liquefaction (heating to 200-300 ⁰ F), when the slurry is allowed to cool to atmospheric temperature. This cooling step can be 30 minutes to 180 minutes (3 hours), e.g. 90 minutes to 120 minutes (2 hours).

As used herein, the term "minutes of secondary liquefaction" refers to the time that has elapsed from the start of secondary liquefaction, to the time that the DE is measured.

Another aspect contemplates the additional use of a β-amylase in the composition comprising Bacillus sp. strain TS-23 α-amylase or variant thereof, β-amylases (EC 3.2.1.2) are exo-acting maltogenic amylases, which catalyze the hydrolysis of 1,4-α-glucosidic linkages in to amylose, amylopectin, and related glucose polymers, thereby releasing maltose.

β-amylases have been isolated from various plants and microorganisms (W. M. Fogarty and C. T. Kelly, Progress in Industrial Microbiology, vol. 15, pp. 112- 115, 1979). These β-amylases are characterized by having optimum temperatures in the range from 40 ⁰ C to 65°C, and optimum pH in the range from about 4.5 to about 7.0. Contemplated β-amylases include, but are not limited to, β-amylases from barley Spezyme® BBA 1500, Spezyme® DBA, Optimalt® ME, Optimalt® BBA (Genencor International Inc.) and Novozym™ WBA (Novozymes A/S).

Another enzyme contemplated for use in the composition is a glucoamylase (EC 3.2.1.3). Glucoamylases are derived from a microorganism or a plant. Exemplary glucoamylases are of fungal or bacterial origin. Exemplary bacterial

glucoamylases are Aspergillus glucoamylases, in particular A niger Gl or G2 glucoamylase (Boel et al., EMBO J. 3(5) : 1097-1102 (1984), or variants thereof, such as disclosed in WO 92/00381; and WO 00/04136; the A. awamori glucoamylase (WO 84/02921); A. oryzae (Agric. Biol. Chem., 55(4) : 941-949 (1991)), or variants or fragments thereof.

Other contemplated Aspergillus glucoamylase variants include variants to enhance the thermal stability: G137A and G139A (Chen et al., Prot. Eng. 9: 499-505 (1996)); D257E and D293E/Q (Chen et al., Prot. Eng. 8: 575-582 (1995)); N182 (Chen et al., Biochem. J. 301 : 275-281 (1994)); disulfide bonds, A246C (Fierobe et al., Biochemistry, 35: 8698-8704 (1996)); and introduction of Pro residues in positions A435 and S436 (Li et al., Protein Eng. 10: 1199-1204 (1997)). Other contemplated glucoamylases include and Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (U.S. Patent No. RE 32,153), Talaromyces duponti, Talaromyces thermophilus (U.S. Patent No. 4,587,215). Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135138) and C. thermohydrosulfuricum (WO 86/01831). Exemplary glucoamylases include the glucoamylases derived from Aspergillus oryzae. Also contemplated are the commercial glucoamylases such as AMG 200L; AMG 300 L; SAN™ SUPER and AMG™ E (Novozymes);

OPTIDEX®300 (from Genencor International, Inc.); AMIGASE® and AMIGASE® PLUS (DSM); G-ZYME® G900 (Enzyme Bio-Systems); G-ZYME® G990 ZR (A niger glucoamylase and low protease content).

Glucoamylases may be added in an amount of 0.02-2.0 AGU/g DS, or 0.1-1.0 AGU/g DS, such as 0.2 AGU/g DS.

Additional enzymes and enzyme variants are also contemplated for inclusion in the composition. One or more α-amylases can be used in addition to a Bacillus sp. strain TS-23 α-amylase or variant thereof, or can further include other enzymes discussed herein.

Another enzyme that can optionally be added is a debranching enzyme, such as an isoamylase (EC 3.2.1.68) or a pullulanase (EC 3.2.1.41). Isoamylase hydrolyses α-l,6-D-glucosidic branch linkages in amylopectin and β-limit dextrins and can be distinguished from pullulanases by the inability of isoamylase to attack pullulan, and by the limited action on α-limit dextrins. Debranching enzymes may be added in effective amounts well known to the person skilled in the art.

The exact composition of the products of the process depends on the combination of enzymes applied as well as the type of granular starch processed. For example, the soluble hydrolysate can be maltose with a purity of at least about 85%, at least about 90%, at least about 95.0%, at least about 95.5%, at least about 96.0%, at least about 96.5%, at least about 97.0%, at least about 97.5%, at least about 98.0%, at least about 98.5, at least about 99.0% or at least about 99.5%. Alternatively, the soluble starch hydrolysate can be glucose or the starch hydrolysate has a DX (glucose percent of total solubilized dry solids) of at least 94.5%, at least 95.0%, at least 95.5%, at least 96.0%, at least 96.5%, at least 97.0%, at least 97.5%, at least 98.0%, at least 98.5, at least 99.0% or at least 99.5%. The process can include a product which is a specialty syrup, such as a specialty syrup containing a mixture of glucose, maltose, DP3 and DPn for use in the manufacture of ice creams, cakes, candies, canned fruit.

Two milling processes are: wet and dry milling. In dry milling, the whole kernel is milled and used. Wet milling gives a good separation of germ and meal (starch granules and protein), and is with a few exceptions, applied at locations where the starch hydrolysate is used in production of syrups. Both dry and wet milling are well known in the art of starch processing and are equally contemplated for use with the compositions and methods disclosed. The process may be conducted in an ultrafiltration system where the retentate is held under recirculation in presence of enzymes, raw starch and water and where the permeate is the soluble starch hydrolysate. Equally contemplated is the process conducted in a continuous membrane reactor with ultrafiltration membranes and where the retentate is held under recirculation in presence of enzymes, raw

starch and water, and where the permeate is the soluble starch hydrolysate. Also contemplated is the process conducted in a continuous membrane reactor with microfiltration membranes and where the retentate is held under recirculation in presence of enzymes, raw starch and water, and where the permeate is the soluble starch hydrolysate.

In one regard, the soluble starch hydrolysate of the process is subjected to conversion into high fructose starch-based syrup (HFSS), such as high fructose corn syrup (HFCS). This conversion can be achieved using a glucose isomerase, and by an immobilized glucose isomerase supported on a solid support. Contemplated isomerases include the commercial products Sweetzyme® , IT (Novozymes A/S); G-zyme® IMGI, and G-zyme® G993, Ketomax™ , G-zyme® G993 (Rhodia); G-zyme® G993 liquid, GenSweet® IGI (Genencor International, Inc.).

In another aspect, the soluble starch hydrolysate produced by these methods can be used in the production of fuel or potable ethanol. In the process of the third aspect the fermentation may be carried out simultaneously or separately/sequential to the hydrolysis of the granular starch slurry. When the fermentation is performed simultaneous to the hydrolysis, the temperature is between 30 ⁰ C and 35°C, or between 31°C and 34°C. The process may be conducted in an ultrafiltration system where the retentate is held under recirculation in presence of enzymes, raw starch, yeast, yeast nutrients and water and where the permeate is an ethanol containing liquid. Equally contemplated is the process conducted in a continuous membrane reactor with ultrafiltration membranes and where the retentate is held under recirculation in presence of enzymes, raw starch, yeast, yeast nutrients and water and where the permeate is an ethanol containing liquid.

The soluble starch hydrolysate of the process may also be used for production of a fermentation product comprising fermenting the treated starch into a fermentation product, such as citric acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta lactone, or sodium erythorbate.

The amylolytic activity of a Bacillus sp. strain TS-23 α-amylase or variant thereof may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue color is formed, but during the break-down of the starch, the blue color gets weaker and gradually turns into a reddish-brown, which is compared to a colored glass standard.

4.2 Compositions and Methods for Baking and Food Preparation

For the commercial and home use of flour for baking and food production, it is important to maintain an appropriate level of α-amylase activity in the flour. A level of activity that is too high may result in a product that is sticky and/or doughy and unmarketable; but flour with insufficient α-amylase activity may not contain enough sugar for proper yeast function, resulting in dry, crumbly bread. To augment the level of endogenous α-amylase activity in flour, an α-amylase may be added to flour in the form of a Bacillus sp. strain TS-23 α-amylase or variant thereof. Therefore, the ability to determine the level of activity of both endogenous (natural) and fungal α-amylase, or other α-amylase, in a flour sample would benefit the food production process and promote more efficient use of flour in food production.

In addition to the use of grains and other plant products in baking, grains such as barley, oats, wheat, as well as plant components such as corn, hops, and rice are used for brewing, both in industry and for home brewing. The components used in brewing may be unmalted or malted, which means partially germinated resulting in an increase in the levels of enzymes including α-amylase. For successful brewing, adequate levels of α-amylase enzyme activity are necessary to ensure the appropriate levels of sugars for fermentation.

As used herein, the term "flour" means milled or ground cereal grain. The term "flour" may also mean Sago or tuber products that have been ground or mashed. In some embodiments, flour may also contain components in addition to the milled or mashed cereal or plant matter. An example of an additional

component, although not intended to be limiting, is a leavening agent. Cereal grains include: wheat, oat, rye, and barley. Tuber products can include tapioca flour, cassava flour, and custard powder. The term "flour" also includes ground corn flour, maize-meal, rice flour, whole-meal flour, self-rising flour, tapioca flour, cassava flour, ground rice, enriched flour, and custard powder.

As used herein, the term "stock" means grains and plant components that are crushed or broken. For example, barley used in beer production is a grain that has been coarsely ground or crushed to yield a consistency appropriate for producing a mash for fermentation. As used herein, the term "stock" includes any of the aforementioned types of plants and grains in crushed or coarsely ground forms. The methods described herein may be used to determine α- amylase activity levels in flours, and also in stock, which includes the aforementioned types of grains, tubers, and other plant products that have been crushed.

As described above, in one aspect, the present invention relates to a method of preparing a food product, comprising incorporating into the food product an AmyTS-23 alpha-amylase. The food product may be any food product, such as an animal feed or a dough, such as a dough used for preparing a baked product. When an AmyTS-23 alpha-amylase or a further enzyme such as a further amylase is added to a food product, the enzyme(s) may be refered to as a "food additive".

When an AmyTS-23 alpha-amylase, optionally in combination with a further amylase is used in a food product to improve any feature of the food product, the amylase(s) or the composition comprising the amylase(s) may be refered to as a "food improver". The term "food improver" includes bread improving compositions and dough improving compositions. These compositions may optionally be together with any further ingredient, such as a further enzyme.

In some embodiments, the AmyTS-23 alpha-amylase, optionally in combination with a further amylase such as an anti-staling amylase is used as a food additive

in an isolated form. In some embodiments, the AmyTS-23 alpha-amylase alone or in combination with a further amylase is in a purified form.

The dough product may be any processed dough product, including fried, deep fried, roasted, baked, steamed and boiled doughs, such as steamed bread and rice cakes. In some embodiments, the food product is a bakery product.

Preferably, the food product is a bakery product. Typical bakery (baked) products include bread - such as loaves, rolls, buns, pizza bases etc. pastry, pretzels, tortillas, cakes, cookies, biscuits, krackers etc.

In some embodiments, the food product used according to the present invention is selected from one or more of the following : eggs, egg-based products, including but not limited to mayonnaise, salad dressings, sauces, ice creams, egg powder, modified egg yolk and products made therefrom; baked goods, including breads, cakes, sweet dough products, laminated doughs, liquid batters, muffins, doughnuts, biscuits, crackers and cookies; confectionery, including chocolate, candies, caramels, halawa, gums, including sugar free and sugar sweetened gums, bubble gum, soft bubble gum, chewing gum and puddings; frozen products including sorbets, preferably frozen dairy products, including ice cream and ice milk; dairy products, including cheese, butter, milk, coffee cream, whipped cream, custard cream, milk drinks and yoghurts; mousses, whipped vegetable creams, meat products, including processed meat products; edible oils and fats, aerated and non-aerated whipped products, oil-in-water emulsions, water-in-oil emulsions, margarine, shortening and spreads including low fat and very low fat spreads; dressings, mayonnaise, dips, cream based sauces, cream based soups, beverages, spice emulsions and sauces.

Suitably the food product in accordance with the present invention may be a "fine foods", including cakes, pastry, confectionery, chocolates, fudge and the like.

In one aspect the food product in accordance with the present invention may be a dough product or a baked product, such as a bread, a fried product, a snack, cakes, pies, brownies, cookies, noodles, instant noodles, tortillas, snack items

such as crackers, graham crackers, pretzels, and potato chips, and pasta, and breakfast cereals.

In a further aspect, the food product in accordance with the present invention may be a plant derived food product such as flours, pre-mixes, oils, fats, cocoa butter, coffee whitener, salad dressings, margarine, spreads, peanut butter, shortenings, ice cream, cooking oils.

In another aspect, the food product in accordance with the present invention may be a dairy product, including butter, milk, cream, cheese such as natural, processed, and imitation cheeses in a variety of forms (including shredded, block, slices or grated), cream cheese, ice cream, frozen desserts, yoghurt, yoghurt drinks, butter fat, anhydrous milk fat, other dairy products. The enzyme used according to the present invention may improve fat stability in dairy products.

In another aspect, the food product in accordance with the present invention may be a food product containing animal derived ingredients, such as processed meat products, cooking oils, shortenings.

In a further aspect, the food product in accordance with the present invention may be a beverage, a fruit, mixed fruit, a vegetable or wine. In some cases the beverage may contain up to 20 g/l of added phytosterols derived from the invention.

In another aspect, the food product in accordance with the present invention may be an animal feed. The animal feed may be enriched with phytosterol and/or phytostanols, preferably with beta-sitosterol/stanol. Suitably, the animal feed may be a poultry feed. When the food product is poultry feed, the present invention may be used to lower the cholesterol content of eggs produced by poultry fed on the food product according to the present invention.

In one aspect preferably the food product is selected from one or more of the following : eggs, egg-based products, including mayonnaise, salad dressings,

sauces, ice cream, egg powder, modified egg yolk and products made therefrom.

Preferably the food product according to the present invention is a water containing food product. Suitably the food product may be comprised of 10-98% water, suitably 14-98%, suitably of 18-98% water, suitably of 20-98%, suitably of 40-98%, suitably of 50-98%, suitably of 70-98%, suitably of 75-98%.

Also disclosed are methods for measuring α-amylase activity in flour and grain or tuber products and stock. As used herein, the term "α-amylase" means endogenous α-amylase (present in the flour or stock) or a Bacillus sp. strain TS- 23 α-amylase or variant thereof that has been added to the flour or stock.

A Bacillus sp. strain TS-23 α-amylase or variant thereof alone or in a combination with other amylases can be added to prevent staling. The anti- staling amylases used may be any amylase that is effective in retarding the staling (crumb firming) of baked products.

The amylase can have a temperature optimum in the presence of starch in the ranges for example of 30-90 ⁰ C, 50-80 ⁰ C, 55-75°C, 60-70 ⁰ C. The temperature optimum may be measured in a 1% solution of soluble starch at pH 5.5.

Additional anti-staling amylases that can be used in combination with a Bacillus sp. strain TS-23 α-amylase include an endo-amylase, e.g., a bacterial endo- amylase from Bacillus. For example, the additional amylase can be a maltogenic alpha-amylase (EC 3.2.1.133), e.g. from Bacillus. Novamyl® is a maltogenic alpha-amylase from B. stearothermophilus strain NCIB 11837 and is described in C. Christophersen et al., 1997 Starch 50(1): 39-45.

Other examples of anti-staling endo-amylases can include other bacterial alpha- amylases, derived e.g. from Bacillus, such as B. licheniformis or B. amyloliquefaciens .

The anti-staling amylase may be an exo-amylase such as β-amylase, e.g. from plant (e.g., soy bean) or from microbial sources (e.g., Bacillus).

The α-amylase of Bacillus sp. strain TS-23 or variant thereof can be added alone or with other amylases in an amount effective for retarding the staling (crumb firming) of the baked product. The amount of anti-staling amylase, such as AmyTS-23 alpha-amylase or a further anti-staling amylase will typically be in the range of 0.01-10 mg of enzyme protein per kg of flour, such as e.g. 1-10 mg/kg.

Methods to determine enzyme protein amounts are known to the person skilled in the art and may determined as described in e.g. Bradford, M. M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 72: 248- 254.

The further amylase may be added in an amount effective for retarding the staling (crumb firming) of the baked product. The amount of further amylase will typically be in the range of 0.01-10 mg of enzyme protein per kg of flour, e.g. 1-10 mg/kg. The effective amounts of the further amylase, such as PS4 or variants thereof or Novamyl, may alternatively be identified in corresponding BMK units (1000 Betamyl units).

The effective amounts of α-amylase of Bacillus sp. strain TS-23 or variant thereof described elsewhere and used according to the invention may also alternatively be identified in corresponding BMK units (1000 Betamyl units).

The baking composition comprising an α-amylase of Bacillus sp. strain TS-23 can further comprise a phospholipase. The phospholipase may have Ai or A ₂ activity to remove fatty acid from the phospholipid and form a lyso-phospholipid. It may or may not have lipase activity, i.e. activity on triglycerides. The phospholipase can have a temperature optimum in the range of 30-90 ⁰ C, e.g. 30-70 ⁰ C. The added phospholipases can be of animal origin, e.g. from pancreas (e.g., bovine or porcine pancreas), snake venom or bee venom. Alternatively, the phospholipase may be of microbial origin, e.g. from filamentous fungi, yeast or bacteria, such as the genus or species Aspergillus, A. niger; Dictyostelium, D. discoideum; Mucor, M. javanicus, M. mucedo, M. subtilissimus; Neurospora, N. crassa; Rhizomucor, R. pusillus; Rhizopus, R. arrhizus, R. japonicus, R.

stolonifer; Sclerotinia, S. libertiana; Trichophyton, T. rubrum; Whetzelinia, W. sclerotiorum; Bacillus, B. megaterium , B. subtilis; Citrobacter, C. freundii; Enterobacter, E. aerogenes, E. cloacae; Edwardsiella, E. tarda; Erwinia, E. herbicola; Escherichia, E. coli; Klebsiella, K. pneumoniae; Proteus, P. vulgaris; Providencia, P. stuartii; Salmonella, S. typhimurium; Serratia, S. liquefasciens, S. marcescens; Shigella, S. flexneri; Streptomyces, S. violeceoruber; Yersinia, Y. enter ocolitica; Fusarium, F. oxysporum (e.g., strain DSM 2672).

The phospholipase is added in an amount that improves the softness of the bread during the initial period after baking, particularly the first 24 hours. The amount of phospholipase will typically be in the range of 0.01-10 mg of enzyme protein per kg of flour (e.g. 0.1-5 mg/kg) or 200-5000 LEU/kg of flour (e.g. 500- 2000 LEU/kg). A phospholipase with lipase activity is generally added in an amount corresponding to a lipase activity of 20-1000 LU/kg of flour, particularly 50-500 LU/kg. One LU (Lipase Unit) is defined as the amount of enzyme required to release 1 μmol butyric acid per minute at 30.0 ⁰ C; pH 7.0; with gum arabic as emulsifier and tributyrin as substrate.

Compositions of dough generally comprise wheat meal or wheat flour and/or other types of meal, flour or starch such as corn flour, cornstarch, rye meal, rye flour, oat flour, oatmeal, soy flour, sorghum meal, sorghum flour, potato meal, potato flour or potato starch. The dough may be fresh, frozen or par-baked.

The dough is normally a leavened dough or a dough to be subjected to leavening. The dough may be leavened in various ways, such as by adding chemical leavening agents, e.g., sodium bicarbonate or by adding a leaven (fermenting dough). For example, the dough can be leavened by adding a suitable yeast culture, such as a culture of Saccharomyces cerevisiae (baker's yeast), e.g. a commercially available strain of S. cerevisiae.

The dough may also comprise other conventional dough ingredients, e.g., proteins, such as milk powder, gluten, and soy; eggs (either whole eggs, egg yolks or egg whites); an oxidant such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate; an amino

acid such as L-cysteine; a sugar; a salt such as sodium chloride, calcium acetate, sodium sulfate or calcium sulfate.

The dough may comprise fat (triglyceride) such as granulated fat or shortening.

The dough may further comprise an emulsifier such as mono- or diglycerides, diacetyl tartaric acid esters of mono- or diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetic acid esters of monoglycerides, polyoxyethylene stearates, or lysolecithin, but is applicable to a dough which is made without addition of emulsifiers (other than optionally phospholipid).

Optionally, an additional enzyme may be used together with the anti-staling amylase and the phospholipase. The additional enzyme may be a second amylase, such as an amyloglucosidase, a β-amylase, a cyclodextrin glucanotransferase, or the additional enzyme may be a peptidase, in particular an exopeptidase, a transglutaminase, a lipase, a cellulase, a hemicellulase, in particular a pentosanase such as xylanase, a protease, a protein disulfide isomerase, e.g., a protein disulfide isomerase as disclosed in WO 95/00636, a glycosyltransferase, a branching enzyme (1,4-α-glucan branching enzyme), a 4- α-glucanotransferase (dextrin glycosyltransferase) or an oxidoreductase, e.g., a peroxidase, a laccase, a glucose oxidase, a pyranose oxidase, a lipoxygenase, an L-amino acid oxidase or a carbohydrate oxidase.

The additional enzyme may be of any origin, including mammalian and plant origin, and as well as of microbial (bacterial, yeast or fungal) origin and may be obtained by techniques conventionally used in the art.

The xylanase can be microbial origin, e.g. derived from a bacterium or fungus, such as a strain of Aspergillus, in particular of A. aculeatus, A. niger (cf. WO

91/19782), A. awamori (WO 91/18977), or A. tubigensis (WO 92/01793); from a strain of Trichoderma , e.g. T. reesei, or from a strain of Humicola, e.g. H. insolens (WO 92/17573). Pentopan® and Novozym 384® are commercially available xylanase preparations produced from Trichoderma reesei.

The amyloglucosidase may be an A. niger amyloglucosidase (such as AMG®). Other useful amylase products include Grindamyl® A 1000 or A 5000 (available from Grindsted Products, Denmark) and Amylase® H or Amylase® P (available from DSM Gist Brocades, The Netherlands).

The glucose oxidase may be a fungal glucose oxidase, in particular an Aspergillus niger glucose oxidase (such as Gluzyme®).

Exemplary proteases are Neutrase® (Novozymes) and Protex OxG (Genencor International, Inc.).

Exemplary lipase can be derived from strains of Thermomyces (Humicola), Rhizomucor, Candida, Aspergillus, Rhizopus, or Pseudomonas, in particular from Thermomyces lanuginosus (Humicola lanuginosa), Rhizomucor miehei, Candida antarctica, Aspergillus niger, Rhizopus delemar or Rhizopus arrhizus or Pseudomonas cepacia. In specific embodiments, the lipase may be Lipase A or Lipase B derived from Candida antarctica as described in WO 88/02775, or the lipase may be derived from Rhizomucor miehei as described in EP 238,023, or Humicola lanuginosa described in EP 305,216, or Pseudomonas cepacia as described in EP 214,761 and WO 89/01032.

The process may be used for any kind of baked product prepared from dough, either of a soft or a crisp character of a white, light, or dark type. Examples are bread (in particular white, whole-meal or rye bread), typically in the form of loaves or rolls, French baguette-type bread, pita bread, tortillas, cakes, pancakes, biscuits, cookies, pie crusts, crisp bread, steamed bread, pizza and the like.

Another aspect contemplates the use of the Bacillus sp. strain TS-23 α-amylase or variant thereof in a pre-mix comprising flour together with an anti-staling amylase, a phospholipase and a phospholipid. The pre-mix may contain other dough-improving and/or bread-improving additives, e.g. any of the additives, including enzymes, mentioned above.

Another aspect provided is an enzyme preparation comprising an anti-staling amylase and a phospholipase, for use as a baking additive. The enzyme preparation can be in the form of a granulate or agglomerated powder. It can have a narrow particle size distribution with more than 95% (by weight) of the particles in the range from 25 to 500 μm.

Granulates and agglomerated powders may be prepared by conventional methods, e.g. by spraying the amylase onto a carrier in a fluid-bed granulator. The carrier may consist of particulate cores having a suitable particle size. The carrier may be soluble or insoluble, e.g. a salt (such as NaCI or sodium sulfate), a sugar (such as sucrose or lactose), a sugar alcohol (such as sorbitol), starch, rice, corn grits, or soy.

Another aspect contemplates the enveloping of a Bacillus sp. strain TS-23 α- amylase. To prepare the enveloped alpha-amylase particles, the enzymes are contacted with a food grade lipid, discussed in further detail below, in sufficient quantity to suspend all of the alpha-amylase particles.

Food grade lipids, as used herein, may be any naturally organic compound that is insoluble in water but is soluble in non-polar organic solvents such as hydrocarbon or diethyl ether. The food grade lipids utilized can include, but are not limited to, triglycerides either in the form of fats or oils that are either saturated or unsaturated. Examples of fatty acids and combinations thereof which make up the saturated triglycerides utilized include, but are not limited to, butyric (derived from milk fat), palmitic (derived from animal and plant fat), and/or stearic (derived from animal and plant fat). Examples of fatty acids and combinations thereof which make up the unsaturated triglycerides utilized include, but are not limited to, palmitoleic (derived from animal and plant fat), oleic (derived from animal and plant fat), linoleic (derived from plant oils), and/or linolenic (derived from linseed oil). Other food grade lipids contemplated and within the scope include, but are not limited to, monoglycerides and diglycerides derived from the triglycerides discussed above, phospholipids and glycolipids.

The food grade lipid, in the liquid form, is contacted with a powdered form of the alpha-amylase particles in such a fashion that the lipid material covers at least a portion of the surface of at least a majority, and for example 100% of the α- amylase particles. Thus, each alpha-amylase particle is individually enveloped in a lipid. For example, all or substantially all of the particles of α-amylase are provided with a thin, continuous, enveloping film of lipid. This can be accomplished by first pouring a quantity of lipid into a container, and then slurrying the α-amylase so that the lipid thoroughly wets the surface of each α- amylase particle. After a short period of stirring, the enveloped α-amylase particles, carrying a substantial amount of the lipids on their surfaces, are recovered. The thickness of the coating so applied to the particles of α-amylase can be controlled by selection of the type of lipid used and by repeating the operation in order to build up a thicker film, when desired.

The storing, handling and incorporation of the loaded delivery vehicle can be accomplished by means of a packaged mix. The packaged mix can comprise the enveloped α-amylase. However, the packaged mix may further contain additional ingredients as required by the manufacturer or baker. After the enveloped α-amylase is incorporated into the dough, the baker continues through the normal production process for that product.

The advantages of enveloping the α-amylase are two-fold. First, the food grade lipid protects the enzyme from thermal denaturation during the baking process for those enzymes that are heat labile. Consequently, while the α-amylase is stabilized and protected during the proving and baking stages, it is released from the protective coating in the final baked good product, where it hydrolyzes the glucosidic linkages in polyglucans. The loaded delivery vehicle also provides a sustained release of the active enzyme into the baked good. That is, following the baking process, active α-amylase is continually released from the protective coating at a rate that counteracts, and therefore reduces the rate of, staling mechanisms.

In general, the amount of lipid applied to the α-amylase particles can vary from a few percent of the total weight of the α-amylase to many times that weight,

depending upon the nature of the lipid, the manner in which it is applied to the α-amylase particles, the composition of the dough mixture to be treated, and the severity of the dough-mixing operation involved.

The loaded delivery vehicle {i.e., the lipid-enveloped enzyme) is added to the ingredients used to prepare a baked good in an effective amount to extend the shelf-life of the baked good. The baker computes the amount of enveloped α- amylase, prepared as discussed above, that will be required to achieve the desired anti-staling effect. The amount of the enveloped alpha-amylase required is calculated based on the concentration of enzyme enveloped and on the proportion of α-amylase to flour specified. A wide range of concentrations has been found to be effective, although, as has been discussed, observable improvements in anti-staling do not correspond linearly with the α-amylase concentration, but above certain minimal levels, large increases in α-amylase concentration produce little additional improvement. The α-amylase concentration actually used in a particular bakery production could be much higher than the minimum necessary in order to provide the baker with some insurance against inadvertent under-measurement errors by the baker. The lower limit of enzyme concentration is determined by the minimum anti-staling effect the baker wishes to achieve.

A typical method of preparing a baked good according to the method comprises: a) preparing lipid-coated alpha-amylase particles, wherein substantially 100 percent of the α-amylase particles are coated; b) mixing a dough containing flour; c) adding the lipid-coated α-amylase to the dough before the mixing is complete and terminating the mixing before the lipid coating is removed from the α-amylase; d) proofing the dough; and e) baking the dough to provide the baked good, wherein the α-amylase is inactive during the mixing, proofing and baking stages and is active in the baked good.

Thus, the enveloped α-amylase can be added to the dough near the end of the mix cycle. A feature of the method is that the enveloped α-amylase is added at a point in the mixing stage that allows sufficient distribution of the enveloped α- amylase though-out the dough, however, the mixing stage is terminated before

the protective coating becomes stripped from the α-amylase particle(s). Depending on the type and volume of dough, and mixer action and speed, anywhere from one to six minutes or more might be required to mix the enveloped α-amylase into the dough, but two to four minutes is average. Thus, there are several variables that may determine the precise procedure. First, the quantity of enveloped α-amylase must have a total volume sufficient to allow the enveloped α-amylase to be spread throughout the dough mix. If the preparation of enveloped alpha-amylase is highly concentrated, additional oil may need to be added to the pre-mix before the enveloped α-amylase is added to the dough. Recipes and production processes may require specific modifications. However, good results generally can be achieved when 25% of the oil specified in a bread dough formula is held out of the dough and is used as a carrier for a concentrated enveloped alpha-amylase when added near the end of the mix cycle. In bread or other baked goods, recipes which have extremely low fat content (such as French-style breads), it has been found that an enveloped α- amylase mixture of approximately 1% of the dry flour weight is sufficient to properly admix the enveloped α-amylase with the dough, but the range of percentages that may work is extremely wide and is dependent on the formula, finished product, and production methodology requirements of the individual baker rather than upon any known limitations. Second, the enveloped α- amylase suspension must be added to the mix with enough time remaining in the mix cycle for complete mixture into the dough, but not so early that excessive mechanical action will strip the protective lipid coating from a large proportion of the enveloped α-amylase particles.

In another embodiment, bacterial α-amylase (BAA) is added to the lipid-coated enzyme particles. BAA is known to reduce bread to a gummy mass due to its excessive thermostability and retained activity in the fully baked loaf of bread. However, it has been found that when BAA is incorporated into the protected enzyme product, substantial additional anti-staling protection is obtained, even at very low BAA dosage levels. For example, BAA dosages of 150 RAU

(Reference Amylase Units) per 100 pounds of flour have been found to be effective. In one aspect, between about 50 to 2000 RAU of BAA is added to the lipid-coated enzyme product. This low BAA dosage level, combined with the

ability of the protective coating to keep enzyme in the fully-baked loaf from free contact with the starches, (except when water vapor randomly releases the enzyme from its coating) helps to achieve very high levels of anti-staling activity without the negative side-effects of BAA.

Improved Handling Properties

The AmyTS-23 alpha amylase described here preferably provides one or more improved handling properties compared to the use without the AmyTS-23 alpha amylase. The improved handling properties may in preferred embodiments comprise improved baking properties.

The AmyTS-23 alpha amylase optionally used in combination with a further amylase described here preferably provides one or more improved handling properties compared to the use without the AmyTS-23 alpha amylase. In a further aspect, the AmyTS-23 alpha amylase when used in combination with a further amylase described here preferably provides one or more improved handling properties compared to the use without the further amylase. The improved handling properties may in preferred embodiments comprise improved baking properties.

Thus, the AmyTS-23 alpha amylase when used optionally in combination with a further amylase are such that a food product treated with either the AmyTS-23 alpha amylase or the combination has an improved handling or preferably baking property compared to a food product which has not been treated with the AmyTS-23 alpha amylase or with the further amylase alone. The handling or baking property may be selected from the group consisting of: firmness, resilience, cohesiveness, crumbliness and foldability.

These handling properties may be tested by any means known in the art. For example, firmness, resilience and cohesiveness may be determined by analysing bread slices by Texture Profile Analysis using for example a Texture Analyser, as described in the Examples.

Firmness

The AmyTS-23 alpha amylase are preferably such that a food product treated with the AmyTS-23 alpha amylase optionally in combination with a further amylase described here has lower firmness compared to a food product which has not been treated with the AmyTS-23 alpha amylase.

The firmness is in preferred embodiments inversely correlated with the softness of the food product; thus, a higher softness may reflect a lower firmness, and vice versa.

Firmness is preferably measured by the "Firmness Evaluation Protocol" set out in Example 8.

Thus, the AmyTS-23 alpha amylase when used optionally in combination with a further amylase described here are preferably such that a food product treated has a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more lower firmness compared to a food product which has not been treated with the AmyTS-23 alpha amylase. A food product treated with the AmyTS-23 alpha amylase optionally in combination with a further amylase described here may have a l .lx, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x or more lower firmness compared to a food product which has not been treated with the AmyTS-23 alpha amylase.

Resilience

The AmyTS-23 alpha amylase are preferably such that a food product treated with the AmyTS-23 alpha amylase optionally in combination with a further amylase described here has a higher resilience compared to a food product which has not been treated with the AmyTS-23 alpha amylase.

Resilience is preferably measured by the "Resilience Evaluation Protocol" set out in Example 9.

Thus, the AmyTS-23 alpha amylase are preferably such that a food product treated with the AmyTS-23 alpha amylase optionally in combination with a further amylase described here has a 10%, 20%, 30%, 40%, 50%, 60%, 70%,

80%, 90%, 100%, 200% or more higher resilience compared to a food product which has not been treated with the AmyTS-23 alpha amylase. A food product treated with the AmyTS-23 alpha amylase optionally in combination with a further amylase described here may have a l. lx, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 1Ox or more higher resilience compared to a food product which has not been treated with the AmyTS-23 alpha amylase.

Cohesiveness

The AmyTS-23 alpha amylase are preferably such that a food product treated with the AmyTS-23 alpha amylase optionally in combination with a further amylase described here has a higher cohesiveness compared to a food product which has not been treated with the AmyTS-23 alpha amylase.

Cohesiveness is preferably measured by the "Cohesiveness Evaluation Protocol" set out in Example 10.

Thus, the AmyTS-23 alpha amylase are preferably such that a food product treated with the AmyTS-23 alpha amylase optionally in combination with a further amylase described here has a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more higher cohesiveness compared to a food product which has not been treated with the AmyTS-23 alpha amylase. A food product treated with the AmyTS-23 alpha amylase optionally in combination with a further amylase described here may have a l. lx, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x or more higher cohesiveness compared to a food product which has not been treated with the AmyTS-23 alpha amylase.

Crumbliness

The AmyTS-23 alpha amylase are preferably such that a food product treated with the AmyTS-23 alpha amylase optionally in combination with a further amylase described here has a lower crumbliness compared to a food product which has not been treated with the AmyTS-23 alpha amylase.

Crumbliness is preferably measured by the "Crumbliness Evaluation Protocol" set out in Example 11.

Thus, the AmyTS-23 alpha amylase are preferably such that a food product treated with the AmyTS-23 alpha amylase optionally in combination with a further amylase described here has a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more lower crumbliness compared to a food product which has not been treated with the AmyTS-23 alpha amylase. A food product treated with the AmyTS-23 alpha amylase optionally in combination with a further amylase described here may have a l.lx, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 1Ox or more lower crumbliness compared to a food product which has not been treated with the AmyTS-23 alpha amylase.

Foldability

The AmyTS-23 alpha amylase are preferably such that a food product treated with the AmyTS-23 alpha amylase optionally in combination with a further amylase described here has a higher foldability compared to a food product which has not been treated with the AmyTS-23 alpha amylase.

Foldability is preferably measured by the "Foldability Evaluation Protocol" set out in Example 12.

Thus, the AmyTS-23 alpha amylase are preferably such that a food product treated with the AmyTS-23 alpha amylase optionally in combination with a further amylase described here has a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more higher foldability compared to a food product which has not been treated with the AmyTS-23 alpha amylase. A food product treated with the AmyTS-23 alpha amylase optionally in combination with a further amylase described here may have a l.lx, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x or more higher foldability compared to a food product which has not been treated with the AmyTS-23 alpha amylase.

More specifically the use of the AmyTS-23 alpha amylase optionally when used in combination with a further amylase described here in combination with a xylanase provides for improving fodability.

5. Methods

5.1 Filter Screening Assays

The assays discussed below may be used in the screening of AmyTS-23 alpha- amylase variants having altered stability at high or low pH and/or under Ca ²⁺ depleted conditions compared to the parent alpha-amylase enzyme.

5.2 High pH Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany)--and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates with 10 micro g/ml kanamycin at 37 ⁰ C for at least 21 hours. The cellulose acetate layer is located on the TY agar plate.

Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with glycin- NaOH buffer, pH 8.6-10.6 and incubated at room temperature (can be altered from 10-60 ⁰ C) for 15 min. The cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on plates containing 1% agarose, 0.2% starch in glycin- NaOH buffer, pH 8.6-10.6. The assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours at room temperature. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are rescreened twice under the same conditions as the first screen.

5.3 Low Calcium Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany)--and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates with a relevant antibiotic, e.g., kanamycin or chloramphenicol, at 37 ⁰ C for at least 21 hours. The cellulose-acetate layer is located on the TY agar plate.

Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with carbonate/bicarbonate buffer pH 8.5-10 and with different EDTA concentrations (0.001 mM-100 mM). The filters are incubated at room temperature for 1 hour. The cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on plates containing 1% agarose, 0.2% starch in carbonate/bicarbonate buffer pH 8.5-10. The assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours at room temperature. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are rescreened twice under the same conditions as the first screen.

5.4 Low pH Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany)--and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dasseli Germany) on TY agar plates with 10 micro g/ml chloramphenicol at 37 ⁰ C for at least 21 hours. The cellulose acetate layer is located on the TY agar plate.

Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter, and the nitrocellulose filter with bound variants is transferred to a container with citrate

buffer, pH 4.5 and incubated at 8O ⁰ C for 20 minutes (when screening for variants in the wild type backbone) or 85 ⁰ C for 60 minutes (when screening for variants of the parent alpha-amylase). The cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on assay plates containing 1% agarose, 0.2% starch in citrate buffer, pH 6.0. The assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours at 5O ⁰ C. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are re-screened twice under the same conditions as the first screen.

5.5 Secondary Screening

Positive transformants after rescreening are picked from the storage plate and tested in a secondary plate assay. Positive transformants are grown for 22 hours at 37 ⁰ C in 5 ml LB+chloramphenicol. The Bacillus culture of each positive transformant and as a control a clone expressing the corresponding backbone are incubated in citrate buffer, pH 4.5 at 9O ⁰ C and samples are taken at 0, 10, 20, 30, 40, 60 and 80 minutes. A 3 micro liter sample is spotted on an assay plate. The assay plate is stained with 10% Lugol solution. Improved variants are seen as variants with higher residual activity (detected as halos on the assay plate) than the backbone. The improved variants are determined by nucleotide sequencing.

5.6 Stability Assay of Unpurified Variants

The stability of the variants may be assayed as follows: Bacillus cultures expressing the variants to be analyzed are grown for 21 hours at 37 ⁰ C in 10 ml LB+chloramphenicol. 800 micro liter culture is mixed with 200 microliters citrate buffer, pH 4.5. A number of 70 microliter aliquots corresponding to the number of sample time points are made in PCR tubes and incubated at 7O ⁰ C or 9O ⁰ C for various time points (typically 5, 10, 15, 20, 25 and 30 minutes) in a PCR machine. The 0 min sample is not incubated at high temperature. Activity in the

sample is measured by transferring 20 microliter to 200 microliter of the alpha- amylase PNP-G ₇ substrate MPR3 ((Boehringer Mannheim Cat. no. 1660730) as described below under "Assays for Alpha-Amylase Activity". Results are plotted as percentage activity (relative to the 0 time point) versus time, or stated as percentage residual activity after incubation for a certain period of time.

5.7 Fermentation and Purification of Alpha-Amylase Variants

A B. subtilis strain harboring the relevant expression plasmid may be fermented and purified as follows: The strain is streaked on a LB-agar plate with 10 micro g/ml kanamycin from -8O ⁰ C stock, and grown overnight at 37 ⁰ C. The colonies are transferred to 100 ml PS-I media supplemented with 10 micro g/ml chloramphinicol in a 500 ml shaking flask.

Composition of PS-I medium

Pearl sugar 100 g/l

Soy Bean Meal 40 g/l Na ₂ HPO ₄ , 12 H ₂ O 10 g/l

Pluronic ™ PE 6100 0.1 g/l

CaCO ₃ 5 g/l

The culture is shaken at 37 ⁰ C at 270 rpm for 5 days.

Cells and cell debris are removed from the fermentation broth by centrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatant is filtered to obtain a completely clear solution. The filtrate is concentrated and washed on a UF-filter (10000 cut off membrane) and the buffer is changed to 20 mM Acetate pH 5.5. The UF-filtrate is applied on a S-sepharose F. F. and elution is carried out by step elution with 0.2M NaCI in the same buffer. The eluate is dialysed against 10 mM Tris, pH 9.0 and applied on a Q-sepharose F. F. and eluted with a linear gradient from 0-0.3M NaCI over 6 column volumes. The fractions that contain the activity (measured by the Phadebas assay) are pooled, pH was adjusted to pH 7.5 and

remaining color was removed by a treatment with 0.5% W/vol. active charcoal in 5 minutes.

5.8 Specific Activity Determination

The specific activity is determined using the Phadebas® assay (Pharmacia) as activity/mg enzyme. The manufacturer's instructions are followed (see also below under "Assay for Alpha-Amylase Activity").

5.9 Determination of Isoelectric Point

The pi is determined by isoelectric focusing (ex: Pharmacia, Ampholine, pH 3.5- 9.3).

5.10 Accelerated Stability Assay

In 50 ml Propylene tubes, 10 ml of detergent of interest was added. Appropriate dilution was made to both AmyTS-23t and AmyTS-23tδRS so that 180 ppm of each was measured with a pippette into separate tubes containing the detergent. The detergent with each mutant enzyme was vortex for 30 sec and then placed on a RotaMix (ATR RKVS Model) for 10 minutes. 100 micro-liters of the detergent with the mutant enzyme were measured with a pipette and diluted 1 :651. The initial activity of the mutants was assayed using Blocked P-Nitro- Phenyl-Maltoheptaose (Blocked PBNPG7) substrate on a Konelab, Model 20XT. The detergent samples were then incubated in a constant temperature incubator set at 37 ⁰ C. Samples were removed at 1, 2, 4, 7 and 17 days and the enzyme activity assayed.

5.11 Assays for Alpha-Amylase Activity

In some embodiments, the AmyTS-23 alpha-amylase used according to the invention is an AmyTS-23 alpha-amylase variant having alpha-amylase activity. As used herein, the phrase "AmyTS-23 alpha-amylase variant having alpha-

amylase activity" refers to an AmyTS-23 alpha-amylase that has alpha-amylase activity as measured in any Assays for Alpha-Amylase Activity as described herein.

5.11.1 Phadebas A Assay

Alpha-amylase activity is determined by a method employing Phadebas® tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, supplied by Pharmacia Diagnostic) contain a cross-linked insoluble blue-colored starch polymer, which has been mixed with bovine serum albumin and a buffer substance and tabletted.

For every single measurement one tablet is suspended in a tube containing 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mM phosphoric add, 50 mM boric acid, 0.1 mM CaCI ₂ , pH adjusted to the value of interest with NaOH). The test is performed in a water bath at the temperature of interest. The alpha- amylase to be tested is diluted in x ml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylase solution is added to the 5 ml 50 mM Britton-Robinson buffer. The starch is hydrolyzed by the alpha-amylase giving soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of the alpha-amylase activity.

It is important that the measured 620 nm absorbance after 10 or 15 minutes of incubation (testing time) is in the range of 0.2 to 2.0 absorbance units at 620 nm. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law). The dilution of the enzyme must therefore be adjusted to fit this criterion. Under a specified set of conditions (temp., pH, reaction time, buffer conditions) 1 mg of a given alpha-amylase will hydrolyze a certain amount of substrate and a blue color will be produced. The color intensity is measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity/mg of pure alpha-amylase protein) of the alpha- amylase in question under the given set of conditions.

5.11.2 Alternative Method

Alpha-amylase activity may be determined by a method employing the PNP-G ₇ substrate. PNP-G ₇ which is a abbreviation for p-nitrophenyl-alpha-D- maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo- amylase. Following the cleavage, the alpha-glucosidase included in the kit digest the substrate to liberate a free PNP molecule which has a yellow color and thus can be measured by visible spectophometry at λ=405 nm (400-420 nm). Kits containing PNP-G ₇ substrate and alpha-glucosidase are manufactured by Boehringer-Mannheim (cat. No.1054635).

To prepare the reagent solution 10 ml of substrate/buffer solution is added to 50 ml enzyme/buffer solution as recommended by the manufacturer. The assay is performed by transferring a 20 microliter sample to a 96 well microtitre plate and incubating at 25 ⁰ C. 200 microliter reagent solution pre-equilibrated to 25 ⁰ C is added. The solution is mixed and pre-incubated 1 minute and absorption is measured every 30 seconds over 4 minutes at OD 405 nm in an ELISA reader.

The slope of the time dependent absorption-curve is directly proportional to the activity of the alpha-amylase in question under the given set of conditions.

5.12

It is known that some non-maltogenic exoamylases can have some degree of endoamylase activity. In some cases, this type of activity may need to be reduced or eliminated since endoamylase activity can possibly negatively effect the quality of the food product such as a final bread product by producing a sticky or gummy crumb due to the accumulation of branched dextrins.

Exo-specificity can usefully be measured by determining the ratio of total amylase activity to the total endoamylase activity. This ratio is referred to in this document as a "Exo-specificity index". In preferred embodiments, an enzyme is considered an exoamylase if it has a exo-specificity index of 20 or more, i.e., its total amylase activity (including exo-amylase activity) is 20 times or more greater than its endoamylase activity. In highly preferred embodiments, the

exo-specificity index of exoamylases is 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more. In highly preferred embodiments, the exo-specificity index is 150 or more, 200 or more, 300 or more, 400 or more, 500 or more or 600 or more.

The total amylase activity and the endoamylase activity may be measured by any means known in the art. For example, the total amylase activity may be measured by assaying the total number of reducing ends released from a starch substrate. Alternatively, the use of a Betamyl assay is described in further detail in the Examples, and for convenience, amylase activity as assayed in the Examples is described in terms of "Betamyl Units".

Endoamylase activity may be assayed by use of a Phadebas Kit (Pharmacia and Upjohn). This makes use of a blue labelled crosslinked starch (labelled with an azo dye); only internal cuts in the starch molecule release label, while external cuts do not do so. Release of dye may be measured by spectrophotometry. Accordingly, the Phadebas Kit measures endoamylase activity, and for convenience, the results of such an assay (described in the Examples) are referred to in this document as "Phadebas B units".

In a highly preferred embodiment, therefore, the exo-specificity index is expressed in terms of Betamyl Units / Phadebas B Units, also referred to as "B/Phad".

Exo-specificity may also be assayed according to the methods described in the prior art, for example, in our International Patent Publication Number WO99/50399. This measures exo-specificity by way of a ratio between the endoamylase activity to the exoamylase activity. Thus, in a preferred aspect, the anti-staling amylases described here will have less than 0.5 endoamylase units (EAU) per unit of exoamylase activity. Preferably the non-maltogenic exoamylases which are suitable for use according to the present invention have less than 0.05 EAU per unit of exoamylase activity and more preferably less than 0.01 EAU per unit of exoamylase activity.

The anti-staling amylases described here will preferably have exospecificity, for example measured by exo-specificity indices, as described above, consistent with their being exoamylases.

Specific embodiments of the invention:

As described above the present invention relates to a method of preparing a food product, comprising incorporating into the food product an AmyTS-23 alpha-amylase, as well as a composition comprising an AmyTS-23 alpha- amylase and optionally a further amylase such as an anti-staling amylase.

In some embodiments, the AmyTS-23 alpha-amylase is an AmyTS-23 alpha- amylase variant having at least 80% identity to SEQ ID NO 1.

In some embodiments, the AmyTS-23 alpha-amylase is an AmyTS-23 alpha- amylase variant comprising at least two of the following :

(a) a truncation of the C-terminus,

(b) a substitution of residue 201, or

(c) a deletion of residues R180 and S181,

wherein said amino acid residues refer to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the AmyTS-23 alpha-amylase is an AmyTS-23 alpha- amylase variant having alpha-amylase activity.

In some embodiments, the AmyTS-23 alpha-amylase is an AmyTS-23 alpha- amylase variant having at least 90%, such as at least 95% identity to SEQ ID NO: 1.

In some embodiments, the AmyTS-23 alpha-amylase is a AmyTS-23 alpha- amylase variant further comprising a substitution at one or more residues

selected from the group consisting of residue 87, residue 225, residue 272, and residue 282 wherein said residue refer to the amino acid residue at a position corresponding to the identical position of SEQ ID NO: 1.

In some embodiments, the AmyTS-23 alpha-amylase is an AmyTS-23 alpha- amylase variant having a higher thermostability compared to the parent TS-23 alpha-amylase having the amino acid sequence of SEQ ID NO: 1, when tested under the same conditions.

In some embodiments, the AmyTS-23 alpha-amylase is a parent AmyTS-23 alpha-amylase having the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the further amylase is an exo-amylase.

In some embodiments, the further amylase is selected from the group consisting of a Bacillus stearothermophilus having SEQ ID No 10 or a variant, homologue, or mutant thereof and a Pseudomonas saccharophila (PS4) variant polypeptide.

In some embodiments, the further amylase is an exo-amylase.

In some embodiments, the further amylase is a non-maltogenic exo-amylase.

In some embodiments, the further amylase is a EC 3.2.1.60 amylase.

In some embodiments, the further amylase is a maltogenic exo-amylase.

In some embodiments, the further amylase is a EC 3.2.1.133 amylase.

In some embodiments, the the further amylase has at least 60% sequence identity to the amino acid sequence of SEQ ID No:8.

In some embodiments, the further amylase comprises the amino acid sequence of SEQ ID No: 7.

In some embodiments, the further amylase has the amino acid sequence of SEQ ID No: 7.

In some embodiments, the further amylase comprises the amino acid sequence of SEQ ID No: 7 without the M in position 1.

In some embodiments, the further amylase has the amino acid sequence of SEQ ID No: 7 without the M in position 1.

In some embodiments, the further amylase compared to SEQ ID No:8 comprises a substitution at one or more residues selected from the group consisting of residue 33, 34, 70, 121, 134, 141, 146, 157, 161, 178, 179, 223, 229, 272, 303, 307, 309 and 334 wherein said amino acid residues refer to the amino acid residue at a position corresponding to the identical position of SEQ ID NO:8.

In some embodiments, the further amylase compared to SEQ ID No:8 comprises a substitution at one or more residues selected from the group consisting of residue 121, 134, 141, 146, 157, 161, 178, 179, 223, 229, 307, 309 and 334 wherein said amino acid residues refer to the amino acid residue at a position corresponding to the identical position of SEQ ID NO:8.

In some embodiments, the one or more substitutions are selected from the group consisting 33Y, 34N, 7OD, 7OK, 121F, 121Y, 121D, 134R, 141P, 146G, 157L, 157M, 161A, 178F, 179T, 223A, 223E, 223S, 229P, 272Q, 303E, 307K, 307R, 309P and 334P wherein said amino acid residues refer to the amino acid residue at a position corresponding to the identical position of SEQ ID NO:8.

In some embodiments, the one or more substitutions are selected from the group consisting 121F, 134R, 141P, 146G, 157L, 161A, 178F, 179T, 223A, 223E, 223S, 229P, 307K, 309P and 334P, wherein said amino acid residues refer to the amino acid residue at a position corresponding to the identical position of SEQ ID NO:8.

In some embodiments, the further amylase has a higher thermostability compared to wild-type PS4 having SEQ ID N0:9 when tested under the same conditions.

In some embodiments, the further amylase has a half life (tl/2), which is increased by 15% or more, preferably by 50% or more, most preferably by 100% or more, relative to wild-type PS4 having SEQ ID NO:9 when measured under same conditions, preferably at a temperature of 60 degrees C.

In some embodiments, the food product is a dough.

In some embodiments, the food product comprises a dough or a dough product, preferably a processed dough product.

In some embodiments, the food product according to the invention is a bakery product.

In some embodiments, the dough is used for preparing a baked product.

In some embodiments, the AmyTS-23 alpha-amylase has anti-staling amylase activity.

In some embodiments, the methods according to the invention further comprises incorporating into the food product a further amylase such as an anti- staling amylase.

In some embodiments, the further amylase used according to the invention is an anti-staling amylase.

In some embodiments, a baked product is prepared from the dough and the obtained baked product has any one or more, preferably all of the following properties: (a) lower firmness; (b) higher resilience; (c) higher cohesiveness; (d) lower crumbliness; and (e) higher foldability compared to a baked product which has not been treated with the AmyTS-23 alpha-amylase.

In some embodiments, a baked product is prepared from the dough and the obtained baked product has lower firmness compared to a baked product which has not been treated with the AmyTS-23 alpha-amylase.

In some embodiments, a baked product is prepared from the dough and the obtained baked product has higher resilience or higher cohesiveness compared to a baked product which has not been treated with the AmyTS-23 alpha- amylase.

In some embodiments, a baked product is prepared from the dough and the resilience, cohesiveness or foldability of the obtained baked product is independently increased by 15% or more, preferably by 50% or more, most preferably by 100% or more, relative to a baked product which has not been treated with the AmyTS-23 alpha-amylase.

In some embodiments, a baked product is prepared from the dough and each of resilience, cohesiveness and foldability of the obtained baked product is increased compared to a baked product which has not been treated with the AmyTS-23 alpha-amylase.

In some embodiments, a baked product is prepared from the dough and the firmness or the crumbliness of the obtained baked product is independently decreased by 15% or more, preferably by 50% or more, most preferably by 100% or more, relative to a baked product which has not been treated with the AmyTS-23 alpha-amylase.

In some embodiments, a baked product is prepared from the dough and each of the firmness and crumblines of the obtained baked product treated is decreased compared to a baked product which has not been treated with the AmyTS-23 alpha-amylase.

In some embodiments, the preparation according to the invention further comprises a lipase.

In some embodiments, the preparation according to the invention further comprises a hemicellulase, preferably a pentosanase, more preferably a xylanase.

In some embodiments, the preparation according to the invention is a granulate or an agglomerated powder.

EXAMPLE 1

Expression of AmyTS-23 in B. subtilis

To test expression of AmyTS-23 full length, the synthetic DNA sequence depicted in Fig. 3 (made by Geneart, Regensburg, Germany) was cloned behind the LAT (licheniformis amylase) promoter and fused in frame to a sequence encoding the LAT signal peptide (Fig. 5) into vector pHPLT (see e.g. WO2005111203 and [Solingen et al. (2001) Extremophiles 5:333-341]) and transformed into a 9 protease deleted B. subtilis strain (degU ^Hy 32,oppA, δspoII3501, amyE: :xylRPxylAcomK- ermC, δaprE, δnprE, δepr, δispA, δbpr, δvpr, δwprA, δmpr-ybfJ, δnprB) (see US20050202535A1). Neomycin (10 μg/ml) resistant transformants secrete AmyTS-23 amylase as judged by halo formation on starch plates after iodine staining (see WO2005111203). One of these amylase positive transformants was selected and designated BG6006 (pHPLT-AmyTS-23). Cultures of this strain were typically grown at 37 deg for 60 to 72 hours at 250 rpm in the following medium (per liter): 10 g Soytone, 75 g glucose, 7.2 g urea, 40 mM MOPS, 4 mM Tricine, 3 mM dibasic potassium phosphate, 21.4 mM KOH, 50 mM NaCI, 276 μM potassium sulfate, 528 μM magnesium chloride, 50 μM trisodium citrate dihydrate, 100 μM calcium chloride dihydrate, 14 μM ferrous sulfate heptahydrate, 5.9 μM manganese sulfate dihydrate, 5.7 μM zinc sulfate monohydrate, 2.9 μM cupric chloride dihydrate, 4.2 μM cobalt chloride hexahydrate, 4.5 μM sodium molybdate dihydrate. For a IL volume, all components except for Soytone were mixed in 500 mL, sterile filtered, and added to an equal part of 2X Soytone, which had been sterilized by autoclaving. Trace metals and citrate can be made up as a IOOX or IOOOX stock solutions. Buffers, potassium hydroxide, sodium chloride, potassium sulfate,

and magnesium chloride and trace metals can be made up as a 1OX stock solutions. After all components were mixed, the pH was adjusted to 7.3. Prior to use this medium was supplemented with 20 mM calcium chloride.

The culture expressed the amylase in two major forms. A high molecular weight form was observed at the 66 kDa marker on a 10% SDS-PAGE gel. A shorter form was observed at 55 kDa.

The high molecular weight component was isolated from the culture broth by treating 500 ml_ of the broth with 10 ml_ settled volume of β-cyclodextrin- sepharose affinity matrix resin, synthesized in-house by standard protocol from β-cyclodextrin (Sigma Aldrich Cat. No. c4767) and epoxy-activated-sepharose- 6B (GE Healthcare, NJ. Cat. No. 17-0480-01), over night at 4°C with gentle agitation, collecting the resin, and washing with 25 mM bis-Tris propane buffer (pH 8.5) containing 2 mM calcium chloride (CaCI ₂ ) The high molecular weight enzyme was eluted by washing the resin with the same buffer supplemented with 50 mM β-cyclodextrin. Fractions were analyzed by SDS-PAGE and those containing enzyme were pooled and dialyzed to remove β -cyclodextrin. Enzyme protein concentration was estimated by gel densitometry with OxAm amylase (Genencor) serving as the protein standard.

EXAMPLE 2

Expression of AmyTS-23t in B. subtilis

To test expression of genetically truncated AmyTS-23 (AmyTS-23t) the synthetic DNA fragment depicted in Figure 4 was cloned into pHPLT and transformed into the 9 protease deleted B. subtilis strain as described in Example 1. Neomycin resistant transformants secrete AmyTS-23t amylase as judged by halo formation on starch plates after iodine staining. One of these amylase positive transformants was selected and designated BG6006(pME622.1). This strain was cultured to produce AmyTS-23t amylase as described in example 1. Culture supernatant was examined by SDS-PAGE and shown to produce a product of the expected size of 55 kDa.

The amylase protein was partially purified by the addition of NH ₄ SO ₄ to 500 ml_ of culture to a final concentration of IM. Next, 10 ml_ settled volume of Phenyl- sepharose resin was added and the mixture was gently agitated overnight at 4 deg C. The resin was collected and washed with 25 mM bis-Tris propane buffer (pH 8.5) containing IM NH ₄ SO ₄ and 2 mM calcium chloride (CaCI ₂ ). Enzyme activity was eluted in the same buffer without NH ₄ SO ₄ . Fractions were analyzed by SDS-PAGE and those containing enzyme were pooled and dialyzed to remove residual NH ₄ SO ₄ . Enzyme protein concentration was estimated by gel densitometry with OxAm amylase (Genencor International, Inc.) serving as the protein standard.

EXAMPLE 3

Expression of AmyTS-23 variants in B. subtilis

In this example, the construction of Bacillus subtilis strains expressing variants of AmyTS-23t is described. Synthetic DNA fragment 056426 (produced by Geneart GmbH, Josef- Engert-strasse 11, D-93053 Regensburg, Germany), containing the codon optimized AmyTS-23 gene (Figure 3) served as template DNA. The pHPLT vector (Solingen et al., Extremophiles 5: 333-341 [2001]) which contains the Bacillus licheniformis alpha-amylase (LAT) promoter and the LAT signal peptide (pre LAT) followed by Pstl and Hpal restriction sites for cloning, was used for expression of the AmyTS-23t variants.

Three DNA fragments were produced by PCR using the DNA primers listed below:

1. AmyTS-23t with CGG of codon 180 and AGC of codon 181 deleted (AmyTS-23tδRS)

2. AmyTS-23t with ATG of codon 201 replaced by CTG (AmyTS-23t(M201L))

3. AmyTS-23t with both ATG of codon 201 replaced by CTG, and CGG of codon 180 and AGC of codon 181 deleted (AmyTS-23t(M201L + δRS)

Primer name DNA sequence pHPLT-Pstl-FW CTCATTCTGCAGCTTCAGCAAATACGGCG p H PLT- H pa I - RV CTCTGTTAACTCATTTGGCG ACCCAGATTGAAACG

TS-delRS-FW CTATAAATTTACGGGCAAAGCATGGGATTGG

TS-delRS-RV TGCTTTGCCCGTAAATTTATAGATCCGGTTCAG

TS-M201L-FW CTATGACTATCTGCTGTTTGCCGATCTG

TS- M 20 IL- RV CAGATCGGCAAACAGCAGATAGTCATAG

TS-delRS/M201L-

FW GCATGGGATTGGGAAGTCGATACGGAAAACGGCAACTATGACTATCTGCTGTTTGCCG

TS-delRS/M201L-

RV CGTATCGACTTCCCAATCCCATGCTTTGCCCGTAAATTTATAGATCCGGTTC

These DNA primers were synthesized and desalted by Sigma (Sigma-Aldrich Chemie B. V., Postbus 27, 3330 AA Zwijndrecht, The Netherlands).

For all the PCR reactions described below, a final concentration of 0.2 μM DNA primer was used (forward and reverse primer), and 0.1 - 10 ng of DNA template was used (DNA fragment 056426 or pDNA pHPLT). In addition, all PCR reactions were completed in a volume of 50 μl, using Finnzymes (Finnzymes OY, Keilaranta 16 A, 02150 Espoo, Finland) Phusion High-Fidelity DNA Polymerase (Cat. no. F-530L). Also, all PCR reaction mixes contained 10 μl_ of 5 x Phusion HF buffer, 1 μl_ of 10 mM dNTP mixture, 0.75 μl_ of Phusion DNA polymerase (2 units/ μl_), 1 μl_ of 100% DMSO and deionized, autoclaved water making up a final volume of 50 μl. The PCR programs, using a MJ Research PTC-200 Peltier thermal cycler (MJ Research, 590 Lincoln Street, Waltham, MA 02451, USA) were run as described by Finnzymes (protocol of manufacturer) : 30 sec. at 98°C, 3Ox(IO sec. at 98°C, 20 sec. at 55°C, 22 sec./kb at 72°C), 5 min.72°C.

1. Generation of AmyTS-23tδRS:

Two PCR reactions were performed using primers TS-delRS-FW and pHPLT-Hpal- RV on synthetic DNA fragment 056426 and primers TS-delRS-RV and pHPLT- Pstl-FW on synthetic DNA fragment 056426. In order to fuse these two generated DNA fragments, 1 μl unpurified PCR mix from both reactions was

added to a third PCR reaction sample in which primers pHPLT-Pstl-FW and pHPLT-Hpal-RV were added.

The amplified linear 1.5 kb DNA fragment was purified (using Qiagen ^® Qiaquick PCR purification kit Cat. no. 28106) and digested with Pstl and Hpal restriction enzymes. Subsequently, the AmyTS-23tδRS (also referred to herein as AmyTS- 23tδRS) DNA fragment and pHPLT pDNA (50 ng/μl range, digested with Pstl and Hpal zymes) were both purified (using Qiagen ^® Qiaquick PCR purification kit Cat. no. 28106) and then ligated at the Pstl and Hpal ends. Reaction conditions are:

4 μl of purified and, Pstl and Hpal digest of the AmyTS-23tδRS DNA fragment, 2 μl of purified and, Pstl and Hpal digested pHPLT DNA fragment, 8 μl_ T4 DNA

Ligase buffer (Invitrogen ^® Cat. no. 46300-018), 25 μl distilled, autoclaved water and 1 μl_ T4 DNA Ligase, 1 unit/μL (Invitrogen ^® Cat. no. 15224-017). Ligation reaction took place for 16-20 hours at 20 ⁰ C.

Subsequently, the ligation mixture was transformed into a B. subtilis strain (δaprE, δnprE, δepr, δispA, δbpr) and (degU ^Hy 32, oppA, δspoIIE3501, amyEwxylRPxylAcomK-ermC, {δvpr, δwprA,δmpr-ybfJ, δnprB). Transformation into B. subtilis was performed as described in WO 02/14490. The B. subtilis transformants were selected on agar plates containing Heart infusion agar (Difco, Cat.no 244400) and 10 mg/L Neomycin. Selective growth of B. subtilis transformants harboring the pHPLT- AmyTS-23tδRS vector was performed in shake flasks as described in Example 1. This growth resulted in the production of secreted AmyTS-23tδRS amylase with starch hydrolyzing activity as visualized by spotting culture supernatant on a starch agar plate followed by iodine staining.

2. Generation of AmyTS-23t(M201L):

The same protocol was performed as described for the "Generation of AmyTS- 23tδRS", except for the first two PCR reactions:

Two PCR reactions were performed using primers TS-M201L-FW and pHPLT- Hpal-RV on synthetic DNA fragment 056426 and primers TS-M201L-RV and pHPLT-Pstl-FW on synthetic DNA fragment 056426.

3. Generation of AmyTS-23t(M201L)-RSdelete:

The same protocol was performed as described for the "Generation of AmyTS- 23tδRS", except for the first two PCR reactions:

Two PCR reactions were performed using primers TS-delRS/M201L-FW and pHPLT-Hpal-RV on synthetic DNA fragment 056426 and primers TS- delRS/M201L-RV and pHPLT-Pstl-FW on synthetic DNA fragment 056426.

EXAMPLE 4

Amylase Production in B. subtilis

In this Example, production of Bacillus sp. TS-23t and variants thereof in B. subtilis are described. Transformation was performed as known in the art (See e.g., WO 02/14490). Briefly, the gene encoding the parent amylases was cloned into the pHPLT expression vector, which contains the LAT promoter (PLAT), a sequence encoding the LAT signal peptide (preLAT), followed by Pstl and Hpal restriction sites for cloning.

The coding region for the LAT signal peptide is shown below:

atgaaacaacaaaaacggctttacgcccgattgctgacgctgttatttgcgctcatc ttcttgctgcctcattctgca gcttcagca (SEQ ID NO:5).

The amino acid sequence of the LAT signal peptide is shown below:

MKQQKRLYARLLTLLFALIFLLPHSAASA (SEQ ID NO:6)

The coding region for the mature AmyTS-23t amylase is shown in Figure 4.

The amino acid sequence of the mature AmyTS-23t amylase was used as the basis for making the variant libraries described herein is shown in Figure 2.

The PCR products were purified using Qiaquik columns from Qiagen, and resuspended in 50μl_ of deionized water. 50μl_ of the purified DNA was digested with Hpal (Roche) and Pstl (Roche) and the resultant DNA resuspended in 30μl_ of deionized water. 10-20ng/μl_ of the DNA was cloned into plasmid pHPLT using Pstl and Hpal cloning sites. The ligation mixtures were directly transformed into competent B. subtilis cells (genotype: δvpr, δwprA, δmpr-ybfJ, δnprB). The B. subtilis cells have a competency gene (comK) which is placed under a xylose inducible promoter, so xylose was used to induce competency for DNA binding and uptake (see Hahn et al., MoI. Microbiol., 21 :763-775 [1996]).

The elements of plasmid pHPLT-AmyS include: pUBllO = DNA fragment from plasmid pUBllO (McKenzie et al., Plasmid 15: 93-103 [1986]). Plasmid features include: ori-pUB110 = origin of replication from pUBllO, neo = neomycin resistance gene from pUBllO, Plat = transcriptional promoter from B. licheniformis amylase, Pre LAT = signal peptide from B. licheniformis amylase, SAMY 425ss = The coding region for truncated Amy TS-23 gene sequence (replaced by the coding regions for each truncated Amy TS-23 variant expressed in this study), Terminator = transcriptional terminator from B. licheniformis amylase.

Amylase Expression - 2ml scale

B. subtilis clones containing AmyTS-23t expression vectors were replicated with a steel 96-well replicator from glycerol stocks into 96-well culture plates (BD, 353075) containing 150 μl of LB media + 10 μg/ml neomycin, grown overnight at 37°C, 220 rpm in a humidified enclosure. A 100 μl aliquot from the overnight culture was used to inoculate 2000 μl defined media + lOμg/ml neomycin in 5ml plastic culture tubes. The cultivation media was an enriched semi-defined media based on MOPs buffer, with urea as major nitrogen source, glucose as the main carbon source, and supplemented with 1% soytone and 5 mM calcium for robust cell growth. Culture tubes were incubated at 37°C, 250 rpm, for 72 hours.

Following this incubation, the culture broths were centrifuged for lOminutes at 3000 x g. The supernatant solution was decanted into 15ml polypropylene conical tubes and 80 μl_ of each sample were aliquoted into 96 well plates for protein quantitation.

Generation of Bacillus sp. AmyTS-23t Combinatorial Charge Library

Multiple protein variants spanning a range of a physical properties of interest are selected from existing libraries or are generated by site-directed mutagenesis techniques as known in the art (See e.g., US Pat. Appln. Ser. Nos., 10/576,331, 11/581,102, and 11/583,334). This defined set of probe proteins is then assayed in a test of interest.

AmyTS-23t is a truncated form of Bacillus sp. TS-23 alpha amylase (see Lin et al., 1998, Production and properties of a raw-starch-degrading amylase from the thermophilic and alkaliphilic Bacillus sp. TS-23, Biotechnol. Appl. Biochem. 28:61-68). Expression of AmyTS-23t in a multiple-protease deleted B. subtilis strain (degU ^Hy 32, oppA, δspoII3501, amyE: :xylRPxylAcomK- ermC, δaprE, δnprE, δepr, δispA, δbpr, δvpr, δwprA, δmpr-ybfJ, δnprB) (See, e.g., US2005/0202535A1) has been described in copending application (US Prov. Appln. Serial Number 60/026,056, Genencor Docket Number 31066USP0, filed 4 February 2008). The AmyTS-23t plasmid DNA isolated from transformed B. subtilis cells was sent to DNA2.0 Inc. (Menlo Park, CA) as the template for CCL construction. DNA 2.0 was requested to prepare a parent construct for the CCL by introducing the following seven mutations into AmyTS-23t, which was consequently termed AmyTS-23t-7mut: Q98R, M201L, S243Q R309A, Q320R, Q359E, and K444E. Variants were supplied as glycerol stocks in 96-well plates. Subsequently a request was made to DNA2.0 Inc. for the generation of positional libraries at each of the four sites in AmyTS-23t-7mut amylase that are shown in Table 9-1.

The AmyTS-23t-7mut combinatorial charge library was designed by identifying the following four residues in AmyTS-23t-7mut: GIn 87, Asn 225, Asn 272, and

Asn 282. A four site, 81-member CCL was created by making all combinations of three possibilities at each site: wild-type, arginine, or aspartic acid.

Table 9-1. AmyTS-23t-7mut CCL Variants

EXAMPLE 5

BAKING

Amylase Assays

Betamyl assay

One Betamyl unit is defined as activity degrading 0,0351 mmole per 1 min. of PNP-coupled maltopentaose so that 0,0351 mmole PNP per 1 min. can be released by excess a-glucosidase in the assay mix. The assay mix contains 50 ul 50 mM Na-citrate, 5 mM CaCI2, pH 6,5 with 25 ul enzyme sample and 25 ul

Betamyl substrate (Glc5-PNP and a-glucosidase) from Megazyme, Ireland (1 vial dissolved in 10 ml water). The assay mix is incubated for 30 min. at 40C and then stopped by adding 150 ul 4% Tris. Absorbance at 420 nm is measured using an ELISA-reader and the Betamyl activity is calculate based on Activity = A420 * d in Betamyl units/ml of enzyme sample assayed. 1 BMK is defined as 1000 Betamyl units.

Baking Trial Test

Baking trials were carried out with a standard white bread sponge and dough recipe for US toast. The sponge dough is prepared from 1600 g of flour "All Purpose Classic" from Sisco Mills, USA", 950 g of water, 40 g of soy bean oil and 32 g of dry yeast. The sponge is mixed for 1 min. at low speed and subsequently 3 min. at speed 2 on a Hobart spiral mixer. The sponge is subsequently fermented for 2,5 hours at 35°C, 85% RH followed by 0,5 hour at 5°C.

Thereafter 400 g of flour, 4 g of dry yeast, 40 g of salt, 2,4 g of calcium propionate, 240 g of high fructose corn sirup ( Isosweet), 5 g of the emulsifier PANODAN 205, 5 g of enzyme active soy flour, 30 g of non-active soy flour, 220 g of water and 30 g of a solution of ascorbic acid (prepared from 4 g ascorbic acid solubilised in 500 g of water) are added to the sponge. The resulting dough is mixed for 1 min. at low speed and then 6 min. on speed 2 on a Diosna mixer. Thereafter the dough is rested for 5 min. at ambient temperature, and then 550 g dough pieces are scaled, rested for 5 min. and then sheeted on Glimek sheeter with the settings 1 :4, 2 :4, 3 : 15, 4: 12 and 10 on each side and transferred to a baking form. After 60 min. proofing at 43°C at 90% RH the doughs are baked for 29 min. at 218°C

Firmness and resilience were measured with a TA-XT 2 texture analyser. The Softness, cohesiveness and resilience is determined by analysing bread slices by Texture Profile Analysis using a Texture Analyser From Stable Micro Systems, UK. The following settings were used :

Pre Test Speed : 2 mm/s

Test Speed : 2 mm/s

Post Test Speed : 10 mm/s

Rupture Test Distance: 1%

Distance: 40% Force: 0.098 N

Time: 5.00 sec

Count: 5

Load Cell : 5 kg

Trigger Type: Auto - 0.01 N

Firmness Effect in Baking Trial

TS-23t RS del does reduce firmness, and the effect increases with increasing dosage from 0,1 to 0,4 BMK/kg flour (Fig. 8).

In conclusion, TS-23t RSdel can be used to reduce the firmness development of bread.

EXAMPLE 6

Baking Composition

This example demonstrate the use of TS-23 in a baking composition.

Amylase Assays

Ceralpha assay

One Ceralpha unit is defined as activity degrading 0,0351 mmole per 1 min. of PNP-coupled non-reducing end blocked maltoheptaose so that 0,0351 mmole PNP per 1 min. can be released by excess glucoamylase and alpha-glucosidase in the assay mix. The assay mix contains 50 ul 50 mM Na-citrate, 5 mM CaCI2, pH 6,5 with 25 ul enzyme sample and 25 ul Ceralpha substrate (non-reducing end blocked Glc7-PNP, glucoamylase and alpha-glucosidase) from Megazyme, Ireland (1 vial dissolved in 10 ml water). The assay mix is incubated for 30 min. at 40C and then stopped by adding 150 ul 4% Tris. Absorbance at 420 nm is measured using an ELISA-reader and the Ceralpha activity is calculate based on Activity = A420 * d in Ceralpha units/ml of enzyme sample assayed.

Betamyl assay

The Betamyl assay was carried out as described in Example 5.

Baking Trial Test

Baking trials were carried out as described in Example 5.

Firmness and Cohessiveness Effects in Baking Trial

TS-23fl in combination with a variant of the wild type maltotetraohydrolase (PS4wt) from Pseudomonas saccharophila does reduce firmness relative to the variant PS4 alone, whereas TS-23fl alone does not reduce firmness significantly (Fig. 6).

The variant PS4 amylase has the following sequence (SEQ ID NO:7)

MDQAGKSPAG VRYHGGDEII LQGFHWNVVR EAPYNWYNIL RQQASTIAAD GFSAIWMPVP WRDFSSWTDG DKSGGGEGYF WH DFNKNGRY GSDAQLRQAA GALGGAGVKV LYDVVPN HM N RFYPDKEIN L PAGQRFWRND CPDPGNGPND

CDDGDRFLGG EADLNTGHPQ IYG M FRDEFT N LRSGYGAGG FRFDFVRGYA PERVDSWMSD SADSSFCVG E LWKEPSEYPP WDWRNTASWQ QIIKDWSDRA KCPVFDFALK ERMQNGSVAD WKHGLNGN PD PRWREVAVTF VDN H DTGYSP

GQNGGQHKWP LQDGLIRQAY AYILTSPGTP VVYWPHMYDW GYGDFIRQLI QVRRTAGVRA DSAISFHSGY SGLVATVSGS QQTLVVALNS DLANPGQVAS GSFSEAVNAS NGQVRVWRSG SGDGGGNDGG

Figures 6 and 7 show the results of a baking trial comparing doughs with and without TS-23fl. TS-23H in combination with variant PS4 does improve cohesiveness relative to variant PS4 alone, whereas TS-23fl alone does not change cohesiveness significantly (Fig. 6). In conclusion, TS-23H can be used to enhance the firmness reducing and cohesiveness improving effects of variant PS4 (Fig. 7).

EXAMPLE 7

Protocol for Evaluation of Firmness, Resilience and Cohesiveness

Texture Profile Analysis of Bread

Firmness, resilience and cohesiveness are determined by analysing bread slices by Texture Profile Analysis using a Texture Analyser From Stable Micro Systems, UK. Calculation of firmness and resilience is done according to preset standard supplied by Stable Micro System, UK. The probe used is aluminium 50 mm round.

Bread is sliced with the width of 12.5 mm. The slices are stamped out to a circular piece with a diameter of 45 mm and measured individually.

The following settings are used :

Pre Test Speed : 2 mm/s Test Speed : 2 mm/s

Post Test Speed : 10 mm/s Rupture Test Distance: 1%

Distance: 40%

Force: 0.098 N

Time: 5.00 sec

Count: 5

Load Cell : 5 kg

Trigger Type: Auto - 0.01 N

The mode of compression is a modification to the one used in Standard method AACC 74-09. The sample is compressed twice in the test. Figure 9 shows an example of a curve from the Texture Analyser.

EXAMPLE 8

Protocol for Evaluation of Firmness

Firmness is determined at 40% compression during the first compression. The figure is the force needed to compress the slice to 40% of the total thickness. The lower the value, the softer the bread. The firmness is expressed as a pressure, for example, in hPa.

This assay may be referred to as the "Firmness Evaluation Protocol".

EXAMPLE 9

Protocol for Evaluation of Resilience

Area under the curve is a measure of work applied during the test. The area under the curve in the compression part (Al) and the withdrawal part (A2) during the first compression are shown in Figure 9.

The ratio between Al and A2 is defined as the resilience of the sample, and is expressed as Resilience Units. True elastic material will give a symmetric curve, as the force applied during the first part will be equal to the force in the second part. For bread and bread-like material, A2 is normally smaller than A2 due to

disturbance of the structure during compression. Hence, resilience is always lower than 1.

This assay may be referred to as the "Resilience Evaluation Protocol".

EXAMPLE 10

Protocol for Evaluation of Cohesiveness

The cohesiveness is defined as the ratio between the area under second compression to the area under first compression (A3/A1+A2), and is expressed as Cohesiveness Units. It is a measure of the decay of the sample during compression. The higher the ability of the sample to regain its shape after first compression the closer the value will be to 1. For bread and bread-like material cohesiveness is always lower than 1.

This assay may be referred to as the "Cohesiveness Evaluation Protocol".

EXAMPLE 11

Protocol for Evaluation of Crumbliness (Resistance to Crumbling)

Two slices of bread are placed on a piece of paper. Each slice is divided into 4 squares by vertical and subsequent horizontal tears of the slice.

Tearing is done by pulling the crumb apart by the fingers. First the slice is torn from the middle of the top bread surface to the middle of the bottom bread surface. Thereafter, each half of the original slice is torn from the crust side to the inside of the slice. The small crumb pieces, which are separated from the 4 squares, are removed by shaking each piece after a tear at least 3 times by moving the hand up and down.

The weight of the separated small crumb pieces is determined as a measure of crumbliness. This assay may be referred to as the "Crumbliness Evaluation Protocol".

EXAMPLE 12

Protocol for Evaluation of Foldability

The toast bread is sliced using an automatic bread slicer with set slice thickness of 15 mm. The slice is folded by hand from the top of the slice towards the bottom, so that the direction of the crease is from side to side.

The foldability is visually assessed using the following scoring system :

This assay may be referred to as the "Foldability Evaluation Protocol".

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly

limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.

SEQUENCE LISTING :

SEQUENCE OF AMYTS-23 (FULL-LENGTH MOLECULE, MATURE CHAIN; 583 AMINO ACIDS; NO SIGNAL SEQUENCE) (SEQ ID NO: 1) : NTAPINETMMQYFEWDLPNDGTLWTKVKNEAANLSSLGITALWLPPAYKGTSQSDVGYG VYDLYDLGEFNQKGTIRTKYGTKTQYIQAIQAAKAAGMQVYADVVFNHKAGADGTEFVDA VEVDPSNRNQETSGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGTDWDESRKLNRIY KFRSTGKAWDWEVDTENGNYDYLMFADLDMDHPEVVTELKNWGTWYVNTTNIDGFRLD AVKHIKYSFFPDWLTYVRNQTGKNLFAVGEFWSYDVNKLHNYITKTNGSMSLFDAPLHNN FYTASKSSGYFDMRYLLNNTLMKDQPSLAVTLVDNHDTQPGQSLQSWVEPWFKPLAYAFI LTRQEGYPCVFYGDYYGIPKYNIPGLKSKIDPLLIARRDYAYGTQRDYIDHQDIIGWTRE GI DTKPNSGLAALITDGPGGSKWMYVGKKHAGKVFYDLTGNRSDTVTINADGWGEFKVNG GSVSIWVAKTSNVTFTVNNATTTSGQNVYVVANIPELGNWNTANAIKMNPSSYPTWKATI ALPQGKAIEFKFIKKDQAGNVIWESTSNRTYTVPFSSTGSYTASWNVP

SEQUENCE OF AMYTS-23T (TRUNCATED MOLECULE, MATURE CHAIN (SEQ ID NO: 2) :

NTAPINETMMQYFEWDLPNDGTLWTKVKNEAANLSSLGITALWLPPAYKGTSQSDVG YG VYDLYDLGEFNQKGTIRTKYGTKTQYIQAIQAAKAAGMQVYADVVFNHKAGADGTEFVDA VEVDPSNRNQETSGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGTDWDESRKLNRIY KFRSTGKAWDWEVDTENGNYDYLMFADLDMDHPEVVTELKNWGTWYVNTTNIDGFRLD AVKHIKYSFFPDWLTYVRNQTGKNLFAVGEFWSYDVNKLHNYITKTNGSMSLFDAPLHNN FYTASKSSGYFDMRYLLNNTLMKDQPSLAVTLVDNHDTQPGQSLQSWVEPWFKPLAYAFI LTRQEGYPCVFYGDYYGIPKYNIPG LKSKIDPLLIARRDYAYGTQRDYIDHQDIIGWTREGI DTKPNSGLAALITDGPGGSKWMYVGKKHAGKVFYDLTGNRSDTVTINADGWGEFKVNG GSVSIWVAK

DNA SEQUENCE OF OPTIMIZED AMYTS-23 GENE (SEQ ID NO: 3) :

AATACGGCGCCGATCAACGAAACGATGATGCAGTATTTTGAATGGGATCTGCCGAAT G ATGGAACGCTGTGGACGAAAGTCAAAAACGAAGCGGCGAATCTTAGCAGCCTGGGAAT CACAGCACTTTGGCTTCCGCCGGCATATAAAGGAACGAGCCAAAGCGATGTCGGCTAT GGCGTCTATGATCTGTATGACCTGGGCGAATTTAACCAAAAAGGCACGATCCGGACGA AATATGGCACGAAAACACAGTATATCCAAGCGATCCAGGCAGCAAAAGCAGCAGGCAT GCAAGTCTATGCCGACGTCGTCTTTAATCATAAAGCGGGAGCGGATGGCACAGAATTT GTCGATGCCGTCGAAGTTGATCCGAGCAACAGAAACCAAGAAACGAGCGGCACGTATC AAATCCAAGCGTGGACGAAATTTGATTTTCCGGGCAGAGGCAATACGTATAGCAGCTTT AAATGGCGCTGGTATCATTTTGACGGCACGGATTGGGATGAAAGCAGAAAACTGAACC GGATCTATAAATTTCGGAGCACGGGCAAAGCATGGGATTGGGAAGTCGATACGGAAAA CGGCAACTATGACTATCTGATGTTTGCCGATCTGGATATGGATCATCCGGAAGTCGTCA CGGAACTGAAAAATTGGGGCACGTGGTATGTTAATACGACGAACATCGATGGCTTTAG ACTGGATGCCGTCAAACATATCAAATATAGC I I I I I I CCGGACTGGCTGACGTATGTCA GAAACCAGACGGGCAAAAACCTTTTTGCCGTCGGCGAATTTTGGAGCTATGACGTCAA CAAACTTCATAACTATATCACGAAAACGAACGGCAGCATGAGCCTTTTTGATGCCCCGC TTCATAACAACTTTTATACGGCGAGCAAAAGCTCAGGCTATTTTGATATGAGATATCTGC TGAACAACACGCTGATGAAAGATCAACCGAGCCTGGCAGTCACACTGGTCGATAACCA TGATACACAACCGGGCCAAAGCCTTCAAAGCTGGGTCGAACCGTGGTTTAAACCGCTG GCGTATGCCTTTATCCTGACGAGACAAGAAGGGTATCCTTGCGTCTTTTATGGCGACTA TTATGGCATCCCGAAATATAATATCCCGGGCCTGAAAAGCAAAATCGATCCGCTGCTGA

TCGCCAGACGGGATTATGCCTATGGCACACAGCGGGATTATATCGACCATCAGGACA T CATCGGCTGGACAAGAGAAGGCATCGATACGAAACCGAATAGCGGACTGGCAGCACT GATTACAGATGGACCGGGCGGAAGCAAATGGATGTATGTCGGCAAAAAACATGCCGGC AAAGTCTTTTATGATCTGACGGGCAACAGAAGCGATACGGTCACGATCAATGCTGATG GCTGGGGAGAATTTAAAGTCAATGGCGGCAGCGTTTCAATCTGGGTCGCCAAAACGAG CAATGTCACGTTTACGGTCAACAATGCCACGACAACGAGCGGCCAAAATGTCTATGTCG TCGCCAATATCCCGGAACTGGGCAATTGGAATACGGCGAACGCAATCAAAATGAACCC GAGCAGCTATCCGACATGGAAAGCGACAATCGCTCTGCCGCAAGGAAAAGCGATCGAA TTTAAATTTATCAAAAAAGACCAGGCGGGCAATGTTATTTGGGAAAGCACGAGCAATAG AACGTATACGGTCCCGTTTAGCAGCACAGGAAGCTATACAGCGAGCTGGAATGTTCCG TGA

DNA SEQUENCE OF OPTIMIZED AMYTS-23T GENE (SEQ ID NO: 4) :

AATACGGCGCCGATCAACGAAACGATGATGCAGTATTTTGAATGGGATCTGCCGAAT G

ATGGAACGCTGTGGACGAAAGTCAAAAACGAAGCGGCGAATCTTAGCAGCCTGGGAA T CACAGCACTTTGGCTTCCGCCGGCATATAAAGGAACGAGCCAAAGCGATGTCGGCTAT GGCGTCTATGATCTGTATGACCTGGGCGAATTTAACCAAAAAGGCACGATCCGGACGA AATATGGCACGAAAACACAGTATATCCAAGCGATCCAGGCAGCAAAAGCAGCAGGCAT GCAAGTCTATGCCGACGTCGTCTTTAATCATAAAGCGGGAGCGGATGGCACAGAATTT GTCGATGCCGTCGAAGTTGATCCGAGCAACAGAAACCAAGAAACGAGCGGCACGTATC AAATCCAAGCGTGGACGAAATTTGATTTTCCGGGCAGAGGCAATACGTATAGCAGCTTT AAATGGCGCTGGTATCATTTTGACGGCACGGATTGGGATGAAAGCAGAAAACTGAACC GGATCTATAAATTTCGGAGCACGGGCAAAGCATGGGATTGGGAAGTCGATACGGAAAA CGGCAACTATGACTATCTGATGTTTGCCGATCTGGATATGGATCATCCGGAAGTCGTCA CGGAACTGAAAAATTGGGGCACGTGGTATGTTAATACGACGAACATCGATGGCTTTAG ACTGGATGCCGTCAAACATATCAAATATAGCTTTTTTCCGGACTGGCTGACGTATGTCA GAAACCAGACGGGCAAAAACCTTTTTGCCGTCGGCGAATTTTGGAGCTATGACGTCAA CAAACTTCATAACTATATCACGAAAACGAACGGCAGCATGAGCCTTTTTGATGCCCCGC TTCATAACAACTTTTATACGGCGAGCAAAAGCTCAGGCTATTTTGATATGAGATATCTGC TGAACAACACGCTGATGAAAGATCAACCGAGCCTGGCAGTCACACTGGTCGATAACCA TGATACACAACCGGGCCAAAGCCTTCAAAGCTGGGTCGAACCGTGGTTTAAACCGCTG GCGTATGCCTTTATCCTGACGAGACAAGAAGGGTATCCTTGCGTCTTTTATGGCGACTA TTATGGCATCCCGAAATATAATATCCCGGGCCTGAAAAGCAAAATCGATCCGCTGCTGA TCGCCAGACGGGATTATGCCTATGGCACACAGCGGGATTATATCGACCATCAGGACAT CATCGGCTGGACAAGAGAAGGCATCGATACGAAACCGAATAGCGGACTGGCAGCACT GATTACAGATGGACCGGGCGGAAGCAAATGGATGTATGTCGGCAAAAAACATGCCGGC AAAGTCTTTTATGATCTGACGGGCAACAGAAGCGATACGGTCACGATCAATGCTGATG GCTGGGGAGAATTTAAAGTCAATGGCGGCAGCGTTTCAATCTGGGTCGCCAAATGA

The coding region for the LAT signal peptide is shown below (SEQ ID NO: 5) : atgaaacaacaaaaacggctttacgcccgattgctgacgctgttatttgcgctcatcttc ttgctgcctcattctgca gcttcagca.

The amino acid sequence of the LAT signal peptide is shown below (SEQ ID

NO: 6) :

MKQQKRLYARLLTLLFALIFLLPHSAASA

The variant PS4 amylase has the following sequence (SEQ ID N0:7) :

MDQAGKSPAGVRYHGGDEIILQGFHWNVVREAPYNWYNILRQQASTIAAD GFSAIWMPVPWRDFSSWTDGDKSGGGEGYFWHDFNKNGRYGSDAQLRQAA

GALGGAGVKVLYDVVPNHMNRFYPDKEINLPAGQRFWRND CPDPGNG PN D CDDGDRFLGG EADLNTGHPQ IYGM FRDEFT N LRSGYGAGG FRFDFVRGYA PERVDSWMSD SADSSFCVG E LWKEPSEYPP WDWRNTASWQ QIIKDWSDRA KCPVFDFALK ERMQNGSVAD WKHGLNGN PD PRWREVAVTF VDN H DTGYSP

GQNGGQHKWP LQDGLIRQAY AYILTSPGTP VVYWPHMYDW GYGDFIRQLI

QVRRTAGVRA DSAISFHSGY SGLVATVSGS QQTLVVALNS DLANPGQVAS GSFSEAVNAS NGQVRVWRSG SGDGGGN DGG

Mature protein sequence of G4-amylase from Pseudomonas saccharophila with the starch binding domain removed SEQ ID NO:8:

DQAGKSPAGVRYHGGDEIILQGFHWNVVREAPNDWYNILRQQASTIAADGFSAIWMP VP WRDFSSWTDGGKSGGGEGYFWHDFNKNGRYGSDAQLRQAAGALGGAGVKVLYDVVPN HMNRGYPDKEINLPAGQGFWRNDCADPGNYPNDCDDGDRFIGGESDLNTGHPQIYGMF RDELANLRSGYGAGGFRFDFVRGYAPERVDSWMSDSADSSFCVGELWKGPSEYPSWDW RNTASWQQIIKDWSDRAKCPVFDFALKERMQNGSVADWKHGLNGNPDPRWREVAVTFV DNHDTGYSPGQNGGQHHWALQDGLIRQAYAYILTSPGTPVVYWSHMYDWGYGDFIRQL IQVRRTAGVRADSAISFHSGYSGLVATVSGSQQTLVVALNSDLANPGQVASGSFSEAVNA SNGQVRVWRSGSGDGGGNDGG

>gi |77787| pir| |S05667 glucan 1, 4-alpha-maltotetraohydrolase (EC 3.2.1.60) mature protein without the signal sequence - Pseudomonas saccharophila SEQ ID NO: 9:

DQAGKSPAGVRYHGGDEIILQGFHWNVVREAPNDWYNILRQQASTIAADGFSAIWMP VP WRDFSSWTDGGKSGGGEGYFWHDFNKNGRYGSDAQLRQAAGALGGAGVKVLYDVVPN HMNRGYPDKEINLPAGQG FWRN DCADPGNYPNDCDDGDRFIGGESDLNTGHPQIYGMF RDELANLRSGYGAGGFRFDFVRGYAPERVDSWMSDSADSSFCVGELWKGPSEYPSWDW RNTASWQQIIKDWSDRAKCPVFDFALKERMQNGSVADWKHGLNGNPDPRWREVAVTFV DNHDTGYSPGQNGGQHHWALQDGLIRQAYAYILTSPGTPVVYWSHMYDWGYGDFIRQL IQVRRTAGVRADSAISFHSGYSGLVATVSGSQQTLVVALNSDLANPGQVASGSFSEAVNA SNGQVRVWRSGSGDGGGNDGGEGGLVNVNFRCDNGVTQMGDSVYAVGNVSQLGNWS PASAVRLTDTSSYPTWKGSIALPDGQNVEWKCLIRNEADATLVRQWQSGGNNQVQAAA GASTSGSF

Novamyl (SEQ ID NO: 10) :

SSSASVKGDVIYQIIIDRFYDGDTTNNNPAKSYGLYDPTKSKWKMYWGGDLEGVRQK LPY

LKQLGVTTIWLSPVLDNLDTLAGTDNTGYHGYWTRDFKQIEEHFGNWTTFDTLVNDA HQ

NGIKVIVDFVPNHSTPFKANDSTFAEGGALYNNGTYMGNYFDDATKGYFHHNGDISN WD DRYEAQWKNFTDPAGFSLADLSQENGTIAQYLTDAAVQLVAHGADGLRIDAVKHFNSGFS KSLADKLYQKKDIFLVGEWYGDDPGTANHLEKVRYANNSGVNVLDFDLNTVIRNVFGTFT QTMYDLNNMVNQTGNEYKYKENLITFIDNHDMSRFLSVNSNKANLHQALAFILTSRGTPS I YYGTEQYM AGG N DPYN RG M M PA FDTTTTA FK EVST LAG LRRN NAAI QYGTTTQRWI NND VYIYERKFFNDVVLVAINRNTQSSYSISGLQTALPNGSYADYLSGLLGGNGISVSNGSVA S FTLAPG AVSVWQYSTSASAPQIGSVAPN MGIPG N VVTI DG KG FGTTQGTVTFGGVTATVK SWTSNRIEVYVPNMAAGLTDVKVTAGGVSSNLYSYNILSGTQTSVVFTVKSAPPTNLGDK I

YLTGNIPELGNWSTDTSGAVNNAQGPLLAPNYPDWFYVFSVPAGKTIQFKFFIKRAD GTIQ WENGSNHVATTPTGATGNITVTWQN

DNA Primers:

pHPLT-Pstl-FW (SEQ NO: 11) : CTCATTCTGCAGCTTCAGCAAATACGGCG

pHPLT-Hpal-RV (SEQ NO: 12) : CTCTGTTAACTCATTTGGCGACCCAGATTGAAACG

TS-delRS-FW (SEQ NO: 13) : CTATAAATTTACGGGCAAAGCATGGGATTGG

TS-delRS-RV (SEQ NO: 14) :

TGCTTTGCCCGTAAATTTATAGATCCGGTTCAG

TS-M201L-FW (SEQ NO: 15) : CTATGACTATCTGCTGTTTGCCGATCTG

TS-M201L-RV (SEQ NO: 16) : CAGATCGGCAAACAGCAGATAGTCATAG

TS-delRS/M201L-FW (SEQ NO: 17) : GCATGGGATTGGGAAGTCGATACGGAAAACGGCAACTATGACTATCTGCTGTTTGCCG

TS-delRS/M201L-RV (SEQ NO: 18) : CGTATCGACTTCCCAATCCCATGCTTTGCCCGTAAATTTATAGATCCGGTTC