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Title:
LIPASE ENZYMES
Document Type and Number:
WIPO Patent Application WO/2018/209018
Kind Code:
A1
Abstract:
Lipase enzymes, methods of making lipase enzymes, methods of using lipase enzymes in food, feed, personal care, detergents, grain processing, pulp and paper processing, biofuels, ethanol production, textiles, dairy processing, cocoa butter processing, cocoa extraction, dietary supplements, coffee processing, coatings, water treatment, and oil processing.

Inventors:
LISZKA, Michael (3550 John Hopkins Ct, San Diego, CA, 92121, US)
KUTSCHER, Jochen (Robert-Hansen-Strasse 1, 25A, Illertissen, Illertissen, DE)
PRASHAR, Aditi (3550 John Hopkins Ct, San Diego, CA, 92121, US)
POP, Cristina (3550 John Hopkins Ct, San Diego, CA, 92121, US)
Application Number:
US2018/031956
Publication Date:
November 15, 2018
Filing Date:
May 10, 2018
Export Citation:
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Assignee:
BASF SE (Carl-Bosch-Strasse 38, Ludwigshafen am Rhein, Rhein, DE)
International Classes:
A21D8/04; A61K38/43; C12N9/18; C12N9/20; C12N15/55; C12P7/64
Domestic Patent References:
WO2017142904A12017-08-24
WO2002000852A22002-01-03
WO2009133177A12009-11-05
Foreign References:
US20030003561A12003-01-02
Other References:
COLEMAN ET AL.: "The Genome of Nectria haematococca: Contribution of Supernumerary Chromosomes to Gene Expansion", PLOS GENETICS, vol. 5, no. 8, 28 August 2009 (2009-08-28), pages e1000618, XP055214133
DATABASE Protein [O] 14 August 2010 (2010-08-14), "hypothetical protein NECHADRAFT_49364 [[Nectria] haematococca mpVI 77-13-4]", XP055548806, retrieved from ncbi Database accession no. XP_003050606
SCHAFFARCZYK ET AL.: "Lipases in Wheat Breadmaking: Analysis and Functional Effects of Lipid Reaction Products", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, vol. 62, no. 32, 17 July 2014 (2014-07-17), pages 8229 - 8237, XP002767672
Attorney, Agent or Firm:
SIDDONS, Brian, W. et al. (3550 John Hopkins Ct, San Diego, CA, 92121, US)
Download PDF:
Claims:
Claims

1. A variant polypeptide comprising an amino acid sequence that is at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, 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% identical to the amino acid sequence of SEQ ID NO: 1, and the variant polypeptide has lipase activity.

2. The variant polypeptide of claim 1, comprising an amino acid residue insertion, deletion, or substitution to the amino acid sequence of SEQ ID NO: l, and the variant polypeptide has lipase activity.

3. The variant polypeptide of claim 2, wherein the amino acid residue insertion, deletion, or substitution is at the amino acid residue position number 23, 33, 82, 83, 84, 85, 160, 199, 254, 255, 256, 258, 263, 264, 265, 268, 308, 311, or any combination thereof to the amino acid sequence of SEQ ID NO: 1, and the variant polypeptide has lipase activity.

4. The variant polypeptide of claim 2, wherein the amino acid substitution is selected from the group consisting of: Y23A, K33N, S82T, S83D, S83H, S83I, S83N, S83R, S83T, S83Y, S84S, S84N, 84Ύ, 84'L, 84' S, I85A, I85C, I85F, I85H, I85L, I85M, I85P, I85S, I85T, I85V, I85Y, K160N, P199I, P199V, I254A, I254C, I254E, I254F, I254G, I254L, I254M, I254N, I254R, I254S, I2454V, I254W, I254Y, I255A, I255L, A256D, L258A, L258D; L258E, L258G, L258H, L258N, L258Q, L258R, L258S, L258T, L258V, D263G, D263K, D263P, D263R, D263S; T264A, T264D, T264G, T264I, T264L, T264N, T264S, D265A, D265G, D265K, D265L, D265N, D265S, D265T, T268A, T268G, T268K, T268L, T268N, T268S, D308A, and Y311E, or any combination thereof to the amino acid sequence of SEQ ID NO: 1, and the variant polypeptide has lipase activity

5. A variant polypeptide comprising an amino acid sequence that is at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, 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% identical to the amino acid sequence as set forth in SEQ ID NO: l, wherein the variant polypeptide has at least one single amino acid substitution to the amino acid sequence of SEQ ID NO: l, and the one single amino acid substitution is selected from the group consisting of: Y23A, K33N, S82T, S83D, S83H, S83I, S83N, S83R, S83T, S83Y, S84S, S84N, 84Ύ, 84'L, 84' S, 185 A, I85C, I85F, I85H, I85L, I85M,

I85P, I85S, I85T, I85V, I85Y, K160N, P199I, P199V, I254A, I254C, I254E, I254F, I254G, I254L, I254M, I254N, I254R, I254S, I2454V, I254W, I254Y, I255A, I255L, A256D, L258A, L258D; L258E, L258G, L258H, L258N, L258Q, L258R, L258S, L258T, L258V, D263G, D263K, D263P, D263R, D263S; T264A, T264D, T264G, T264I, T264L, T264N, T264S, D265A, D265G, D265K, D265L, D265N, D265S, D265T, T268A, T268G, T268K, T268L, T268N, T268S, D308A, and Y31 IE, to the amino acid sequence of SEQ ID NO: 1; wherein the variant polypeptide has lipase activity.

6. A variant polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence as set forth in SEQ ID NO: l, wherein the variant polypeptide has a modification as set forth in Table: 1, and the variant polypeptide has lipase activity.

7. A variant polypeptide as in any of Claims 1-6, wherein the variant polypeptide is encoded by a nucleic acid sequence that is at least 80% identical the nucleic acid sequence as set forth in SEQ ID NO:2, and the variant polypeptide has lipase activity.

8. A variant polypeptide as in any of claims 1-7, wherein the variant polypeptide is a fragment of the full length amino acid sequence and the fragment has lipase activity.

9. A variant polypeptide comprising a hybrid of at least one variant polypeptide as in any of claims 1-8, and a second polypeptide having lipase activity, wherein the hybrid has lipase activity.

10. A compositions comprising the variant polypeptide as in any of Claims 1-9.

11. A composition comprising the variant polypeptide as in any of Claims 1-9, and at least a second enzyme.

12. The composition of Claim 11, wherein the second enzyme is selected from the group consisting of: a second lipase, an amylase, a beta-amylase, a xylanase, a protease, a cellulase, a glucoamylase, an oxidoreductase, a phospholipase, and a cyclodextrin glucanotransferase.

13. A variant nucleotide of the nucleic acid sequence as set forth in SEQ ID NO:2, wherein the variant nucleotide is a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence as set forth in SEQ ID NO:2, wherein the variant nucleotide encodes a polypeptide having lipase activity.

14. A method of making a variant polypeptide comprising: providing a template nucleic acid sequence of Claim 13, transforming the template nucleic acid sequence into an expression host, cultivating the expression host to produce the variant polypeptide, and purifying the variant polypeptide.

15. The method of claim 14, wherein the expression host is selected from the group consisting of: a bacterial expression system, a yeast expression system, a fungal expression system, and a synthetic expression system.

17. The method of claim 15, wherein the bacterial expression system is selected from an E. coli, a Bacillus, a Pseudomonas, and a Streptomyces.

18. The method of claim 15, wherein the yeast expression system is selected from a Candida, a Pichia, a Saccharomyces, a Schizosaccharomyces.

19. The method of claim 15, wherein the fungal expression system is selected from a Penicillium, an Aspergillus, a Fusarium, a Myceliopthora, a Rhizomucor, a Rhizopus, a Thermomyces, and a Trichoderma.

20. A method of preparing a dough or a baked product prepared from the dough, without the addition of an emulsifier, the method comprising adding one of the variant polypeptides as in any of Claims 1-9 to the dough and baking it.

21. The method of claim 21, wherein the emulsifier is selected from the group consisting of: calcium stearoyl lactylate (CSL), diacetyl tartaric acid esters of monoglycerides (DATEM), ethoxylated mono- and diglycerides (EMG), polysorbates (PS), sodium stearoyl lactylate (SSL), and succinylated monoglycerides (SMG).

22. A pre-mix for dough or a baked product prepared from a dough, comprising at least one of the variant polypeptides as in any of Claims 1-9.

23. The composition as in any of claims 10-12, further comprises a carrier, a stabilizer, a buffer, a preservative, or any combination thereof.

Description:
LIPASE ENZYMES

SEQUENCE LISTING

This application includes a nucleotide and amino acid sequence listing in computer readable form (CRF) as an ASC II text (.txt) file according to "Standard for the Presentation of Nucleotide and Amino Acid Sequence Listings in International Patent Applications Under the Patent Cooperation Treaty (PCT)" ST.25. The sequence listing is identified below and is hereby incorporated by reference into the specification of this application in its entirety and for all purposes.

TECHNICAL FIELD

Bread has been a staple of human nutrition for thousands of years. Bread is usually made by combining a flour, water, salt, yeast, and/or other food additives to make a dough or paste; then the dough is baked to make bread. Enzymes are known to be useful in baking because of the enzymes effects on the baking process can be similar or better than chemical alternatives, enzymes can be useful for antistaling, and increasing bread volume. Several different enzymes can be used for making bread, for example lipases have been known to improve the stability and volume of the bread; however, the industry still needs a lipase that improves volume, stability, tolerance, reduces or eliminates the additive diacetyl tartaric acid esters of monoglycerides (DATEM). This disclosure is directed to variant lipase enzymes that meets or exceeds these industrial requirements.

BRIEF SUMMARY OF THE INVENTION

A variant polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1, and the variant polypeptide has lipase activity.

A variant polypeptide comprising an amino acid residue insertion, deletion, or substitution to the amino acid sequence of SEQ ID NO: 1, and the variant polypeptide has lipase activity.

A variant polypeptide comprising an amino acid residue insertion, deletion, or substitution is at the amino acid residue position number 23, 33, 82, 83, 84, 85, 160, 199, 254, 255, 256, 258, 263, 264, 265, 268, 308, 311, or any combination thereof to the amino acid sequence of SEQ ID NO: 1, and the variant polypeptide has lipase activity.

A variant polypeptide comprising an amino acid substitution is selected from the group consisting of: Y23A, K33N, S82T, S83D, S83H, S83I, S83N, S83R, S83T, S83Y, S84S, S84N, 84Ύ, 84'L, 84' S, I85A, I85C, I85F, I85H, I85L, I85M, I85P, I85S, I85T, 185 V, 185 Y, K160N, PI 991, PI 99V, I254A, I254C, I254E, I254F, I254G, I254L, I254M, I254N, I254R, I254S, I2454V, I254W, I254Y, I255A, I255L, A256D, L258A, L258D; L258E, L258G, L258H, L258N, L258Q, L258R, L258S, L258T, L258V, D263G, D263K, D263P, D263R, D263S; T264A, T264D, T264G, T264I, T264L, T264N, T264S, D265A, D265G, D265K, D265L, D265N, D265S, D265T, T268A, T268G, T268K, T268L, T268N, T268S, D308A, and Y311E, or any combination thereof to the amino acid sequence of SEQ ID NO: 1, and the variant polypeptide has lipase activity.

A variant polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence as set forth in SEQ ID NO: 1, wherein the variant polypeptide has at least one single amino acid substitution to the amino acid sequence of SEQ ID NO: 1, and the one single amino acid substitution is selected from the group consisting of: Y23 A, K33N, S82T, S83D, S83H, S83I, S83N, S83R, S83T, S83Y, S84S, S84N, 84Ύ, 84'L, 84' S, 185 A, I85C, I85F, I85H, I85L, I85M, I85P, I85S, I85T, I85V, I85Y, K160N, P199I, P199V, I254A, I254C, I254E, I254F, I254G, I254L, I254M, I254N, I254R, I254S, I2454V, I254W, I254Y, I255A, I255L, A256D, L258A, L258D; L258E, L258G, L258H, L258N, L258Q, L258R, L258S, L258T, L258V, D263G, D263K, D263P, D263R, D263S; T264A, T264D, T264G, T264I, T264L, T264N, T264S, D265A, D265G, D265K, D265L, D265N, D265S, D265T, T268A, T268G, T268K, T268L, T268N, T268S, D308A, and Y31 IE, to the amino acid sequence of SEQ ID NO: 1; wherein the variant polypeptide has lipase activity.

A variant polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence as set forth in SEQ ID NO: l, wherein the variant polypeptide has a modification as set forth in Table: 1, and the variant polypeptide has lipase activity.

A variant polypeptide wherein the variant polypeptide is encoded by a nucleic acid sequence that is at least 80% identical the nucleic acid sequence as set forth in SEQ ID NO:2, and the variant polypeptide has lipase activity.

A variant nucleotide of the nucleic acid sequence as set forth in SEQ ID NO:2, wherein the variant nucleotide is a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence as set forth in SEQ ID NO:2, wherein the variant nucleotide encodes a polypeptide having lipase activity.

A variant polypeptide comprising a fragment of the full length amino acid sequence of SEQ ID NO: 1, and the fragment is the variant polypeptide having lipase activity.

A variant polypeptide comprising a hybrid of at least one variant polypeptide disclosed herein, and a second polypeptide having lipase activity, wherein the hybrid has lipase activity.

A composition comprising the variant polypeptide as disclosed herein.

A composition comprising the variant polypeptide as disclosed herein, and at least a second enzyme. The composition, further comprising the second enzyme is selected from the group consisting of: a second lipase, an amylase, a xylanase, a protease, a cellulase, a glucoamylase, an Oxidoreductases, a Phospholipase and a cyclodextrin glucanotransferase.

The composition comprising the variant polypeptide as disclosed herein and further comprising a carrier, a stabilizer, a buffer, a preservative, or any combination thereof. The composition comprising the variant polypeptide as disclosed herein, wherein the carrier is a wheat flour. The composition comprising the variant polypeptide as disclosed herein, wherein the stabilizer is calcium acetate, calcium chloride, magnesium chloride, sodium chloride, sodium sulfate, guar gum, or any combination thereof. The composition comprising the variant polypeptide as disclosed herein wherein the buffer is calcium acetate, sodium acetate, sodium citrate, sodium phosphate, potassium phosphate, or any combination thereof. The composition comprising the variant polypeptide as disclosed herein wherein the preservatives are calcium acetate, sodium acetate, sodium propionate, calcium propionate, propionic acid, potassium sorbate, sorbic acid, sodium benzoate, benzoic acid, acetic acid, or any combination thereof. The composition comprising the variant polypeptide as disclosed herein wherein composition. The composition comprising the variant polypeptide as disclosed herein and one or more components selected from the group consisting of sugars like sucrose, trehalose, lactose; milk powder, gluten, granulated fat, an amino acid, a salt, an oxidant such as ascorbic acid, bromate and azodicabonamide, a reducing agent such as L-cysteine, an emulsifier such as mono-glycerides, di- glycerides, clycerol monstearate, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids and diacetyl tartaric acid esters of mono- and diglycerides, gums such as guar gum and xanthangum, flavors, acids such as citric acid and propionic acid, starch, modified starch, humectants such as glycerol, and preservatives.

A method of making a variant polypeptide comprising: providing a template nucleic acid sequence of SEQ ID NO:2, or disclosed herein, transforming the template nucleic acid sequence into an expression host, cultivating the expression host to produce the variant polypeptide, and purifying the variant polypeptide. The method further comprising an expression host is selected from the group consisting of: a bacterial expression system, a yeast expression system, a fungal expression system, and a synthetic expression system. The method wherein the bacterial expression system is selected from a E. coli, a Bacillus, a Pseudomonas, and a Streptomyces. The method wherein the yeast expression system is selected from a Candida, a Pichia, a Saccharomyces, a Schizosaccharomyces. The method wherein the fungal expression system is selected from a Penicillium, an Aspergillus, a Fusarium, a Myceliopthora, a Rhizomucor, a Rhizopus, a Thermomyces, and a Trichoderma. A method of preparing a dough or a baked product prepared from the dough, without the addition of an emulsifier, the method comprising adding one of the variant polypeptides as disclosed herein to the dough and baking it. The method wherein the emulsifier is selected from the group consisting of: calcium stearoyl lactylate (CSL), diacetyl tartaric acid esters of monoglycerides (DATEM), ethoxylated mono- and diglycerides (EMG), polysorbates (PS), sodium stearoyl lactylate (SSL), and succinylated monoglycerides (SMG).

A pre-mix for dough or a baked product prepared from a dough, comprising at least one of the variant polypeptides as disclosed herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

Fig. 1 A and Fig. IB, shows results for baking trials.

DETAILED DESCRIPTION OF THE INVENTION

Bread includes, but is not limited to: rolls, buns, pastries, cakes, flatbreads, pizza bread, pita bread, wafers, pie crusts naan, lavish, pitta, focaccia, sourdoughs, noodles, cookies, tortillas, pancakes, crepes, croutons, and biscuits. Baking bread generally involves mixing ingredients to form dough, kneading, rising, shaping, baking, cooling and storage. The ingredients used for making dough generally include flour, water, salt, yeast, and other food additives.

Flour is generally made from wheat and can be milled for different purposes such as making bread, pastries, cakes, biscuits pasta, and noodles. Alternatives to wheat flour include, but are not limited to: almond flour, coconut flour, chia flour, corn flour, barley flour, spelt flour, soya flour, hemp flour, potato flour, quinoa, teff flour, rye flour, amaranth flour, arrowroot flour, chick pea (garbanzo) flour, cashew flour, flax meal, macadamia flour, millet flour, sorghum flour, rice flour, tapioca flour, and any combination thereof. Flour type is known to vary between different regions and different countries around the world.

Yeast breaks down sugars into carbon dioxide and water. A variety of Baker' s yeast, which are usually derived from Saccharomyces cerevisiae, are known to those skilled in the art including, but not limited to: cream yeast, compressed yeast, cake yeast, active dry yeast, instant yeast, osmotolerant yeasts, rapid-rise yeast, deactivated yeast. Other kinds of yeast include nutritional yeast, brewer's yeast, distiller's and wine yeast.

Sweeteners include but are not limited to: liquid sugar, syrups, white (granulated) sugars, brown (raw) sugars, honey, fructose, dextrose, glucose, high fructose corn syrup, molasses, and artificial sweeteners

Emulsifiers include but are not limited to diacetyl tartaric acid esters of monoglycerides (DATEM), sodium stearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), ethoxylated mono- and diglycerides (EMG), polysorbates (PS), and succinylated monoglycerides (SMG). Other food additives that can be used with the methods of this disclosure include: Lipids, oils, butter, margarine, shortening, butterfat, glycerol, eggs, diary, non-diary alternatives, thickeners, preservatives, colorants, and enzymes.

The ingredients or additives for baking can be added individually to during the baking process. The ingredients or additives can also be combined with more than one ingredient or additive to form pre-mixes and then the pre-mixes are added during the baking process. In addition, enzymes can be added directly to the flour prior to the baking process.

An enzyme is a biological molecule comprising a sequence of amino acids, wherein the enzyme can catalyze a reaction. Enzyme names are known to those skilled in the art based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Enzyme names include: an EC (Enzyme Commission) number, recommended name, alternative names (if any), catalytic activity, and other factors. Enzymes are also known as a polypeptide, a protein, a peptide, an amino acid sequence, or is identified by a SEQ ID NO. In this disclosure, the alternative names for enzyme can be used interchangeably.

Different classes of enzymes are known to be useful in baking, including: Lipases E.C. 3.1.3; Alpha-amylase (E.C. 3.2.1.1); beta-amylase (E.C. 3.2.1.2); Glucan 1, 4-alpha- maltotetraohydrolase (E.C. 3.2.1.60), also known as exo-maltotetraohydrolase, G4-amylase; Glucan 1,4-alpha-maltohydrolase (E.C. 3.2.1.133), also known as maltogenic alpha-amylase; Endo-l,4-beta-xylanase (E.C. 3.2.1.8); Oxidoreductases; Phospholipase Al (E.C. 3.1.1.32) Phospholipase A2 (E.C. 3.1.1.4); Phospholipase C (E.C. 3.1.4.3); Phospholipase D (E.C. 3.1.4.4); Galactolipase (E.C. 3.1.1.26), Cellulase (EC 3.2.1.4), Transglutaminases (EC 2.3.2.13), Phytase (EC 3.1.3.8; 3.1.3.26; and 3.1.1.72) and Protease. Enzymes are used as food ingredients, food additives, and/processing aids.

Lipases (E.C. 3.1.1.3) are hydrolytic enzymes that are known to cleave ester bonds in lipids. Lipases include phospholipases, triacylglycerol lipases, and galactolipases. Lipases have been identified from plants; mammals; and microorganisms including but not limited to: Pseudomonas, Vibrio, Acinetobacter, Burkholderia, Chromobacterium, Cutinase from Fusarium solani (FSC), Candida antarctica A (CalA), Rhizopus oryzae (ROL), Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Aspergillus Niger, Fusarium heterosporum, Fusarium oxysporum, Fusarium culmorum lipases.

In addition, many lipases, phospholipases, and galactolipases have been disclosed in patents and published patent applications including, but not limited to: WO 1993/000924, WO2003/035878, WO2003/089620, WO2005/032496, WO2005/086900, WO2006/031699, WO2008/036863, and WO2011/046812. Commercial lipases used in food processing and baking including, but not limited to: LIPOPAN™, NOOPAZYME, LIPOPAN MAX, LIPOPAN Xtra (available from Novozymes); PANAMORE, CAKEZYME, and BAKEZYME (available from DSM); and GRINDAMYL EXEL 16, GRINDAMYL POWERBAKE, and TS-E 861 (available from Dupont/Danisco).

A "parent" sequence (of a parent protein or enzyme, also called "parent enzyme") is the starting sequence for introduction of changes (e.g. by introducing one or more amino acid substitutions, insertions, deletions, or a combination thereof) to the sequence, resulting in "variants" of the parent sequences. The term parent enzyme (or parent sequence) includes

1. wild-type enzymes (sequences) and

2. Synthetically generated sequences (enzymes) which are used as starting sequences for introduction of (further) changes.

"Enzyme variants" or "sequence variants" or "variant enzymes" refers to an enzyme that differs from its parent enzyme in its amino acid sequence to a certain extent. If not indicated otherwise, variant enzyme "having enzymatic activity" means that this variant enzyme has the same type of enzymatic activity as the respective parent enzyme.

In an embodiment, the variant polypeptide having an amino acid substitution can be a conservative amino acid substitution. A "conservative amino acid substitution" means replacement of one amino acid residue in an amino acid sequence with a different amino acid residue having a similar property at the same position compared to the parent amino acid sequence. Some examples of a conservative amino acid substitution include but are not limited to replacing a positively charged amino acid residue with a different positively charged amino acid residue; replacing a polar amino acid residue with a different polar amino acid residue; replacing a non-polar amino acid residue with a different non-polar amino acid residue, replacing a basic amino acid residue with a different basic amino acid residue, or replacing an aromatic amino acid residue with a different aromatic amino acid residue.

WIPO Standard ST.25 (1998) provides that the amino acid residues should be represented in the sequence listing using the following three-letter symbols with the first letter as a capital. The table below provides an overview of the amino acid identifiers as well as the corresponding DNA codons that encode the amino acid using the standard genetic standard. The DNA condons that encode amino acid residues can be different depending organism that is used and slightly different tables for translation of the genetic code may apply. A compilation of such non-standard code translation tables is maintained at the NCBI. For reference see e.g. https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi. Amino Acids

Name 3 letter code 1 letter code DNA codons

Alanine Ala A GCA, GCC, GCG, GCT

Arginine Arg AGA, AGG, CGA, CGC, CGG, CGT

Asparagine Asn N AAC, AAT

Aspartic acid; (Aspartate) Asp D GAC, GAT

Cysteine Cys C TGC, TGT

Glutamic acid; (Glutamate) Glu E GAA, GAG

Glutamine Gin Q CAA, CAG

Glycine Gly G GGA, GGC, GGG, GGT

Histidine His H CAC, CAT

Isoleucine lie 1 ATA, ATC, ATT

Leucine Leu L CTA, CTC, CTG, CTT, TTA, TTG

Lysine Lys K AAA, AAG

Methionine Met M ATG

Phenylalanine Phe F TTC, TTT

Proline Pro P CCA, CCC, CCG, CCT

Serine Ser S AGC, AGT, TCA, TCC, TCG, TCT

Threonine Thr T ACA, ACC, ACG, ACT

Tryptophan Trp w TGG

Tyrosine Tyr Y TAC TAT

Valine Val V GTA, GTC, GTG, GTT

In a further embodiment, the variant polypeptide having lipase activity is a "mature polypeptide." A mature polypeptide means an enzyme in its final form including any post- translational modifications, glycosylation, phosphorylation, truncation, N-terminal modifications, C-terminal modifications, signal sequence deletion. A mature polypeptide can vary depending upon the expression system, vector, promoter, and/or production process.

In a further embodiment, a lipase is active over a broad pH at any single point within the range from about pH 4.0 to about pH 12.0. In an embodiment, the lipase is active over a range of pH 4.0 to pH 11.0, pH 4.0 to pH 10.0, pH 4.0 to pH 9.0, pH 4.0 to pH 8.0, pH 4.0 to pH 7.0, pH 4.0 to pH 6.0, or pH 4.0 to pH 5.0. In another embodiment the lipase is active at pH 4.0, pH 4.1, pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6 pH 8.7, pH 8.8 pH 8.9, pH 9.0, pH 9.1, pH 9.2, pH 9.3, pH 9.4, pH 9.5, pH 9.6, pH 9.7, pH 9.8, pH 9.9, pH 10.0, pH 10.1, pH

10.2, pH 10.3, pH 10.4, pH 10.5, pH 10.6, pH 10.7, pH 10.8, pH 10.9, pH 11.0, pH 11.1, pH 11.2, pH 11.3, pH 11.4, pH 11.5, pH 11.6, pH 11.7, pH 11.8, pH 11.9, pH 12.0, pH 12.1, pH 12.2, pH

12.3, pH 12.4, and pH 12.5, pH 12.6, pH 12.7, pH 12.8, pH 12.9, and higher. In a further embodiment, a lipase is active over a broad temperature used in at any time during a baking process, wherein the temperature is any point in the range from about 20 °C to about 60°C. In another embodiment, the lipase is active at a temperature range from 20 ° C to 55 ° C, 20 ° C to 50 ° C, 20 ° C to 45 ° C, 20 ° C to 40 ° C, 20 ° C to 35 ° C, 20 ° C to 30 ° C, or 20 ° C to 25° C. In another embodiment the lipase is active at a temperature of at least 19 ° C, 20 ° C, 21 ° C, 22 ° C, 23 ° C, 24 ° C, 25 ° C, 26 ° C, 27 ° C, 28 ° C, 29 ° C, 30 ° C, 31 ° C, 32 ° C, 33 ° C, 34 ° C, 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C, 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 ° C, 45 ° C, 46 ° C, 47 ° C, 48 ° C, 49 ° C, 50 ° C, 51 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C, 56 ° C, 57 ° C, 58 ° C, 59 ° C, 60 ° C, 61 ° C, 62 ° C, or higher temperatures.

"Sequence Identity," "% sequence identity." "% identity," or "Sequence alignment" means a comparison of a first amino acid sequence to a second amino acid sequence, or a comparison of a first nucleic acid sequence to a second nucleic acid sequence and is calculated as a percentage based on the comparison. The result of this calculation can be described as "percent identical" or "percent ID."

Generally, a sequence alignment can be used to calculate the sequence identity by one of two different approaches. In the first approach, both, mismatches at a single position and gaps at a single position are counted as non-identical positions in final sequence identity calculation. In the second approach, mismatches at a single position are counted as non-identical positions in final sequence identity calculation; however, gaps at a single position are not counted (ignored) as non- identical positions in final sequence identity calculation. In other words, in the second approach gaps are ignored in final sequence identity calculation. The differences between these two approaches, counting gaps as non-identical positions vs ignoring gaps, at a single position can lead to variability in sequence identity value between two sequences.

In an embodiment of this disclosure, sequence identity is determined by a program, which produces an alignment, and calculates identity counting both mismatches at a single position and gaps at a single position as non-identical positions in final sequence identity calculation. For example program Needle (EMBOS), which has implemented the algorithm of Needleman and Wunsch (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), and which calculates sequence identity by first producing an alignment between a first sequence and a second sequence, then counting the number of identical positions over the length of the alignment, then dividing the number of identical residues by the length of an alignment, then multiplying this number by 100 to generate the % sequence identity [% sequence identity = (# of Identical residues / length of alignment) x 100)]. In another embodiment of this disclosure, sequence identity can be calculated from a pairwise alignment showing both sequences over the full length, so showing the first sequence and the second sequence in their full length ("Global sequence identity"). For example, program Needle (EMBOSS) produces such alignments; % sequence identity = (# of identical residues / length of alignment) x 100)].

In another embodiment of this disclosure, sequence identity can be calculated from a pairwise alignment showing only a local region of the first sequence or the second sequence ("Local Identity"). For example, program Blast (NCBI) produces such alignments; % sequence identity = (# of Identical residues / length of alignment) x 100)].

In another embodiment, a sequence alignment is calculated with mismatches at a single position are counted as non-identical positions in final sequence identity calculation; however, gaps at a single position are not counted (ignored) as non-identical positions in final sequence identity calculation.

In a preferred embodiment the sequence alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, p. 443-453). Preferably, the program "NEEDLE" (The European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the current invention, with using the programs default parameter (gap open=10.0, gap extend=0.5 and matrix=EBLOSUM62). Then, a sequence identity can be calculated from the alignment showing both sequences over the full length, so showing the first sequence and the second sequence in their full length ("Global sequence identity"). For example,; % sequence identity = (# of identical residues / length of alignment) x 100)].

In another preferred embodiment the preferred alignment program is "NEEDLE" with using the programs default parameter (gap open=10.0, gap extend=0.5 and matrix=EDNAFULL).

According to this invention, enzyme variants may be described as an amino acid sequence which is at least n% identical to the amino acid sequence of the respective parent enzyme with "n" being an integer between 10 and 100. In one embodiment, variant enzymes are at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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% identical when compared to the full length amino acid sequence of the parent enzyme, wherein the enzyme variant has enzymatic activity.

The invention further relates to a polynucleotide encoding the variant polypeptides of the invention. The terms "polynucleotide(s)", "nucleic acid sequence(s)", "nucleotide sequence(s)", "nucleic acid(s)", "nucleic acid molecule" are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length. A "gene" is aDNA segment carrying a certain genetic information.

A "parent" or "template nucleic acid sequence" is a polynucleotide acid sequence is the starting sequence for introduction of mutations to the sequence, resulting in "variants" of said parent polynucleotide sequence. A "variant polynucleotide" refers to a polynucleotide that encodes the same enzyme as the parent polynucleotide does. The variant polynucleotide in this case differs from its parent polynucleotide in its nucleic acid sequence, however the polypeptide encoded remains unchanged.

In an embodiment of the disclosure, the lipase can be used in combination with at least one other enzyme. The other enzyme can be from the same class of enzymes, for example, a composition comprising a first lipase and a second lipase. The other enzyme can also be from a different class of enzymes, for example, a composition comprising a lipase and an amylase. The combination with at least one other enzyme can be a composition comprising at least three enzymes. The three enzymes can have enzymes from the same class of enzymes, for example a first lipase, a second lipase, and a third lipase or the enzymes can be from different class of enzymes for example, a lipase, an amylase, and a xylanase. In another embodiment, the second enzyme comprises or is selected from the group consisting of: an Alpha-amylase; a beta-amylase a Glucan 1, 4-alpha-maltotetraohydrolase, also known as exo-maltotetraohydrolase, G4-amylase; a Glucan 1,4-alpha-maltohydrolase, also known as maltogenic alpha-amylase, a cyclodextrin glucanotransferase, a glucoamylase; an Endo-l,4-beta-xylanase; a xylanase, a cellulase, an Oxidoreductases; a Phospholipase Al; a Phospholipase A2; a Phospholipase C; a Phospholipase D; a Galactolipase, triacylglycerol lipase, an arabinofuranosidase, a transglutaminase, a pectinase, a pectate lyase, a a protease, or any combination thereof. In another embodiment, the enzyme combination is the lipase disclosed herein and a maltogenic alpha-amylase, or the enzyme combination is the lipase disclosed herein, a maltogenic alpha-amylase, and a xylanase.

In another embodiment of the disclosure, the lipase can be a hybrid of more than one lipase enzymes. A "hybrid" or "chimeric" or "fusion protein" means that a domain of a first lipase of the disclosure is combined with a domain of a second lipase to form a hybrid lipase and the hybrid has lipase activity. In one embodiment a domain of a lipase of this disclosure is combined with a domain of a commercially available lipase, such as LIPOPAN (available from Novozymes), or PANAMORE (available from DSM) to form a hybrid lipase and the hybrid has lipase activity.

Industrial enzymes are usually recombinant proteins produced using bacteria, fungi, or yeast expression systems. "Expression system" also means a host microorganism, expression hosts, host cell, production organism, or production strain and each of these terms can be used interchangeably for this disclosure. Examples of expression systems include but are not limited to: Aspergillus niger, Aspergillus oryzae, Hansenula polymorpha, Thermomyces lanuginosus, fusarium oxysporum, Fusarium heterosporum, Escherichia coli, Bacillus, preferably Bacillus subtilis, or Bacillus licheniformis, Pseudomonas, preferably Pseudomonas fluorescens, Pichia pastor is (also known as Komagataella phaffii), Thermothelomyces thermophila (also known as Myceliopthora thermophile (CI)), Schizosaccharomyces pombe, Trichoderma, preferably Trichoderma reesei and Saccharomyces, preferably Saccharomyces cerevisiae. In an embodiment the lipase of this disclosure is produced using the expression system listed above.

Lipases are known to be useful for other industrial applications. In an embodiment of this disclosure, the lipase is used in a detergent. In an embodiment of this disclosure, the lipase is used in personal care products such as contact lens solution. In another embodiment, the lipase of this disclosure is used in the processing of textiles such as leather manufacturing. In another embodiment, the lipase of this disclosure can be used in pulp and paper processing. In a further embodiment, the pulp and paper processing is pitch control, or deinking. In another embodiment, a lipase of this disclosure can be used for manufacturing biodiesel. In another embodiment, a lipase of this disclosure can be used for cheese ripening. In another embodiment, lipases of this disclosure can be used in preparing a meat flavor and/or aroma. In another embodiment, a lipase of this disclosure can be used in the modification of oils & fats. In another embodiment, a lipase of this disclosure can be used in enzymatic oil degumming. In another embodiment, a lipase of this disclosure can be used in the production of ethanol.

The term "baked products" as used herein includes baked products such as bread, loaf bread, pan bread, crispy rolls, sandwich bread, buns, baguette, ciabatta, croissants, noodles, as well as fine bakery wares like donuts, brioche, stollen, cakes, muffins, etc.

The term "dough" as used herein is defined as a mixture of flour, salt, yeast and water, which can be kneaded, molded, shaped or rolled prior to baking. In addition, also other ingredients such as sugar, margarine, egg, milk, etc. might be used. The term includes doughs used for the preparation of baked goods, such as bread, rolls, sandwich bread, baguette, ciabatta, croissants, sweet yeast doughs, etc.

The term "bread volume" as used herein is the volume of a baked good determined by using a laser scanner (e.g. Volscan Profiler ex Micro Stable System) to measure the volume as well as the specific volume. The term also includes the volume which is determined by measuring the length, the width and the height of certain baked goods. The term "comprising" as used herein is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

It is understood that aspects and embodiments of the invention described herein include "consisting" and/or "consisting essentially of aspects and embodiments.

Throughout this disclosure, various aspects are presented in a range format. It should be understood the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Other objects, advantages and features of the present disclosure will become apparent from the following specifications taken in conjunction with the accompanying drawings.

In the following description, numerous specific details are set forth to provide a more thorough understanding of the present disclosure. However, it will be apparent to one of skill in the art that the methods of the present disclosure may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described to avoid obscuring the disclosure.

Example 1: Variant Lipase Enzymes

Non-naturally occurring variant lipase enzymes were created in a lab using rational design single site mutagenesis and multisite mutagenesis. The variant lipase enzymes include single point amino acid modifications, insertions, or deletions of a parent enzyme (LIP062, which is the amino acid sequence of SEQ ID 1, and is encoded by nucleic acid sequence of SEQ ID NO:2) at 18 different amino acid residue positions: 23, 33, 82, 83, 84, 85, 160, 199, 254, 255, 256, 258, 263, 264, 265, 268, 308, 311, or any combination thereof, wherein the variant lipase enzymes has lipase activity.

Variant lipase enzymes were also created with various combinations of the single point modifications of a parent enzyme (LIP062), wherein the variant lipase enzymes have lipase activity. For example, the single point modifications and various combinations of single point modifications are listed in Table: 1.

The table shows a variant lipase enzyme of LIP096, which is a variant polypeptide having the amino acid sequence of LIP062 and one amino acid substitution of A256D, wherein the variant polypeptide has lipase activity. This table also shows a variant lipase enzyme of LIP 182, which is a variant polypeptide having an amino acid sequence of LIP062 and a combination of amino acid substitutions of S83H, I85S, I255A, T264A, and D265T, wherein the variant polypeptide has lipase activity. Table 1, also shows lipase variants of the parent lipase, wherein the variant includes an insertion of an amino acid residue. The insertion of an amino acid residue is shown as ('), for example (84')

Table: 1

Lipase Amino Acid Residue Position Numbers

l i nnrn 23 33 82 83 84 84' 85 160 199 254 255 256 258 263 264 265 268 308 311

Y K S S N - I K P I I A L D T D T

LIP182 H - S - - - A - - - A T -

LIP181 H - V - - - A - - - S T -

LIP180 T - H - - - A - - - - A -

LIP179 V - - - A - - - S T -

LIP178 H - L - - - A - - - s A -

LIP177 H - T - - - A - - - - T -

LIP176 Y . A - - - A - - - - T -

LIP175 T - V - - - A - - - - S -

LIP174 A - - - - A -

LIP173 A - - - - S -

LIP172 N - L - - - A - - - N T -

LIP171 A - - - D T -

LIP170 N - L - - - A - - - - T -

LIP169 N - V - - L - - - - S T -

LIP168 H - - - L - - - - A A -

LIP167 H - - - L - - - - - T -

LIP166 V - - L - - - - D T -

LIP165 Y . - - - - - - - A T -

LIP164 V - - - - - - - D T -

LIP163 Y . A - - - - - - - A T -

LIP162 N - V - - - - - - - N T -

LIP161 N - - - - - - - - D T -

LIP160 H - L - - - - - - - - T -

LIP159 H - A - - - - - - - A T -

LIP158 T - V - - - - - - - - T -

LIP157 H - L - - - - - - - - A -

LIP156 H - V - - - - - - - - A -

LIP155 T - A - - - - - - - - T -

LIP154 H - V - - - - - - - N T -

LIP153 V - - - - - - - - G -

LIP152 H - - - - - - - - - A -

LIP151 Y . V - - - - - - - S S -

LIP150 N - V - - - - - - - - G -

LIP149 H - - - - - - - - - S -

LIP148 H - - - - - - - - - G -

LIP147 H - - - - - - - - - S G

LIP146 H - - - - - - - - - G G

LIP145 A - - - - S G

LIP144 H - - - - A - - - - G -

LIP143 H - - - - A - - - - S G

LIP142 H - - - - A - - - - G G

LIP135 L - - - - - - Table: 1

Lipase Amino Acid Residue Position Numbers

23 33 82 83 84 84' 85 160 199 254 255 256 258 263 264 265 268 308 311

LIP062

K N I K I A L D T D T D Y

LIP134

LIP131 I L S

LIP130 I L G

LIP126

LIP124 - T G G

LIP123 - L G G

LIP120 H - L G

LIP119 - T S G

LIP118 H - L G

LIP117 H - T S G

LIP116 H - L G G

LIP115 H - L S G

LIP114 H - L S

LIP113 - L S

LIP111 A

LIP110 S

LIP109 G

LIP108

LIP102 T

LIP101 P

LIP100 L

LIP099 A

LIP096

LIP095

LIP094

LIP090

LIP089

LIP062J909 T A

LIP062J908 T A

LIP062J907 P A

LIP062J906 P A

LIP062J905 A G G

LIP062J904 A G G

LIP062J903 P G

LIP062J902 P S G

LIP062J901 T S

LIP062J900 T

LIP062J899 P

LIP062J898 P

LIP062J897 G

LIP062J896 S G

LIP062J895 G G

LIP062J894 G G

LIP062J893 G

LIP062J892 G

LIP062J891 S

LIP062J890 G

LIP062J889 S

LIP062J888 G

LIP062J887 G

LIP062 1886 G Table: 1

Lipase Amino Acid Residue Position Numbers

23 33 82 83 84 84' 85 160 199 254 255 256 258 263 264 265 268 308 311

LIP062

Y K S S N - I K P I I A L D T D T D Y

LIP062_ J 885 - - - I - - L S G - -

LIP062_ J 884 - - - I - - T G G - -

LIP062_ J 883 - - - I - - L G G - -

LIP062_ J 882 - H - - T G G - -

LIP062_ J 881 -

LIP062_ J 880 - L

LIP062_ J 879 - P

LIP062_ J 878 - G

LIP062_ J 877 - S

LIP062_ J 876 - K

LIP062_ J 875 - V

LIP062_ J 874 - V

LIP062_ J 873 - L

LIP062_ J 872 - A

LIP062_ J 871 - N

LIP062_ J 870 - K

LIP062_ J 869 - S

LIP062_ J 868 - G

LIP062_ J 867 -

LIP062_ J 866 -

LIP062_ J 865 -

LIP062_ J 864 -

LIP062_ J 863 -

LIP062_ J 862 -

LIP062_ J 861 A

LIP062_ J 857 -

LIP062_ J 856 -

LIP062_ J 855 -

LIP062_ J 854 - E

LIP062_ J 853 - Q

LIP062_ J 852 - T

LIP062_ J 851 - H

LIP062_ J 850 - D

LIP062_ J 849 - V

LIP062_ J 848 - R

LIP062_ J 847 - N

LIP062_ J 846 - G

LIP062_ J 845 - A

LIP062_ J 844 - S

LIP062_ J 843 - M

LIP062_ J 842 - G

LIP062_ J 841 - R

LIP062_ J 840 - F

LIP062_ J 839 - E

LIP062_ J 838 - W

LIP062_ J 837 - L

LIP062_ J 836 - Y

LIP062_ J 835 - S Table: 1

Lipase Amino Acid Residue Position Numbers

23 33 82 83 84 84' 85 160 199 254 255 256 258 263 264 265 268 308 311

LIP062

Y K S S N - I K P I I A L D T D T D Y

LIP062J834 C -

LIP062J833 A -

LIP062J832 V -

LIP062J831 N -

LIP062J830 M -

LIP062J829 S -

LIP062J828 C -

LIP062J827 - N N -

LIP062J826 I

LIP062J825 N - - V - - - A A G

LIP062J824 T - - V - - - A - G

LIP062J823 N - - V - - - A S S

LIP062J822 - - - H - - T - - - A S S

LIP062J820 A A T

UP062J818 - - - Y A - T

UP062J817 A G T

UP062J816 A N A

UP062J814 - - - T - - A - - - A - T

UP062J812 - - - N A - A

UP062J810 - - - T A N T

LIP062J807 A D A

LIP062J805 H - - V - - - A - A

LIP062J804 - - - H A A T

LIP062J803 N - - V - - - A S A

LIP062J801 A - G

LIP062J799 A N T

LIP062J798 Y - - V - - - A N T

LIP062J797 - - - H - - T - - - A - A

LIP062J796 - - - H A A S

LIP062J795 N - - V - - - A N T

LIP062J793 A - T

LIP062J792 Y - - V - - - A S T

LIP062J790 A S S

LIP062J788 N - - L - - - A s G

LIP062J782 - - - N L - N T

LIP062J781 - - - H - - A - - L - A T

LIP062J780 - - - H L - - G

LIP062J779 N - - V - - L - D T

LIP062J778 L - A T

LIP062J776 H - - V - - L - - A

LIP062J775 T - - V - - L - S A

LIP062J774 L - D A

LIP062J773 N - - V - - L - A A

LIP062J770 L - N T

LIP062J768 L - D T

LIP062J767 L - S T

LIP062J766 L - N A

LIP062J704 - - - H A A

LIP062J703 - - - H - - T - A A

LIP062J701 - - - T - - V - G T

LIP062 1700 S T Table: 1

Lipase Amino Acid Residue Position Numbers

23 33 82 83 84 84' 85 160 199 254 255 256 258 263 264 265 268 308 311

LIP062

Y K S S N - I K P I I A L D T D T D Y

LIP062_ J 696 - - - - - - - A T

LIP062_ J 695 - N - V - - - - - - A T

LIP062_ J 694 - - - - - - - - G

LIP062_ J 692 - - - - - - - A T

LIP062_ . 1691 - N - V - - - - - - S S

LIP062_ J 686 - H - V - - - - - - A S

LIP062_ J 685 - N - V - - - - - - N A

LIP062_ J 684 - - - - - - - N T

LIP062_ J 683 - - - - - - - D A

LIP062_ . 1681 - T - - - - - - - N T

LIP062_ J 680 - N - A - - - - - - - T

LIP062_ J 678 - N - - - - - - - - A

LIP062_ J 677 - - - - - - - G T

LIP062_ J 676 - Y . - - - - - - G T

LIP062_ J 674 - - - - - - - G T

LIP062_ J 670 - N - - - - - - - - T

LIP062_ J 669 - - - - - - - S G

LIP062_ J 668 - N - - - - - - - - G

LIP062_ J 667 - A - - - - - - - G

LIP062_ J 665 - - - - - - - D T

LIP062_ J 664 - N - - - L - - - A T

LIP062_ _0450 - F - - - - - - - -

LIP062_ _0449 - Y - - - - - - - -

LIP062_ 0391 - - - - - - - - -

Example 2: Expression and Purification of Lipase Enzymes

Expression

The variant lipase enzymes were obtained by constructing expression plasmids containing the encoding polynucleotide sequences, transforming plasmids into Pichia pastoris (Komagataella phaffii) and growing the resulting expression strains in the following way. Fresh Pichia pastoris cells of the expression strains were obtained by spreading the glycerol stocks of sequence- confirmed strains onto Yeast extract Peptone Dextrose (YPD) agar plates containing Zeocin. After 2 days, starter seed cultures of the production strains were inoculated into 100 mL of Buffered Glycerol complex Medium (BMGY) using cells from these plates, and grown for 20-24 hours at 30°C and 225-250 rpm. Seed cultures were scaled up by transferring suitable amounts into 2-4 L of BMMY medium in a baffled Fermentor. Fermentations were carried out at 30°C and under 1100 rpm of agitation, supplied via flat-blade impellers, for 48-72 hours. After the initial batch-phase of fermentation, sterile-filtered Methanol was added as feed whenever the dissolved oxygen level in the culture dipped below 30%. Alternatively, feed was added every 3 hours at 0.5% v/v of the starting batch culture. The final fermentation broth was centrifuged at 7000xg for 30 mins at 4°C to obtain the cell-free supernatant. Expression levels of the variant lipase enzymes are shown in Table 2, determined as follows: supernatant was assayed for protein of interest expression by either SDS-PAGE or capillary electrophoresis and by enzymatic activity using P P-octanoate as substrate. The results are shown below in Table 2, and the data is shown as a percentage as compared to the parent (LIP062) expression. The expression levels were not determined "n.d." for some of the variant lipase enzymes; however, enough material was generated to move the variant lipase enzyme into the Lipase Activity testing in Example 3, and sent for amino acid sequence identification as described above in Example 1, Table 1.

Table 2 Table 2 Table 2

Lipase Expression Lipase Expression Lipase Expression

LIP062 100 LIP143 200 LIP158 n.d.

LIP089 100 LIP144 100 LIP164 n.d.

LIP090 20 LIP145 100 LIP062_1700 n.d.

LIP094 100 LIP062_1664 n.d. LIP062_1701 n.d.

LIP095 100 LIP062_1665 n.d. LIP152 n.d.

LIP096 100 LIP160 n.d. LIP062_1703 n.d.

LIP062_391 20 LIP062_1667 n.d. LIP062_1704 n.d.

LIPIOI 30 LIP062_1668 n.d. LIP062_1766 n.d.

LIP102 30 LIP062_1669 n.d. LIP062_1767 n.d.

LIP099 30 LIP062_1670 n.d. LIP062_1768 n.d.

LIP100 50 LIP161 n.d. LIP166 n.d.

LIP062_449 20 LIP154 100 LIP062_1770 n.d.

LIP062_450 20 LIP162 n.d. LIP167 n.d.

LIP108 80 LIP062_1674 n.d. LIP168 n.d.

LIP109 70 LIP163 n.d. LIP062_1773 n.d.

LIPllO 65 LIP062_1676 n.d. LIP062_1774 n.d.

LIP111 140 LIP062_1677 n.d. LIP062_1775 n.d.

LIP113 100 LIP062_1678 n.d. LIP062_1776 n.d.

LIP114 62 LIP165 n.d. LIP169 n.d.

LIP115 30 LIP062_1680 n.d. LIP062_1778 n.d.

LIP116 71 LIP062_1681 n.d. LIP062_1779 n.d.

LIP117 24 LIP150 n.d. LIP062_1780 n.d.

LIP118 28 LIP062_1683 n.d. LIP062_1781 n.d.

LIP119 34 LIP062_1684 n.d. LIP062_1782 n.d.

LIP120 80 LIP062_1685 n.d. LIP062_1788 100

LIP123 80 LIP062_1686 n.d. LIP170 100

LIP124 26 LIP155 n.d. LIP062_1790 100

LIP126 200 LIP151 n.d. LIP171 200

LIP134 200 LIP156 n.d. LIP062_1792 200

LIP135 150 LIP153 n.d. LIP062_1793 100

LIP130 49 LIP062_1691 n.d. LIP181 100

LIP131 57 LIP062_1692 n.d. LIP062_1795 100

LIP146 n.d. LIP159 n.d. LIP062_1796 100

LIP147 n.d. LIP062_1694 n.d. LIP062_1797 100

LIP148 n.d. LIP062_1695 n.d. LIP062_1798 100

LIP149 n.d. LIP062_1696 n.d. LIP062_1799 200

LIP142 100 LIP157 n.d. LIP172 50 Table 2 Table 2 Table 2

Lipase Expression Lipase Expression Lipase Expression

LIP062_1801 100 LIP062_1838 100 LIP062_1875 30

LIP173 100 LIP062_1839 50 LIP062_1876 60

LIP062_1803 100 LIP062_1840 50 LIP062_1877 130

LIP062_1804 100 LIP062_1841 90 LIP062_1878 100

LIP062_1805 100 LIP062_1842 20 LIP062_1879 150

LIP174 200 LIP062_1843 40 LIP062_1880 200

LIP062_1807 100 LIP062_1844 110 LIP062_1881 150

LIP175 100 LIP062_1845 65 LIP062_1882 360

LIP178 100 LIP062_1846 110 LIP062_1883 6

LIP062_1810 200 LIP062_1847 90 LIP062_1884 10

LIP176 100 LIP062_1848 200 LIP062_1885 41

LIP062_1812 100 LIP062_1849 40 LIP062_1886 360

LIP177 100 LIP062_1850 40 LIP062_1887 40

LIP062_1814 100 LIP062_1851 90 LIP062_1888 20

LIP179 100 LIP062_1852 80 LIP062_1889 26

LIP062_1816 200 LIP062_1853 200 LIP062_1890 20

LIP062_1817 100 LIP062_1854 80 LIP062_1891 14

LIP062_1818 100 LIP062_1855 20 LIP062_1892 50

LIP180 200 LIP062_1856 10 LIP062_1893 30

LIP062_1820 100 LIP062_1857 50 LIP062_1894 n.d.

LIP182 50 LIP062_1858 10 LIP062_1895 n.d.

LIP062_1822 100 LIP062_1859 90 LIP062_1896 n.d.

LIP062_1823 100 LIP062_1860 10 LIP062_1897 n.d.

LIP062_1824 100 LIP062_1861 45 LIP062_1898 n.d.

LIP062_1825 100 LIP062_1862 65 LIP062_1899 n.d.

LIP062_1826 10 LIP062_1863 100 LIP062_1900 n.d.

LIP062_1827 100 LIP062_1864 70 LIP062_1901 n.d.

LIP062_1828 9 LIP062_1865 70 LIP062_1902 n.d.

LIP062_1829 10 LIP062_1866 160 LIP062_1903 n.d.

LIP062_1830 40 LIP062_1867 200 LIP062_1904 100

LIP062_1831 200 LIP062_1868 80 LIP062_1905 100

LIP062_1832 120 LIP062_1869 100 LIP062_1906 100

LIP062_1833 140 LIP062_1870 75 LIP062_1907 100

LIP062_1834 250 LIP062_1871 85 LIP062_1908 100

LIP062_1835 40 LIP062_1872 100 LIP062_1909 100

LIP062_1836 80 LIP062_1873 65

LIP062_1837 67 LIP062_1874 1

Recovery

After filtering through cheese-cloth, the cell-free supernatants were ultrafiltered using a lab-scale tangential flow filtration (TFF) system with a molecular weight cut-off of 5 kD (SpectrumLabs). Samples were first concentrated 10-20X and then buffer-exchanged 5X into 50 mM HEPES pH 7.5. The resultant retentate was centrifuged at 27000xg for 1 hour, and then sterile filtered through 0.2 μπι filters to remove any production organisms or particulate matter. Total protein content of the final samples was determined using the Braford assay. Lipases were lyophilized to form powder.

Example 3 - Lipase Activity

The activity of the variant lipase enzymes was determined using natural substrates in solution. Natural lipid substrates were prepared at 5 mM final concentration in 0.25 % sodium deoxycholate by sonication. Substrate (15 μΐ,) was mixed with 30 uL fluorescein (0.25 μg/mL in 10 mM CaC12) and 10 μΐ ^ recovered lipase (-1-2 μg/mL) pre-diluted in 5 mM Hepes pH 7.5. Products of lipid hydrolysis were monitored by the drop in fluorescence due to pH change (485 nm/525 nm for excitation/emission), recorded kinetically every 30 seconds for 10 min at 26°C . Activity on a log scale was proportional with the fluorescence change per min. The results are shown below in Table 3, and the data is expressed as percentage of parent (LIP062) fluorescence change at same protein concentration. The activity of the variant lipase enzymes was not determined "n.d." for some of the variant lipase enzymes on some of the substrates; however, enough material was created as described in Example 2, and sent for amino acid sequence identification as described above in Example 1, Table 1.

Table 3

Lipase 1-Olein Galactolipids PC C8-PNP TAGs

LIP062 100 100 100 100 100

LIP089 50 80 95 n.d. 45

LIP090 85 60 70 n.d. 55

LIP094 70 80 15 67 75

LIP095 85 95 10 67 110

LIP096 75 80 25 100 65

LIP062_391 65 90 110 100 110

LIPIOI 50 50 10 61 110

LIP102 80 95 60 90 110

LIP099 70 80 75 150 100

LIP100 70 90 100 100 95

LIP062_449 50 70 65 120 50

LIP062_450 35 60 55 120 40

LIP108 106 125 114 80 100

LIP109 115 171 140 130 120

LIPllO 110 150 140 150 110

LIP111 100 145 90 180 110

LIP113 70 125 100 161 70

LIP114 50 110 90 95 80

LIP115 50 100 100 139 200

LIP116 50 100 60 102 60

LIP117 60 130 90 112 70

LIP118 50 125 100 44 200

LIP119 75 130 100 114 50

LIP120 70 115 85 31 50

LIP123 87 103 89 62 117

LIP124 88 118 147 67 80 Table 3

Lipase 1-Olein Galactolipids PC C8-PNP TAGs

LIP126 84 79 111 40 88

LIP134 93 89 127 n.d. 78

LIP135 85 76 116 n.d. 60

LIP130 66 72 89 78 45

LIP131 74 86 140 180 52

LIP146 60 136 94 n.d. 74

LIP147 69 186 142 n.d. 124

LIP148 78 164 100 n.d. 131

LIP149 57 128 44 n.d. 87

LIP142 64 159 52 n.d. 70

LIP143 81 214 86 n.d. 84

LIP144 46 112 41 n.d. 66

LIP145 76 164 85 n.d. 79

LIP062_1664 n.d. n.d. n.d. n.d. n.d.

LIP062_1665 n.d. n.d. n.d. n.d. n.d.

LIP160 51 104 25 n.d. 102

LIP062_1667 n.d. n.d. n.d. n.d. n.d.

LIP062_1668 n.d. n.d. n.d. n.d. n.d.

LIP062_1669 n.d. n.d. n.d. n.d. n.d.

LIP062_1670 n.d. n.d. n.d. n.d. n.d.

LIP161 51 129 5 n.d. 86

LIP154 54 122 5 n.d. 100

LIP162 60 131 10 n.d. 101

LIP062_1674 n.d. n.d. n.d. n.d. n.d.

LIP163 51 106 10 n.d. 79

LIP062_1676 n.d. n.d. n.d. n.d. n.d.

LIP062_1677 n.d. n.d. n.d. n.d. n.d.

LIP062_1678 n.d. n.d. n.d. n.d. n.d.

LIP165 43 105 6 n.d. 39

LIP062_1680 n.d. n.d. n.d. n.d. n.d.

LIP062_1681 n.d. n.d. n.d. n.d. n.d.

LIP150 69 131 75 n.d. 80

LIP062_1683 n.d. n.d. n.d. n.d. n.d.

LIP062_1684 n.d. n.d. n.d. n.d. n.d.

LIP062_1685 n.d. n.d. n.d. n.d. n.d.

LIP062_1686 n.d. n.d. n.d. n.d. n.d.

LIP155 53 94 9 n.d. 69

LIP151 49 90 40 n.d. 70

LIP156 44 111 22 n.d. 67

LIP153 76 119 118 n.d. 82

LIP062_1691 n.d. n.d. n.d. n.d. n.d.

LIP062_1692 n.d. n.d. n.d. n.d. n.d.

LIP159 50 121 9 n.d. 82

LIP062_1694 n.d. n.d. n.d. n.d. n.d.

LIP062_1695 n.d. n.d. n.d. n.d. n.d.

LIP062_1696 n.d. n.d. n.d. n.d. n.d.

LIP157 49 103 31 n.d. 109

LIP158 81 180 61 n.d. 117

LIP164 56 120 10 n.d. 68 Table 3

Lipase 1-Olein Galactolipids PC C8-PNP TAGs

LIP062_1700 n.d. n.d. n.d. n.d. n.d.

LIP062_1701 n.d. n.d. n.d. n.d. n.d.

LIP152 57 116 24 n.d. 85

LIP062_1703 n.d. n.d. n.d. n.d. n.d.

LIP062_1704 n.d. n.d. n.d. n.d. n.d.

LIP062_1766 29 59 0 n.d. 41

LIP062_1767 39 75 8 n.d. 38

LIP062_1768 27 54 0 n.d. 30

LIP166 34 72 2 n.d. 47

LIP062_1770 29 42 10 n.d. 37

LIP167 59 118 8 n.d. 57

LIP168 44 99 0 n.d. 59

LIP062_1773 42 87 12 n.d. 33

LIP062_1774 22 26 8 n.d. 33

LIP062_1775 35 61 7 n.d. 46

LIP062_1776 30 41 27 n.d. 53

LIP169 59 118 3 n.d. 54

LIP062_1778 41 73 10 n.d. 30

LIP062_1779 20 55 4 n.d. 18

LIP062_1780 47 86 19 n.d. 37

LIP062_1781 39 59 5 n.d. 21

LIP062_1782 24 52 1 n.d. 21

LIP062_1788 51 105 17 n.d. n.d.

LIP170 75 160 20 n.d. 100

LIP062_1790 95 123 128 n.d. n.d.

LIP171 65 138 7 n.d. 81

LIP062_1792 87 117 17 n.d. n.d.

LIP062_1793 94 127 91 n.d. n.d.

LIP181 65 139 16 n.d. 104

LIP062_1795 59 103 8 n.d. n.d.

LIP062_1796 78 120 40 n.d. n.d.

LIP062_1797 51 80 17 n.d. n.d.

LIP062_1798 47 72 0 n.d. n.d.

LIP062_1799 72 100 13 n.d. n.d.

LIP172 44 85 5 n.d. 69

LIP062_1801 105 150 276 n.d. n.d.

LIP173 89 153 138 n.d. 98

LIP062_1803 83 127 15 n.d. n.d.

LIP062_1804 87 115 16 n.d. n.d.

LIP062_1805 67 100 87 n.d. n.d.

LIP174 82 153 73 n.d. 94

LIP062_1807 76 106 10 n.d. n.d.

LIP175 90 179 137 n.d. 106

LIP178 60 109 18 n.d. 92

LIP062_1810 59 90 10 n.d. n.d.

LIP176 86 169 12 n.d. 100

LIP062_1812 78 105 43 n.d. n.d.

LIP177 106 185 168 n.d. 101

LIP062_1814 66 91 67 n.d. n.d. Table 3

Lipase 1-Olein Galactolipids PC C8-PNP TAGs

LIP179 87 148 40 n.d. 113

LIP062_1816 110 141 19 n.d. n.d.

LIP062_1817 48 67 4 n.d. n.d.

LIP062_1818 83 119 17 n.d. n.d.

LIP180 68 99 7 n.d. 100

LIP062_1820 87 125 37 n.d. n.d.

LIP182 63 132 5 n.d. 63

LIP062_1822 51 75 3 n.d. n.d.

LIP062_1823 84 108 109 n.d. n.d.

LIP062_1824 83 113 144 n.d. n.d.

LIP062_1825 88 133 24 n.d. n.d.

LIP062_1826 15 25 25 n.d. 25

LIP062_1827 1 60 0 1 0

LIP062_1828 35 50 40 70 24

LIP062_1829 40 50 3 70 4

LIP062_1830 70 80 90 135 100

LIP062_1831 130 100 100 50 80

LIP062_1832 100 90 80 160 100

LIP062_1833 120 110 90 130 120

LIP062_1834 200 90 60 150 80

LIP062_1835 90 70 80 100 80

LIP062_1836 110 60 55 90 20

LIP062_1837 90 50 40 160 90

LIP062_1838 170 60 40 160 70

LIP062_1839 120 65 50 3 0

LIP062_1840 86 60 60 240 120

LIP062_1841 140 80 60 20 0

LIP062_1842 110 70 70 200 100

LIP062_1843 55 40 25 260 90

LIP062_1844 130 100 70 30 0

LIP062_1845 180 80 24 35 0

LIP062_1846 140 100 80 55 20

LIP062_1847 150 80 20 20 15

LIP062_1848 100 40 70 15 0

LIP062_1849 90 55 60 30 120

LIP062_1850 90 15 10 30 0

LIP062_1851 110 50 0 20 0

LIP062_1852 130 80 80 20 40

LIP062_1853 100 70 45 10 0

LIP062_1854 150 90 30 20 5

LIP062_1855 0 0 0 0 0

LIP062_1856 0 0 0 0 0

LIP062_1857 5 5 5 5 5

LIP062_1858 n.d. n.d. n.d. n.d. n.d.

LIP062_1859 45 50 50 60 35

LIP062_1860 n.d. n.d. n.d. n.d. n.d.

LIP062_1861 21 2 1 26 18

LIP062_1862 76 70 80 235 100

LIP062_1863 76 76 46 100 67 Table 3

Lipase 1-Olein Galactolipids PC C8-PNP TAGs

LIP062_1864 77 87 78 127 68

LIP062_1865 35 22 21 70 34

LIP062_1866 76 81 60 53 75

LIP062_1867 46 47 12 148 47

LIP062_1868 111 159 121 76 88

LIP062_1869 107 154 126 74 87

LIP062_1870 98 20 22 61 8

LIP062_1871 144 112 43 88 86

LIP062_1872 103 122 82 89 104

LIP062_1873 80 12 10 138 78

LIP062_1874 3 0 15 0 13

LIP062_1875 68 55 89 299 85

LIP062_1876 75 70 100 125 125

LIP062_1877 70 70 95 70 110

LIP062_1878 n.d. n.d. n.d. n.d. n.d.

LIP062_1879 n.d. n.d. n.d. n.d. n.d.

LIP062_1880 n.d. n.d. n.d. n.d. n.d.

LIP062_1881 n.d. n.d. n.d. n.d. n.d.

LIP062_1882 109 186 157 69 58

LIP062_1883 47 37 57 73 71

LIP062_1884 64 52 51 117 16

LIP062_1885 53 79 65 201 92

LIP062_1886 93 70 88 197 95

LIP062_1887 60 51 58 47 81

LIP062_1888 74 120 109 50 38

LIP062_1889 59 88 73 73 57

LIP062_1890 64 68 66 52 52

LIP062_1891 57 54 59 135 44

LIP062_1892 63 77 57 28 39

LIP062_1893 41 37 16 12 22

LIP062_1894 97 145 31 n.d. 142

LIP062_1895 54 61 55 n.d. 67

LIP062_1896 90 144 104 n.d. 114

LIP062_1897 45 55 34 n.d. 56

LIP062_1898 37 23 0 n.d. 76

LIP062_1899 36 39 2 n.d. 70

LIP062_1900 63 79 46 n.d. 105

LIP062_1901 54 58 48 n.d. 78

LIP062_1902 40 48 22 n.d. 131

LIP062_1903 29 35 5 n.d. 149

LIP062_1904 62 112 77 n.d. 42

LIP062_1905 34 63 18 n.d. 37

LIP062_1906 18 11 3 n.d. 19

LIP062_1907 26 19 3 n.d. 25

LIP062_1908 32 46 8 n.d. 25

LIP062_1909 47 73 19 n.d. 31

Example 4: Lypolytic enzyme activity in dough assessed by HPLC Simplified doughs were treated with several concentrations of variant lipase enzymes to determine their relative specific activity on flour lipids. Dough was prepared from 1 part flour and 2 parts water containing 34 mg/ml sodium chloride and enzymes at six concentrations: 0.02, 0.04, 0.2, 0.4, 2.0, 4.0 μg enzyme/500 μΐ dough. Doughs were mixed for 5 minutes at 3000 rpm then incubated in a humidity controlled chamber at 30°C for a total of 60 minutes. For lipid analysis, 500ul 1-butanol was added to each sample and the dough was homogenized by vortexing at 3000 rpm for 10 min. The solids were then separated by centrifugation at 4000xg for 5 minutes at room temperature. The organic phase was removed and directly injected for lipid analysis. Lipids were separated by HPLC (Agilent 1100 series) with a silica gel column (Chromolith Performance Si 100-4.6mm, Merck) and analyzed by ELSD (Agilent 1260 Infinity). The chromatographic method for lipid separation was derived from Gerits, et. al. "Single run HPLC separation coupled to evaporative light scattering detection unravels wheat flour endogenous lipid redistribution during bread dough making" LWT-Food Science and Technology, 53 (2013) 426-433. The six enzyme doses and a negative control were used to determine if individual lipid classes (Table 4) increased, decreased or showed no change because of the enzyme treatment.

Table 4: Lipid Classes

Table 5 shows the results of the changes in lipid class measurements relative to the parent enzyme. The Enzyme column of Table 5 lists the parent lipase enzymes (LIP062); and 70 different variant lipase enzymes, wherein the variant lipase enzymes have at least one amino acid modification when compared to the parent enzyme. In Table 5 the lipase variant activity on the substrates TAG, MGDG, DGDG, and NAPE is listed as a % relative to the parent lipase enzyme activity (LIP062). Table 5 also shows the accumulation of products (FFA, MAG, MGMG, and DGMG) for the variant lipase enzymes listed as a % relative to the parent lipase enzyme (LIP062). Table 5: Analysis of Enzyme Activity in dough by HPLC

Enzyme TAG FFA MAG MGDG MGMG DGDG NAPE DGMG

LIP062 100% 100% 100% 100% 100% 100% 100% 100%

LIP061 110% 154% 88% 84% 11% 128% 140% 537%

LIP088 69% 81% 72% 56% 6% 37% 82% 208%

LIP089 19% 47% 24% 74% 27% 50% 44% 13%

LIP090 51% 109% 47% 126% 102% 118% 93% 115%

LIP094 20% 34% 24% 48% 20% 27% 27% 46%

LIP095 39% 78% 67% 111% 66% 83% 94% 89%

LIP096 10% 78% 44% 17% 82% 28% 28% 122%

LIP099 104% 127% 96% 132% 120% 108% 103% 96%

LIP100 134% 190% 111% 283% 227% 201% 161% 371%

UP101 4% 31% 2% 80% 50% 44% 31% 20%

LIP102 69% 112% 62% 105% 76% 89% 100% 86%

LIP108 69% 100% 44% 154% 138% 201% 177% 301%

LIP109 68% 210% 79% 179% 147% 325% 166% 137%

UP110 116% 161% 79% 196% 103% 307% 214% 188%

LIP111 117% 135% 107% 120% 113% 201% 87% 335%

LIP113 95% 227% 65% 276% 364% 361% 177% 798%

LIP114 129% 265% 64% 202% 375% 376% 194% 1090%

LIP115 52% 181% 39% 192% 279% 308% 158% 798%

LIP116 71% 264% 37% 276% 409% 372% 189% 1130%

LIP117 97% 191% 24% 278% 194% 460% 237% 647%

LIP118 70% 283% 45% 185% 360% 370% 177% 924%

LIP119 15% 82% 8% 138% 103% 224% 75% 964%

LIP120 18% 104% 6% 362% 510% 267% 145% 298%

LIP122 2% 127% -8% 88% 7% 290% 15% 1413%

LIP123 52% 141% 24% 177% 282% 246% 113% 540%

LIP124 37% 134% 19% 201% 282% 274% 128% 681%

LIP126 77% 69% 69% 94% 52% 66% 78% 44%

LIP130 104% 174% 86% 167% 235% 258% 117% 563%

LIP131 136% 211% 140% 171% 266% 249% 142% 673%

LIP134 57% 72% 38% 108% 91% 86% 74% 108%

LIP135 110% 97% 70% 96% 111% 102% 89% 141%

LIP142 35% 177% 7% 276% 356% 355% 162% 922%

LIP143 27% 180% 42% 238% 315% 328% 167% 1327%

LIP144 15% 155% 15% 141% 140% 251% 140% 628%

LIP145 19% 142% 33% 238% 205% 282% 125% 804%

LIP146 35% 119% 41% 154% 145% 226% 121% 688%

LIP147 74% 205% 80% 238% 209% 321% 172% 1114%

LIP148 51% 173% 77% 238% 208% 296% 161% 901%

LIP149 42% 199% 19% 134% 116% 314% 184% 644%

LIP150 36% 207% 21% 362% 460% 487% 83% 1915%

LIP151 70% 217% 50% 212% 123% 320% 154% 479%

LIP152 121% 237% 82% 216% 171% 418% 213% 688%

LIP153 88% 144% 55% 179% 138% 296% 154% 485%

LIP155 94% 73% 23% 135% 88% 231% 61% 261%

LIP156 6% 102% 7% 33% 44% 90% 28% 320%

LIP158 143% 260% 313% 238% 319% 323% 152% 915%

LIP159 48% 105% 25% 238% 205% 264% 76% 788%

LIP160 79% 137% 86% 238% 205% 283% 146% 967%

LIP161 14% 112% 23% 238% 182% 282% 26% 840%

LIP162 33% 115% 36% 238% 227% 286% 56% 1048%

UP163 46% 75% 25% 172% 163% 264% 32% 658%

LIP164 73% 60% 23% 123% 64% 251% 63% 18% Table 5: Analysis of Enzyme Activity in dough by HPLC

Enzyme TAG FFA MAG MGDG MGMG DGDG NAPE DGMG

LI P165 25% 157% 7% 131% 125% 357% 17% 1378%

U P166 98% 43% 17% 151% 70% 281% 133% 242%

LI P167 43% 287% 19% 213% 204% 535% 183% 2225%

LI P168 130% 72% 8% 271% 171% 200% 153% 177%

LI P169 131% 170% 9% 271% 193% 486% 99% 819%

LI P170 52% 331% 14% 238% 238% 542% 156% 1956%

U P171 59% 109% 11% 129% 104% 312% 34% 540%

LI P172 2% 180% 0% 141% 119% 346% 20% 375%

LI P173 63% 261% 59% 72% 109% 309% 167% 1076%

LI P174 77% 209% 79% 52% 100% 311% 60% 772%

LI P175 102% 483% 14% 249% 386% 536% 247% 3205%

LI P176 35% 349% 8% 313% 313% 557% 225% 3026%

LI P177 144% 359% 68% 223% 217% 488% 237% 880%

LI P178 25% 219% 8% 234% 234% 526% 92% 1574%

LI P179 27% 85% 47% 165% 181% 293% 67% 821%

LI P180 39% 92% 3% 175% 148% 260% 75% 767%

LI P181 16% 286% 6% 253% 237% 548% 123% 1844%

LI P182 5% 131% 1% 283% 191% 401% 93% 1731%

Example 5: Lipase Specific activity at various pH values

The variant lipase enzymes were diluted at the appropriate concentration in 5 mM Hepes pH 7.5, then further diluted 16-fold into 0.4 mM PNP-octanoate prepared in broad range buffer of pH 6.5 to pH 12.0. The broad range buffer contained: 25 mM Phosphoric acid, 25 mM Citric Acid, 25 mM Boric Acid, 25 mM CAPS, and 50 mM NaCl. For pH 8.0, the buffer was supplemented with 10 mM Tris pH 8.0. Activity was measured at 26 °C by recording the absorbance at 405 nm every 40 seconds for 15 minutes. Activity was corrected for the background (no enzyme) and for the absorbance of PNP at each pH under identical conditions. The results are presented in Table 6, and the data is shown as micrograms PNP/min/mg enzyme.

Table 6: Lipase Specific activity at various pH values (micrograms PN P/min/mg enzyme)

PH 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12

LIP062 801 1964 2972 4409 6258 7196 7621 7673 7378 6299 3027 67

LIP108 1223 2699 3352 4889 6265 6952 7000 6970 6483 5300 2321 641

LIP110 1547 3316 3977 5759 7572 7748 8168 7979 7605 6506 2829 0

LIP117 1659 3930 4859 7023 8399 8633 8837 8873 8541 8320 2057 623

LIP120 698 1565 2137 3495 5004 5837 5555 5517 5592 4415 1547 0

LIP147 2148 4745 5294 7279 8469 8626 8903 8803 8174 7058 3290 0

LIP148 1168 2322 2611 3350 4115 4306 4590 4309 4098 3182 1629 0

LIP151 847 2442 3424 4695 6895 8135 7977 8073 7905 6615 1747 0

LIP152 1397 4578 6733 9080 12819 14112 14469 14616 14249 13107 11190 3022

LIP158 1558 5829 9207 13080 16994 17786 20804 20591 19884 17877 16228 3480

LIP159 1597 6049 11246 16254 21623 23367 23500 24613 24755 23503 19089 3266

LIP160 371 1989 3321 4587 7027 7793 6260 6777 6791 6746 5275 2778

LIP161 658 2525 3417 2617 1354 1144 811 901 1006 1198 1563 2259

LIP162 349 1789 3092 3998 6919 8499 7549 7997 8151 5403 2591 2320

LIP167 865 4272 6674 9515 13479 14562 14354 14609 14067 14220 11058 2655

LIP168 732 3227 5770 8165 12593 14811 14549 15625 15305 14363 12634 2594

LIP170 744 2290 3953 5023 9480 11616 10958 11796 12121 10092 5869 0 Table 6: Lipase Specific activity at various pH values (micrograms PN P/min/mg enzyme)

PH 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12

UP171 721 2069 2412 1304 929 907 774 1072 1523 1649 1807 0

LIP173 632 2700 4481 6758 10606 11937 11603 12338 11999 10876 7959 0

LIP174 566 2145 3620 5323 9161 11201 9839 12847 13283 11964 9053 0

LIP175 1339 4241 7361 10793 15578 16856 16903 18052 17557 13316 11183 0

LIP176 1042 3504 5118 7951 12448 14183 13354 15648 15624 13333 7088 0

LIP180 281 1365 2143 2618 4892 7405 6977 10671 12277 11056 4814 31

LIP181 298 930 1229 1652 3163 4865 3739 5566 7607 6046 3805 31

Example 6: Baking trails

The baking performance of the variant lipase enzymes was tested in a fast straight dough system, the Pistolet test. Ingredients using 2000 g of flour type 550 (Vogtmuhlen Ulertissen), 120 g compressed yeast, 40 g salt, 30 g glucose, 22 g wheat starch, 120 ppm ascorbic acid, 5 ppm Nutrilife AM 100 (fungal alpha-amylase), 200 ppm Nutrilife CS 30 (fungal xylanase, cellulase, fungal alpha-amylase) and 1180 g water were mixed in a Kemper SP 15 spiral mixer for 5.5 minutes at speed 1 and 0.5 minutes at speed 2, to a final dough temperature of 28°C. After a resting for 12 minutes, the dough was scaled to a 1500 g piece, rounded and proofed for another 12 minutes. Afterwards, the dough was divided and rounded into 30 pieces of 50 g each by using an automatic dough divider and rounder. Then the dough pieces were proofed for 35 minutes (normal proof) and 45 minutes (extended proof) at 35°C at relative humidity of 85%. After 12 minutes proofing time, a notch was pressed into the middle of the dough pieces. The proofed dough pieces were baked in a deck oven for 12 minutes at 240°C with 15 seconds steam injection.

The variant lipase enzymes, were tested up to six replicates per variant and the results are described in Fig. 1. These results are reported as an average of the replicates tested. Prior to the baking trials, each enzyme was tested for activity, which can vary between different enzymes, then each enzyme was tested to determine the optimum dosages for that enzyme, and finally the enzymes were added at the optimum dosage. For controls, 10-28 replicates have been used to calculate the average. The dosage for Panamore Golden 2.2 (PG2.2) (DSM) is based upon the manufactures recommendations at 0.68 mg lipase/kg flour. The dosage for DATEM, Lametop LT 552 (BASF), is 0.4% as recommended by the manufacturer. LIP062 is parent lipase enzyme for the lipase variants, used at an optimal dosage of 1 mg lipase/kg flour.

The effects of the variant lipase enzymes on the dough properties and on the final baked goods were compared to the parent lipase (LIP062), a negative control (no DATEM), and to a reference containing 0.4% (based on flour) DATEM (Lametop LT 552). The volume effect was determined by measurement of the length, width and height of 15 rolls in relation to the weight.

The negative control is defined as 0%. Dough properties were evaluated by a skilled master baker and described in comparison to the negative control.