Lipase enzymes

This application is a National Stage application of International Application No. PCT/US2018/031956, filed May 10, 2018, which claims priority to U.S. Provisional Patent Application No. 62/505,500, filed on May 12, 2017.

The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a text file. The name of the text file containing the Sequence Listing is “160782_Seqlisting.txt”, which was created on Oct. 31, 2019 and is 4,334 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.

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.

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, 84′Y, 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.

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: Y23A, K33N, S82T, S83D, S83H, S83I, S83N, S83R, S83T, S83Y, S84S, S84N, 84′Y, 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, 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:1, 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 an 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.

FIG. 1A and FIG. 1B , shows results for baking trials.

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-1,4-beta-xylanase (E.C. 3.2.1.8); Oxidoreductases; Phospholipase A1 (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: WO1993/000924, 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 codons 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. www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi.

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)×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)×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)×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)×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 a DNA 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-1,4-beta-xylanase; a xylanase, a cellulase, an Oxidoreductases; a Phospholipase A1; 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 pastoris (also known as Komagataella phaffii ), Thermothelomyces thermophila (also known as Myceliopthora thermophile (C1)), 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.