Lipase variant

Abstract

The present invention relates to the field of bakery materials. More specifically, the present invention relates to variants of a parent polypeptide, wherein the difference between the variant polypeptide and the parent polypeptide is that the KEX2 protease cleavage site present in the parent polypeptide is not present in the variant polypeptide. The present invention further relates to a method for preparing a dough in which the variant polypeptides disclosed herein are used, and a baked product prepared from the dough.

Description

Detailed Description of the Invention

[0001] [Field] The present invention relates to the field of bakery materials. More specifically, the present invention relates to variants of a parent polypeptide, where the difference between the variant polypeptide and the parent polypeptide is that the KEX2 protease cleavage site present in the parent polypeptide is not present in the variant polypeptide. The present invention further relates to a method for preparing a dough using the variant polypeptide disclosed herein and a baked product prepared from the dough.

[0002] [Background] In the baking industry, for example, in the production of industrial doughs and bread, processing aids are generally used to improve the properties of the dough and / or the baked product. The dough properties that can be improved include stability, gas retention capacity, elasticity, extensibility, adhesiveness, machinability, moldability, properties of frozen dough, etc. The baked product properties that can be improved include loaf volume, crispiness of the crust, firmness, cracking, swelling, oven spring, texture of the bread crumb, structure of the bread crumb, softness of the bread crumb, flavor, relative staleness, and shelf life.

[0003] Dough made from whole grain (also called whole wheat) flour is known to be less stable than dough made from refined white flour. As a result, at the end of fermentation, the whole grain dough loses more expanding gas, and the volume of the baked product from the whole grain dough is smaller compared to the volume of the baked product made from refined white flour dough. In particular, during processing, when the dough is slapped or subjected to impact, the volume of the dough decreases and it may partially collapse.

[0004] The flour contains a specific amount of lipids and free fatty acids, and during the storage of the flour, for example, by the lipolysis of endogenous lipids, the amount of free fatty acids in the flour usually increases. This is mainly significant during the storage of whole grain flour (see, for example, Tait and galliard, J Cereal Sci. 1988, 8: 125-137, and Clayton and Morrison, Sci. Food Agric. 1972, 23, 721-735). The amount of free fatty acids in the flour affects dough properties such as dough stability, as well as the properties, taste, and flavor of baked products made from the dough.

[0005] To improve the properties of the dough or baked product, processing aids such as chemical additives and enzymes are added to the flour and / or dough.

[0006] Chemical additives include emulsifiers that act as dough modifiers, such as esters of diacetyl tartaric acid with mono / diglycerides (DATEM), sodium stearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), or (distilled) mono- and diglycerides (MDG / DMG). Emulsifiers such as DATEM can also be used to increase or control the volume of baked products. There is a growing consumer demand for alternatives to chemical emulsifiers, and thus non-chemical emulsifiers are needed.

[0007] As an alternative to chemical emulsifiers, lipolytic enzymes that act on a substrate to generate emulsifying molecules in situ can be used. For example, lipase is used to completely or partially replace DATEM. International Publication No. WO 1998 / 026057 pamphlet describes phospholipases that can be used in the bread manufacturing process. International Publication No. WO 2009 / 106575 pamphlet describes lipolytic enzymes and their use in the bread manufacturing process.

[0008] There is a continuing need for improved lipolytic enzymes that can be used as processing aids in the preparation of baked products such as whole grain baked products.

Brief Description of the Drawings

[0009]

Figure 1

[0010] [Sequence Listing] SEQ ID NO:1 Reference polypeptide M15 SEQ ID NO:2 Polynucleotide encoding reference polypeptide M15 SEQ ID NO:3 Variant polypeptide M15_002 SEQ ID NO:4 Variant polypeptide M15_004 SEQ ID NO:5 Variant polypeptide M15_006 SEQ ID NO:6 Variant polypeptide M15_008 SEQ ID NO:7 Variant polypeptide M15_055 SEQ ID NO:8 Variant polypeptide M15_056 SEQ ID NO:9 Variant polypeptide M15_057 SEQ ID NO:10 Variant polypeptide M15_066 SEQ ID NO:11 Polynucleotide encoding reference polypeptide M15_002 SEQ ID NO:12 Polynucleotide encoding variant polypeptide M15_004 SEQ ID NO:13 Polynucleotide encoding variant polypeptide M15_006 SEQ ID NO:14 Polynucleotide encoding variant polypeptide M15_008 SEQ ID NO:15 Polynucleotide encoding variant polypeptide M15_055 SEQ ID NO:16 Polynucleotide encoding variant polypeptide M15_056 SEQ ID NO:17 Polynucleotide encoding variant polypeptide M15_057 SEQ ID NO:18 Polynucleotide encoding variant polypeptide M15_066 Sequence of loop (linker) amino acids 305 - 320 of SEQ ID NO: 19 M15 Sequence of loop (linker) substituting amino acids 305 - 320 of SEQ ID NO: 20 M15 Sequence of loop (linker) substituting amino acids 305 - 320 of SEQ ID NO: 21 M15 Sequence of loop (linker) substituting amino acids 305 - 320 of SEQ ID NO: 22 M15 Sequence of loop (linker) substituting amino acids 305 - 320 of SEQ ID NO: 23 M15 Sequence of loop (linker) substituting amino acids 305 - 320 of SEQ ID NO: 24 M15 Sequence of loop (linker) substituting amino acids 305 - 320 of SEQ ID NO: 25 M15 Sequence of loop (linker) substituting amino acids 305 - 320 of SEQ ID NO: 26 M15 Sequence of loop (linker) substituting amino acids 305 - 320 of SEQ ID NO: 27 M15 SEQ ID NO: 28 Reference polypeptide LPV06 SEQ ID NO: 29 Mutant polypeptide R3_072 SEQ ID NO: 30 Mutant polypeptide R3_073 SEQ ID NO: 31 Polynucleotide encoding reference polypeptide LPV06 SEQ ID NO: 32 Polynucleotide encoding mutant polypeptide R3_072 SEQ ID NO: 33 Polynucleotide encoding mutant polypeptide R3_073 SEQ ID NO: 34 Parent polypeptide

[0011] [Summary] Mutants of a parent polypeptide are provided, where the difference between the mutant polypeptide and the parent polypeptide is that the KEX2 protease cleavage site present in the parent polypeptide is not present in the mutant polypeptide.

[0012] Furthermore, polynucleotides encoding the mutant polypeptides are provided.

[0013] Furthermore, a recombinant host cell comprising a polynucleotide is provided.

[0014] Furthermore, a method for producing a variant polypeptide is provided.

[0015] Furthermore, a method for producing a mature polypeptide having lipolytic activity is provided.

[0016] Furthermore, a composition comprising a variant polypeptide is provided.

[0017] Furthermore, the use of a variant polypeptide or composition in the manufacture of food is provided.

[0018] Furthermore, a fabric comprising a variant polypeptide is provided.

[0019] Furthermore, a method for producing a baked product comprising baking a fabric is provided.

[0020] Furthermore, a baked product is provided.

[0021] [Definitions] The term "baked product" refers to a baked food prepared from a fabric. Regardless of white, brown, or whole grains (e.g., whole grain or whole wheat type), examples of baked products include bread (usually in the form of a loaf or roll), French baguette type bread, pastry, croissant, brioche, panettone, pasta, noodles (boiled or (fried) fried), pita bread and other flat breads, tortilla, taco, cake, pancake, muffin, cookie, especially biscuit, donuts including yeast donuts, bagel, pie crust, steamed bread, crispbread, brownie, sheet cake, snack foods (e.g., pretzels, tortilla chips, processed snacks, processed potato chips). Baked products are usually made by baking the fabric at a temperature suitable for making baked products, for example, a temperature between 100°C and 300°C. The baked products disclosed herein can be whole grain or whole wheat bread.

[0022] As used herein, the term "dough" is defined as a mixture of flour and other materials. Usually, the dough is firm enough to be kneaded or rolled out. The dough may be raw, frozen, cooked, or partially baked. Dough is typically made from basic dough ingredients including (grain) flour such as wheat flour or rice flour, water, and optionally salt. In the case of fermented products, baker's yeast is mainly used, and optionally, chemical leavening compounds such as a combination of an acid (generating a compound) and a bicarbonate can be used. Grains from which flour can be made include corn, rice, wheat, barley, sorghum, millet, oats, rye, triticale, buckwheat, quinoa, spelt, emmer wheat, einkorn wheat, durum wheat, and kamut. The term "dough" as used herein also includes batter. Batter is a semi-liquid mixture thin enough to be dropped or poured from a spoon made from one or more flours combined with a liquid such as water, milk, or eggs, used to prepare various foods including cakes.

[0023] The term "premix" should be understood in its conventional meaning, i.e., generally as a mixture of baking agents including flour, starch, maltodextrin, and / or salt, which can be used not only in industrial bread baking factories / facilities but also in retail bakeries. The premix contains a polypeptide having lipase activity as disclosed herein. The premix may contain additives referred to herein.

[0024] Additives are, in most cases, added in powder form. Suitable additives include oxidizing agents (including ascorbic acid, bromate, and azodicarbonamide (ADA)), reducing agents (including L-cysteine), emulsifiers (including mono- and diglycerides, monoglycerides such as glyceryl monostearate (GMS), sodium stearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), polyglycerol esters of fatty acids (PGE), and diacetyl tartaric acid esters of mono- and diglycerides (DATEM), propylene glycol monostearate (PGMS), lecithin, but not limited to these), gums (including guar gum, pectin, and xanthan gum), flavorings, acids (including citric acid, propionic acid), starch, modified starch, humectants (including glycerol), and preservatives.

[0025] As used herein, the term "control sequence" refers to a component involved in the regulation of the expression of a coding sequence in a particular organism or in vitro. Examples of control sequences are transcriptional start sequences, termination sequences, promoters, leaders, signal peptides, propeptides, prepropeptides, or enhancer sequences; Shine-Dalgarno sequences, repressor or activator sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and, if necessary, sequences that enhance protein secretion.

[0026] The term "expression" includes any step required in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0027] An expression vector contains a polynucleotide encoding a polypeptide operably linked to appropriate control sequences (e.g., a promoter, as well as transcription and translation stop signals) for expression and / or translation in vitro. An expression vector can be any vector (e.g., a plasmid or virus) that can conveniently undergo recombinant DNA procedures and can effect the expression of the polynucleotide. The choice of vector can usually depend on the compatibility of the vector with the cell into which it is introduced. The vector can be a linear or closed circular plasmid. The vector can be a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. Alternatively, the vector can be a vector that integrates into the genome when introduced into the host cell and is replicated with the chromosome into which it is integrated. An integrating cloning vector can integrate randomly or at a predetermined target position in the chromosome of the host cell. A vector system can be a single vector or plasmid, or two or more vectors or plasmids, which together contain the entire DNA introduced into the genome of the host cell or a transposon. Vectors preferred for use in bacteria are disclosed, for example, in WO 2004 / 074468 pamphlet.

[0028] A host cell as defined herein is an organism suitable for genetic manipulation and can be cultured at a cell density useful for the industrial production of a target product such as a polypeptide according to the present invention. The host cell can be a host cell found in nature or a host cell derived from a parental host cell after genetic manipulation or classical mutagenesis. Advantageously, the host cell is a recombinant host cell. The host cell can be a prokaryotic host cell, an archaeal host cell, or a eukaryotic host cell. The prokaryotic host cell can be, but is not limited to, a bacterial host cell. The eukaryotic host cell can be, but is not limited to, a yeast, fungal, amoeba, algal, plant, animal, or insect host cell.

[0029] A nucleic acid or polynucleotide sequence is defined herein as a nucleotide polymer comprising at least 5 nucleotides or nucleic acid units. Nucleotides or nucleic acids refer to RNA and DNA. The terms "nucleic acid" and "polynucleotide sequence" are used interchangeably herein. A nucleic acid or polynucleotide sequence is defined herein as a nucleotide polymer comprising at least 5 nucleotides or nucleic acid units. Nucleotides or nucleic acids refer to RNA and DNA.

[0030] The term "polypeptide" refers to a molecule containing amino acid residues linked by peptide bonds and containing more than 5 amino acid residues. The term "protein" as used herein is synonymous with the term "polypeptide" and can also refer to two or more polypeptides. Thus, the terms "protein" and "polypeptide" can be used interchangeably. A polypeptide may optionally be modified (e.g., glycosylation, phosphorylation, acylation, farnesylation, prenylation, sulfonation, etc.) to confer functional groups and a polypeptide that exhibits activity under specific conditions in the presence of a specific substrate may also be called an enzyme. It will be understood that due to the degeneracy of the genetic code, multiple nucleotide sequences can be generated that encode a given polypeptide.

[0031] The polypeptides disclosed herein may be fusion or hybrid polypeptides in which another polypeptide is fused to the N-terminus or C-terminus of a polypeptide or a fragment thereof. A fusion polypeptide is generated by fusing a nucleic acid sequence (or a portion thereof) encoding one polypeptide with a nucleic acid sequence (or a portion thereof) encoding another polypeptide.

[0032] Techniques for generating fusion polypeptides are known in the art and include ligating coding sequences encoding polypeptides such that they are in-frame and the expression of the fusion polypeptide is under the control of the same promoter and terminator. Hybrid polypeptides can include combinations of partial or complete polypeptide sequences obtained from at least two different polypeptides, one or more of which can be heterologous to the host cell. Examples of fusion polypeptides and signal sequence fusions are as described, for example, in WO 2010 / 121933 pamphlet.

[0033] As used herein, the term "isolated polypeptide" means a polypeptide removed from at least one component, e.g., other polypeptide materials naturally associated therewith. An isolated polypeptide can be free of any other impurities. An isolated polypeptide can be at least 50% pure, e.g., at least 60% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 80% pure, at least 90% pure, or at least 95% pure, 96%, 97%, 98%, 99%, 99.5%, 99.9% as determined by SDS-PAGE, or any other analytical method suitable for this purpose and known to those of skill in the art. An isolated polypeptide can be produced by a recombinant host cell.

[0034] As used herein, "mature polypeptide" is defined as the polypeptide in its final form, obtained after translation of mRNA into a polypeptide and post-translational modification of said polypeptide. Post-translational modifications include N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and removal of leader sequences such as signal peptides, propeptides, and / or prepropeptides by cleavage.

[0035] "Mature polypeptide coding sequence" means a polynucleotide encoding a mature polypeptide.

[0036] As used herein, the term "promoter" is defined as a DNA sequence that binds to RNA polymerase and induces the polymerase to initiate transcription at the correct downstream transcription start site of a nucleic acid sequence. Suitable bacterial promoters are disclosed, for example, in WO 2004 / 074468 pamphlet.

[0037] As used herein, the term "recombinant" when used in reference to a nucleic acid or protein indicates that the sequence of the nucleic acid or protein has been modified by human intervention as compared to its natural form. The term "recombinant" when used in reference to a cell such as a host cell indicates that the sequence of the cell's genome has been modified by human intervention as compared to its natural form. The term "recombinant" is synonymous with the term "genetically modified".

[0038] Sequence identity or sequence homology are used interchangeably herein. To determine the percentage of sequence homology or sequence identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. To optimize the alignment between two sequences, gaps may be introduced into either of the two sequences being compared. Such an alignment can be performed over the entire length of the sequences being compared. Alternatively, the alignment may be performed over a shorter length, e.g., over about 20, about 50, about 100, or more nucleic acids / bases or amino acids. Preferably, the alignment is performed over the entire length of the sequences being compared. Sequence identity is the percentage of exact matches between two sequences over the reported alignment region. The percent sequence identity between two amino acid sequences or between two nucleotide sequences can be determined using the Needleman and Wunsch algorithm for the alignment of the two sequences. (Needleman, S.B. and Wunsch, C.D. (1970) J. Mol. Biol. 48, 443-453). This algorithm can align both amino acid sequences and nucleotide sequences. The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. For the purposes of the present invention, the NEEDLE program of the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P., Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp276-277, http: / / emboss.bioinformatics.nl / ). In the case of protein sequences, EBLOSUM62 is used for the substitution matrix. For nucleotide sequences, EDNAFULL is used. The optional parameters used are a gap start penalty of 10 and a gap extension penalty of 0.5.All of these different parameters result in slightly different outcomes, but one skilled in the art will understand that the overall percentage of identity between the two arrays will not vary significantly even when different algorithms are used. After alignment by the program NEEDLE described above, the percentage of sequence identity between the query sequence and the sequence of the present invention is calculated as follows: the number of corresponding positions in the alignment that show identical amino acids or identical nucleotides in both sequences is divided by the total length of the alignment after subtracting the total number of gaps in the alignment. The identity as defined herein can be obtained from NEEDLE using the NOBRIEF option and is labeled "longest identity" in the output of the program.

[0039] The nucleic acid and protein sequences of the present invention can further be used, for example, as "query sequences" for performing searches against public databases to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. A BLAST nucleotide search can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. A BLAST protein search can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See the homepage of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0040] "Synthetic molecules" such as synthetic nucleic acids or synthetic polypeptides are produced by chemical synthesis or enzymatic synthesis in vitro. Synthetic molecules include, but are not limited to, mutant nucleic acids made with the optimal codon usage frequency for the optimal host organism.

[0041] The synthetic nucleic acids can preferably be optimized for codon usage according to the methods described in WO 2006 / 077258 and / or WO 2008 / 000632, which are incorporated herein by reference. WO 2008 / 000632 addresses codon pair optimization. Codon pair optimization is a method by which a nucleotide sequence encoding a polypeptide, which is modified with respect to the codon usage frequency of the nucleotide sequence, particularly with respect to the codon pairs being used, is optimized such that the expression of the nucleotide sequence encoding the polypeptide is improved and / or the production of the encoded polypeptide is improved. A codon pair is defined as a set of two consecutive triplets (codons) in a coding sequence. One skilled in the art will need to adapt the codon usage frequency according to the host species, which may result in variants having a homology significantly deviating from SEQ ID NO: 2, but will still understand that they encode the polypeptides according to the invention.

[0042] As used herein, the terms "variant" or "mutant" can be used interchangeably. These can refer to either a polypeptide or a nucleic acid. Variants include substitutions, insertions, deletions, truncations, base conversions, and / or inversions at one or more positions relative to a reference sequence. Variants can be made, for example, by site-saturation mutagenesis, systematic mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed evolution methods, as well as various other recombinant techniques known to those skilled in the art. Mutant genes of nucleic acids can be artificially synthesized by techniques known in the art.

[0043] [Detailed Description] Variants of the parent polypeptide are provided, where the difference between the variant polypeptide and the parent polypeptide is that the KEX2 protease cleavage site present in the parent polypeptide is not present in the variant polypeptide. The parent polypeptide has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, or or The parent polypeptide has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, and the variant polypeptide contains at least one substitution of an amino acid residue at a position corresponding to any of positions 113, 121, 138, 141, 179, 282, 284, 286, 295 of SEQ ID NO: 34.

[0044] A variant polypeptide is referred to herein as a variant polypeptide disclosed herein or a variant polypeptide. A parent polypeptide is referred to herein as a parent polypeptide disclosed herein, or a parent polypeptide, or a reference polypeptide. Absence herein means that the KEX2 protease cleavage site functions in the parent polypeptide but not in the variant polypeptide, or has delayed (temporally) functionality (i.e., reduced functionality); considering the parent polypeptide, the KEX2 protease cleavage site of the variant polypeptide may be completely absent or partially present, resulting in a non-functional or delayed (temporally) functional KEX2 protease cleavage site. Functionality in relation to the KEX2 protease cleavage site means herein that the Kex2 protease cleavage site is recognized by KEX2 protease within the polypeptide and the polypeptide is cleaved by KEX2 protease. Cleavage by KEX2 protease is usually immediately after the KEX2 protease cleavage site. The term delayed (temporally) functionality (or reduced functionality) means that cleavage is slower / delayed compared to the parent polypeptide containing a (fully) functional KEX2 cleavage site. Most preferably, absence herein means that the KEX2 protease cleavage site is functional in the parent polypeptide and has delayed (temporally) functionality (reduced functionality) in the variant polypeptide; compared to the parent polypeptide, the KEX2 protease cleavage site of the variant polypeptide may be partially present, resulting in a delayed (temporally) functional KEX2 protease cleavage site. Functionality in relation to the KEX2 protease cleavage site means herein that the Kex2 protease cleavage site is recognized by KEX2 protease within the polypeptide and the polypeptide is cleaved by KEX2 protease without delay (temporally).A typical functional KEX2 protease cleavage site is dibasic (RR, KK, KR, or RK) in the parent polypeptide, and a delayed (temporally) functional (reduced functionality) KEX2 protease cleavage site is monobasic (K or R at the 1st or 2nd position of a loop / linker sequence) in the mutant polypeptide, i.e., absence most preferably means a monobasic cleavage site (compared to the parent dibasic cleavage site) where protein cleavage by kex2 protease is delayed (temporally) upon secretion.

[0045] In embodiments herein, the KEX2 protease cleavage site can be any KEX2 protease cleavage site known to those skilled in the art. The KEX2 protease cleavage site can be a monobasic cleavage site or a dibasic cleavage site. The KEX2 protease cleavage site can comprise or consist of the amino acids KK, RR, RK, or KR. The KEX2 protease cleavage site can comprise or consist of the amino acid RR. The parent polypeptide may have one or more KEX2 protease cleavage sites. In an embodiment, the parent polypeptide has one or more KEX2 protease cleavage sites. The mutant polypeptide may remain with one or more KEX2 protease cleavage sites. In an embodiment, the mutant polypeptide has no KEX2 protease cleavage site. The term KEX2 protease cleavage site is used interchangeably herein with the terms KEX2 site, KEX2 cleavage site, and KEX2 protease site.

[0046] As described above, the parent polypeptide has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, or the parent polypeptide has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, and the mutant polypeptide further comprises at least one substitution of an amino acid residue at a position corresponding to any of positions 113, 121, 138, 141, 179, 282, 284, 286, 295 of SEQ ID NO: 34.

[0047] Accordingly, the present invention provides a variant of a parent polypeptide, wherein the difference between the variant polypeptide and the parent polypeptide is that the KEX2 protease cleavage site present in the parent polypeptide is not present in the variant polypeptide, and preferably, the KEX2 protease cleavage site consists of the amino acids KK, RR, RK or KR, the parent polypeptide has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 34.

[0048] Furthermore, the present invention provides a variant of a parent polypeptide, wherein the difference between the variant polypeptide and the parent polypeptide is that the KEX2 protease cleavage site present in the parent polypeptide is not present in the variant polypeptide, and preferably, the KEX2 protease cleavage site consists of the amino acids KK, RR, RK or KR, the parent polypeptide has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, and the variant polypeptide comprises at least one substitution of an amino acid residue at a position corresponding to any of positions 113, 121, 138, 141, 179, 282, 284, 286, 295 of SEQ ID NO: 34. References herein to xx% (e.g., 80%) sequence identity are to be understood as sequence identity calculated over the full-length sequence (i.e., full-length sequence comparison).

[0049] In embodiments herein, the KEX2 protease cleavage site may be present immediately prior to the propeptide within the parent polypeptide. In an embodiment, the propeptide having the KEX2 protease cleavage site immediately prior thereto is located at the C-terminus of the parent polypeptide.

[0050] In the embodiments herein, when the variant polypeptide is produced under the same conditions as the parental polypeptide, the variant polypeptide has a higher yield as compared to the parental polypeptide. The production of the variant polypeptide and the parental polypeptide can be carried out according to any method known to those skilled in the art. Preferably, the variant polypeptide and the parental polypeptide are produced according to the methods shown in the examples herein. The yield can be at least 2% higher, for example, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or at least 1000% higher. The yield of the polypeptide can be defined as the amount of the polypeptide produced. Those skilled in the art know how to determine the yield of the polypeptide. This can be determined, for example, by SDS PAGE, protein measurement, and measurement of absolute activity. In the embodiments herein, the parental polypeptide and / or the variant polypeptide can be a secreted polypeptide. Thus, when the variant polypeptide and the parental polypeptide are secreted polypeptides and the variant polypeptide is produced under the same conditions as the parental polypeptide, the secreted variant polypeptide can have a higher yield as compared to the parental polypeptide as described above herein.

[0051] In the embodiments herein, the variant polypeptide may be a fragment of the parental polypeptide, and the variant polypeptide retains the functional characteristics of the parental polypeptide. When the parental polypeptide is an enzyme, the variant polypeptide or fragment retains the enzyme activity of the (mature) parental polypeptide.

[0052] In the embodiments described herein, the variant polypeptide and the parent polypeptide can be enzymes, for example, enzymes having lipolytic activity. Lipolytic activity is also referred to as lipase activity. The variant polypeptides and parent polypeptides having lipase activity disclosed herein can have any suitable lipase activity such as triacylglycerol lipase, galactolipase, and / or phospholipase activity. The variant polypeptides and parent polypeptides disclosed herein can have phospholipase A1 activity. Lipase activity can be determined according to methods known in the art. In an embodiment, the variant polypeptide and the parent polypeptide are EC 3.1.1.3 triacylglycerol lipase. EC 3.1.1.3 triacylglycerol lipase hydrolyzes the ester bond in triglycerides (also known as triacylglycerols or TAGs).

[0053] In the embodiments described herein, the variant polypeptide, preferably the secreted variant polypeptide, can have at least 80%, 85%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, at least 99% or 100% sequence identity to the sequence of amino acids 31 - 304 of SEQ ID NO: 34. In an embodiment, the variant polypeptide can comprise or consist of amino acids 34 - 304, amino acids 34 - 302, amino acids 34 - 303, amino acids 31 - 302, amino acids 31 - 303, or amino acids 31 - 304 of SEQ ID NO: 34. In an embodiment, the secreted variant polypeptide comprises or consists of amino acids 34 - 304 of SEQ ID NO: 34. The length of the polypeptide, for example, the length of the variant polypeptides disclosed herein, can be determined by any method known to those skilled in the art such as LC-MS analysis. The variant polypeptide can be a biologically active fragment of SEQ ID NO: 34. Biologically active fragments herein include polypeptides that contain fewer amino acids than the sequence of amino acids 31 - 304 of SEQ ID NO: 34 but exhibit the lipase activity of the polypeptide consisting of the sequence of amino acids 31 - 304 of SEQ ID NO: 34.

[0054] In the embodiments herein, the KEX2 protease cleavage site can be removed from the parent polypeptide by any method known to those skilled in the art, resulting in a variant polypeptide. Preferably, the method shown in the examples herein is used. In an embodiment, the KEX2 protease cleavage site is (a) deletion or substitution of at least one amino acid residue of the KEX2 protease cleavage site, or (b) substitution of the KEX2 protease cleavage site with an amino acid motif that does not contain the KEX2 protease cleavage site, optionally together with one or more contiguous amino acids adjacent to the KEX2 protease cleavage site to remove it from the parent polypeptide, resulting in a variant polypeptide. One or more contiguous amino acids adjacent to the KEX2 protease cleavage site are preferably located immediately after the protease cleavage site, i.e., in the C-terminal portion of the KEX2 protease cleavage site.

[0055] In the embodiments herein, the KEX2 protease cleavage site is preferably removed from the parent polypeptide by substituting the linker motif adjacent to the C-terminal propeptide with an amino acid motif selected from the group consisting of essentially or exactly SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26 and 27, resulting in a variant polypeptide. In an embodiment, the linker motif adjacent to the C-terminal propeptide of the parent polypeptide has an amino acid sequence essentially or exactly as shown in SEQ ID NO: 19. In an embodiment, the linker motif adjacent to the C-terminal propeptide of the variant polypeptide has an amino acid sequence essentially or exactly as shown in SEQ ID NO: 24. In a more preferred embodiment, the KEX2 protease cleavage site is preferably removed from the parent polypeptide by substituting the linker motif adjacent to the C-terminal propeptide with an amino acid motif selected from the group consisting of essentially or exactly SEQ ID NOs: 24, 25, 26 and 27, resulting in a variant polypeptide.

[0056] In the embodiments herein, the parent polypeptide may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, at least 99% or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 34.

[0057] In the embodiments herein, the variant polypeptide may include additional amino acid substitutions. The variant polypeptide may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, or more additional amino acid substitutions, deletions and / or insertions, whereby the polypeptide still has the same activity and / or function as the parent polypeptide. The variant polypeptide may include at least one substitution of an amino acid residue at a position corresponding to any of positions 113, 121, 138, 141, 179, 282, 284, 286, 295 of SEQ ID NO: 34. The variant polypeptide may further include amino acid substitutions selected from the group consisting of (D295S and V179M), (D295N and V179M), (D295S, I113H and V179M), (D295G and I113T), (D295N and I113N), (D295N, I113T, V179M, and N112D), (D295S, I113H, V179M, and I284M), (D295N and I113T), (D295S, I113T, V179M, and I284M), (D295S, I113T, V179M, N121D), (D295S, I113T and V179M), (D295S and I113N), and (D295N and I284T), wherein the substitutions are defined with respect to SEQ ID NO: 34.

[0058] In the embodiments herein, the variant polypeptide may include, or consist essentially of or exactly of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9 and 10, and may further include at least one substitution of an amino acid residue at a position corresponding to any of positions 113, 121, 138, 141, 179, 282, 284, 286, 295.

[0059] In the embodiments herein, the variant polypeptide can comprise or consist essentially of or exactly of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, and 10, and further can comprise an amino acid substitution selected from the group consisting of (D295S and V179M), (D295N and V179M), (D295S, I113H and V179M), (D295G and I113T), (D295N and I113N), (D295N, I113T, V179M, and N112D), (D295S, I113H, V179M, and I284M), (D295N and I113T), (D295S, I113T, V179M, and I284M), (D295S, I113T, V179M, N121D), (D295S, I113T and V179M), (D295S and I113N), and (D295N and I284T), where the substitution is defined with respect to SEQ ID NO: 34.

[0060] Alternatively, the present invention provides a variant of a parent polypeptide, where the difference between the variant polypeptide and the parent polypeptide is that the KEX2 protease cleavage site present in the parent polypeptide is not present in the variant polypeptide, and preferably, the KEX2 protease cleavage site consists of the amino acids KK, RR, RK, or KR. The parent polypeptide has at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 28. The sequence shown in SEQ ID NO: 19 is substituted by an amino acid motif selected from the group consisting of SEQ ID NOs: 20, 21, 22, 24, 25, 26, and 27, preferably SEQ ID NOs: 24, 25, 26, or 27.

[0061] In this alternative, the parent polypeptide can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, at least 99%, or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 28.

[0062] Furthermore, polynucleotides encoding the variant polypeptides disclosed in the embodiments herein are provided. In an embodiment, the polynucleotide is codon-optimized or codon pair-optimized. Codon optimization is known to those skilled in the art, and any method known to those skilled in the art can be used. Preferably, the method shown in the examples herein is used. The method is described in detail in WO 2008 / 000632 pamphlet. Also provided is an expression vector comprising a polynucleotide disclosed herein operably linked to at least one control sequence that directs the expression of the polypeptide in a host cell. There are several methods known to those skilled in the art for inserting nucleic acids into nucleic acid constructs or expression vectors, see, for example, Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, NY, 2001. It may also be desirable to manipulate the nucleic acid encoding the polypeptide of the present invention using control sequences such as promoter and terminator sequences. Various promoters that can direct transcription in the host cells of the present disclosure can be used. The promoter sequence can be derived from highly expressed genes. Strong constitutive promoters are well known, and an appropriate one can be selected according to the specific sequence controlled in the host cell. Examples of suitable promoters are described in WO 2009 / 106575 pamphlet, including examples of suitable promoters in filamentous fungi. All the promoters mentioned therein are readily available in the art. Any terminator known to those skilled in the art that functions in the cells disclosed herein can be used. Examples of suitable terminator sequences in filamentous fungi include terminator sequences of filamentous fungal genes, for example, those described in WO 2009 / 106575 pamphlet.

[0063] Furthermore, provided are recombinant host cells comprising the polynucleotides disclosed in the embodiments herein or comprising the expression vectors disclosed in the embodiments herein. The host cell can be a prokaryotic host cell, an archaeal host cell, or a eukaryotic host cell. The prokaryotic host cell can be a bacterial host cell. The eukaryotic host cell can be yeast, a fungus, an amoeba, an alga, a plant, an animal cell, for example, a mammalian or insect cell.

[0064] The eukaryotic cell can be a fungal cell, for example, a yeast cell such as a cell of the genus Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia. The yeast cell can be Kluyveromyces lactis, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica, Pichia pastoris, Candida krusei.

[0065] The eukaryotic cell can be a filamentous fungal cell. Some strains of filamentous fungi are publicly and readily available in several culture collections such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Westerdijk Fungal Biodiversity Institute of the former Centraalbureau Voor Schimmelcultures (CBS-KNAW), Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL), and the All-Russian Collection of Microorganisms of the Russian Academy of Sciences (abbreviation in Russian - VKM, abbreviation in English - RCM) (Moscow, Russia), or the Fungal Genetics Stock Center (FGSC).

[0066] Preferred filamentous fungal cells are species of the genera Acremonium, Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Talaromyces, Rasamsonia, Thielavia, Fusarium or Trichoderma, and for example, Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Acremonium alabamense, Talaromyces emersonii, Rasamsonia emersonii, Chrysosporium lucknowense, Fusarium oxysporum, Myceliophthora thermophila, Trichoderma reesei, Thielavia terrestris, or Penicillium chrysogenum. The filamentous fungal host cell belongs to the genus Aspergillus, for example, the species Aspergillus niger.Strains useful in connection with the present disclosure include Aspergillus niger CBS 513.88 (available from the FGSC under accession number A1513), CBS 124.903, Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 1011, CBS 205.89, ATCC 9576, ATCC 14488 - 14491, ATCC 11601, ATCC 12892, P. chrysogenum CBS 455.95, P. chrysogenum Wisconsin 54 - 1255 (ATCC 28089), Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Thielavia terrestris NRRL 8126, Talaromyces emersonii CBS 124.902, Acremonium chrysogenum ATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC 56765 or ATCC 26921, Aspergillus sojae ATCC 11906, Myceliophthora thermophila C1, Garg 27K, VKM - F3500D, Chrysosporium lucknowense C1, Garg 27K, VKM - F3500D, ATCC 44006, and derivatives thereof.

[0067] The filamentous fungal cell can preferably further contain one or more modifications in its genome, and thus, the mutant filamentous fungal host cell, when measured under the same conditions as compared to the parental host cell, has a deficiency in at least one product selected from glucoamylase (glaA), acid-stable alpha-amylase (amyA), neutral alpha-amylase (amyBI and amyBII - WO 2011009700 pamphlet), alpha-1,3-glucan synthase (preferably, AgsE or AgsE and AgsA, WO 2014013074 pamphlet, WO 2016066690 pamphlet), alpha-amylase AmyC (AmyC), toxins, preferably ochratoxin and / or fumonisin (WO 2011009700 pamphlet), protease transcriptional regulator prtT (WO 00 / 20596 pamphlet, WO 01 / 68864 pamphlet, WO 2006 / 040312 pamphlet and WO 2007 / 062936 pamphlet), PepA, the product encoded by gene hdfA and / or hdfB (WO 2005095624 pamphlet), Sec61 polypeptide (WO 2005123763 pamphlet), non-ribosomal peptide synthase npsE (WO 2012001169 pamphlet), zinc binuclear cluster transcriptional regulator - deficient strain (WO 2018166943 pamphlet and references therein) intracellularly.

[0068] In an embodiment, the expression of a polynucleotide encoding a variant polypeptide is increased as compared to the expression of a polynucleotide encoding a reference polypeptide in cells that are otherwise identical when the recombinant cell containing the polynucleotide is cultured under conditions that promote the expression of the polynucleotide (both cells are cultured under the same conditions). Preferably, the increased expression leads to an increased yield of the variant polypeptide as described above herein. Preferably, the increased yield results in the production of more protein when measured, for example, by protein quantification methods such as Bradford, Biuret, Lowry, BCA, HPLC, UV-VIS (280 nm), etc.

[0069] Preferably, the increase in yield results in the production of a more active protein, as measured, for example, by a lipase enzyme assay suitable for the produced lipase enzyme, which is described, for example, in WO 2018 / 114912 or WO 2018 / 114938.

[0070] Furthermore, provided is a method for producing a mutant polypeptide disclosed herein, the method comprising culturing a host cell disclosed herein under conditions that promote the production of the mutant polypeptide and, optionally, recovering the mutant polypeptide. Those skilled in the art know how to carry out the preparation process of a polypeptide having lipase activity according to the host cell used. Suitable fermentation media usually contain carbon and nitrogen sources. Usually, the fermentation medium has a pH value between 3 and 8. A suitable temperature for culturing the host cell is usually between 25°C and 60°C.

[0071] The host cell can be cultured in a shake flask or in a fermenter having a volume of 10 to 100 cubic meters or more, starting from 0.5 or 1 liter or more. The culturing can be carried out aerobically or anaerobically depending on the requirements of the host cell.

[0072] The mutant polypeptide disclosed herein can be recovered or isolated from the fermentation medium. The recovery or isolation of the polypeptide from the fermentation medium can be carried out, for example, by centrifugation, filtration, and / or ultrafiltration.

[0073] Also provided is a method for producing a polypeptide having lipolytic activity, the method comprising culturing a host cell disclosed herein under conditions that promote the production of a mutant polypeptide, recovering the mutant polypeptide, and activating the mutant polypeptide to obtain the mature polypeptide having lipolytic activity. Preferably, the polypeptide having lipolytic activity has at least 80% identity to the mature polypeptide of SEQ ID NO: 1, SEQ ID NO: 34, SEQ ID NO: 28 or SEQ ID NO: 34, and further comprises at least one substitution of an amino acid residue at a position corresponding to any of positions 113, 121, 138, 141, 179, 282, 284, 286, 295 of SEQ ID NO: 34. In other words, the polypeptide having lipolytic activity has at least 80% identity to amino acids 31-304 of SEQ ID NO: 1, SEQ ID NO: 34, SEQ ID NO: 28 or SEQ ID NO: 34, and further comprises at least one substitution of an amino acid residue at a position corresponding to any of positions 113, 121, 138, 141, 179, 282, 284, 286, 295 of SEQ ID NO: 34 (i.e., the polypeptide having lipolytic activity does not include a signal sequence, an N-terminal propeptide and a C-terminal propeptide). Activation of the mutant polypeptide is obtained, for example, by pH treatment or diacetate treatment as described in the examples herein. The purpose of the pH treatment is to obtain a pH near pH 5, for example, pH 5.0 ± 0.5 at 30 °C, preferably pH 5.0 ± 0.3 at 30 °C, or more preferably pH 5.0 ± 0.1 at 30 °C. The appropriate final diacetate concentration is a concentration of at least 15 g / kg broth. As a result of the activation, a mature polypeptide having lipolytic activity is produced.

[0074] Furthermore, provided is a method for producing a mutant polypeptide disclosed herein, the method comprising a) providing a parent polypeptide comprising a KEX2 protease cleavage site; b) removing the KEX2 protease cleavage site from the parent polypeptide; c) optionally, substituting one or more additional amino acids of the parent polypeptide. d) Preparing the mutant polypeptide resulting from steps a) to c); e) Optionally, recovering the mutant polypeptide; and

[0075] Those skilled in the art know methods for removing amino acid sequences from the parent polypeptide. Preferably, the methods shown in the examples herein are used.

[0076] The removal can include substitutions, insertions, deletions, truncations, base conversions, and / or inversions at the KEX protease cleavage site. Such modifications can be carried out, for example, by site-saturation mutagenesis, systematic mutagenesis, insertion mutagenesis, random mutagenesis, site-directed mutagenesis, and directed evolution methods, as well as various other recombinant techniques known to those skilled in the art. The mutant polynucleotide can be artificially synthesized by techniques known in the art. Such processes can include expressing the gene encoding the mutant polypeptide in a suitable recombinant host cell, culturing the host cell, and producing the mutant polypeptide.

[0077] Preferably, step b) includes replacing the KEX2 protease cleavage site from the parent polypeptide by replacing SEQ ID NO: 19 with SEQ ID NO: 24, 25, 26, or 27. As disclosed in the experimental part herein, such substitutions result in surprisingly high expression levels.

[0078] Preferred is the method for producing the mutant polypeptide disclosed herein, the method comprising a) Providing a parent polypeptide comprising a KEX2 protease cleavage site; b) Removing the KEX2 protease cleavage site from the parent polypeptide; c) Optionally, substituting one or more additional amino acids of the parent polypeptide; d) Preparing the mutant polypeptide resulting from steps a) to c); e) Optionally, a step of recovering the mutant polypeptide and comprising The parent polypeptide has at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 34, or The parent polypeptide has at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 34, and the mutant polypeptide comprises at least one substitution of an amino acid residue at a position corresponding to any of positions 113, 121, 138, 141, 179, 282, 284, 286, 295 of SEQ ID NO: 34, or The parent polypeptide has at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 28, or The parent polypeptide has at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0079] In embodiments herein, the KEX2 protease cleavage site is preferably removed from the parent polypeptide by substituting the linker motif adjacent to the C-terminal propeptide with an amino acid motif selected from the group consisting of or consisting essentially of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26 and 27 (step b) of the above method), resulting in a mutant polypeptide. In an embodiment, the linker motif adjacent to the C-terminal propeptide of the parent polypeptide has an amino acid sequence essentially or exactly as set forth in SEQ ID NO: 19. In an embodiment, the linker motif adjacent to the C-terminal propeptide of the mutant polypeptide has an amino acid sequence essentially or exactly as set forth in SEQ ID NOs: 24, 25, 26 or 27.

[0080] Alternatively, a method for generating a mutant polypeptide disclosed herein is provided, the method comprising a) providing a parent polypeptide comprising a KEX2 protease cleavage site, and b) removing the KEX2 protease cleavage site from the parent polypeptide, c) optionally, substituting one or more additional amino acids of the parent polypeptide; d) preparing a mutant polypeptide resulting from steps a) to c); e) optionally, recovering the mutant polypeptide comprising, the parent polypeptide has at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 28; the sequence shown in SEQ ID NO: 19 is substituted by an amino acid motif selected from the group consisting of SEQ ID NOs: 20, 21, 22, 24, 25, 26 and 27, preferably SEQ ID NOs: 24, 25, 26 or 27, most preferably SEQ ID NO: 24 or 26.

[0081] Furthermore, provided is a composition comprising a variant polypeptide disclosed in the embodiments herein, the composition further comprising one or more compounds selected from the group consisting of milk powder, gluten, granulated fat, additional enzymes, amino acids, salts, oxidizing agents, reducing agents, emulsifiers, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids, diacetyl tartaric acid esters of mono- and diglycerides, gums, flavorings, acids, starches, modified starches, humectants, and preservatives. The compositions disclosed herein can be solid or fluid compositions. The compositions disclosed herein can include one or more compounds selected from the group consisting of milk powder, gluten, granulated fat, additional enzymes, amino acids, salts, oxidizing agents, reducing agents, emulsifiers, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids, diacetyl tartaric acid esters of mono- and diglycerides, gums, flavorings, acids, starches, modified starches, humectants, and preservatives. The term composition includes premixes.The compositions disclosed herein may include one or more additional enzymes, such as amylases, e.g., alpha - amylase, e.g., fungal alpha - amylase (which may be useful for providing sugars fermentable by yeast), beta - amylase, etc.; glucanotransferase; peptidases, particularly exopeptidases (which may be useful for enhancing flavor); transglutaminase; cellulase; hemicellulase, particularly pentosanase, e.g., xylanase (which may be useful for the partial hydrolysis of pentosans, more specifically arabinoxylan, to increase the extensibility of the dough); protease (which may be useful for weakening gluten, particularly when using hard flour); protein disulfide isomerase, e.g., the protein disulfide isomerase disclosed in WO 95 / 00636; glycosyltransferase; peroxidase (which may be useful for improving the viscosity of the dough); laccase; oxidases such as hexose oxidase, glucose oxidase, aldose oxidase, pyranose oxidase; lipoxygenase; L - amino acid oxidase (which may be useful for improving the viscosity of the dough), and / or asparaginase.

[0082] Furthermore, provided is the use of a variant polypeptide disclosed herein or a composition disclosed herein for the manufacture of foodstuffs, preferably in the manufacture of dough and / or baked products. Such use may typically involve adding the variant polypeptide disclosed herein or the composition disclosed herein to the dough. In embodiments, such use involves replacing at least a portion of a chemical emulsifier in the manufacture of dough and / or baked products. The at least a portion may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, at least 99%, or 100%.

[0083] Furthermore, there is provided a fabric comprising a variant polypeptide disclosed herein, a variant polypeptide obtained by a method for producing a variant polypeptide disclosed herein, or a composition disclosed herein. The fabric or baked product disclosed in the embodiments herein may comprise any suitable flour, such as whole grain flour, or a mixture of various flours. The flour may also include bran, grains and / or seeds. Grains include corn, rice, wheat, barley, sorghum, millet, oats, rye, triticale, buckwheat, quinoa, spelt, emmer wheat, einkorn wheat, durum wheat and kamut. Whole grain flour, also known as whole wheat flour, is flour made from the entire wheat grain or cereal including the outer portion. The flour may have a free fatty acid content between 0.01 and 0.8 w / w%, for example between 0.05 and 0.6 w / w%, for example between 0.1 and 0.5 w / w%, or between 0.14 and 0.4 w / w%. During storage, the content of free fatty acids in the flour usually increases. The free fatty acids can be, for example, linoleic acid (C18:2), palmitic acid (C16:0), oleic acid (C18:1), linolenic acid (C18:3) (see, for example, MacMurray and Morrison, J. Sci. Food Agr. 21:520-528 (1970)). The free fatty acids in the flour can be determined by methods known to those skilled in the art, as disclosed, for example, in Fierens et al, J. of Cereal Science 65 (2015), p. 81-87.

[0084] Furthermore, there is provided a method for producing a fabric, the method comprising the step of mixing an effective amount of a variant polypeptide disclosed herein, an effective amount of a variant polypeptide obtained by a method disclosed herein, or an effective amount of a composition disclosed herein with at least one fabric material such as the materials described hereinabove.

[0085] Furthermore, a method for manufacturing a baked product is provided, which method includes baking the dough disclosed herein. Also provided is a baked product obtainable by the method disclosed herein. In embodiments herein, the baked product can be a bread, a cake, or a baked product prepared from laminated dough. The baked product can have an improvement in at least one property selected from the group consisting of an increase in volume, an improvement in flavor, an improvement in the structure of the bread body, an improvement in the softness of the bread body, an improvement in crispiness, a reduction in swelling, and an improvement in anti-staling.

[0086] In embodiments herein, the dough can include whole grains, and / or can include flour having a free fatty acid content between 0.01 and 0.8 w / w%, such as between 0.05 and 0.6 w / w%, such as between 0.1 w / w% and 0.5 w / w% or between 0.2 and 4 w / w% free fatty acids. Fatty acids in grains or cereals are known and include palmitic acid, oleic acid, linoleic acid, and / or linolenic acid. The flour can be stored flour, such as flour stored for 1 day to 10 years, such as flour stored for 1 month to 5 years, or flour stored for 2 months to 1 year.

[0087] [Further Embodiments of the Invention] 1. A variant of a parent polypeptide, wherein the difference between the variant polypeptide and the parent polypeptide is that the KEX2 protease cleavage site present in the parent polypeptide is not present in the variant polypeptide.

[0088] 2. The variant polypeptide according to embodiment 1, wherein the KEX2 protease cleavage site consists of the amino acids KK, RR, RK, or KR.

[0089] 3. The variant polypeptide according to embodiment 1 or 2, wherein the KEX2 protease cleavage site is present immediately before the propeptide within the parent polypeptide, and preferably, the propeptide having the KEX2 protease cleavage site immediately before it is located at the C-terminus of the parent polypeptide.

[0090] 4. A variant polypeptide as described in any one of the preceding embodiments, which, when produced under the same conditions as the parent polypeptide, has a higher yield compared to the parent polypeptide.

[0091] 5. A variant polypeptide as described in any one of the preceding embodiments, wherein the variant polypeptide and the parent polypeptide have lipolytic activity, preferably EC 3.1.1.3 triacylglycerol lipase.

[0092] 6. The KEX2 protease cleavage site is (a) a deletion or substitution of at least one amino acid residue of the KEX2 protease cleavage site, or (b) a substitution of the KEX2 protease cleavage site with an amino acid motif that does not contain the KEX2 protease cleavage site, optionally together with one or more consecutive amino acids adjacent to the KEX2 protease cleavage site A variant polypeptide as described in any one of the preceding embodiments, which is removed by

[0093] 7. The variant polypeptide according to embodiment 6, wherein the linker motif adjacent to the C-terminal propeptide is substituted by an amino acid motif selected from the group consisting of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26 and 27.

[0094] 8. The variant polypeptide according to embodiment 7, wherein the linker motif adjacent to the C-terminal propeptide of the parent polypeptide has the amino acid sequence set forth in SEQ ID NO: 19.

[0095] 9. A variant polypeptide as described in any one of the preceding embodiments, wherein the parent polypeptide has at least 60% sequence identity with the amino acid sequence set forth in SEQ ID NO: 34.

[0096] 10. The variant polypeptide is the variant polypeptide according to any one of the preceding embodiments, comprising at least one substitution of an amino acid residue at a position corresponding to any of positions 113, 121, 138, 141, 179, 282, 284, 286, 295 of SEQ ID NO: 34.

[0097] 11. The variant polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9 and 10, and preferably comprises at least one substitution of an amino acid residue at a position corresponding to any of positions 113, 121, 138, 141, 179, 282, 284, 286, 295, which is the variant polypeptide according to any one of the preceding embodiments.

[0098] 12. A polynucleotide encoding the variant polypeptide according to any one of embodiments 1 to 11, preferably a polynucleotide that is codon-optimized.

[0099] 13. A recombinant host cell comprising the polynucleotide according to embodiment 12.

[0100] 14. A method for producing the variant polypeptide according to any one of embodiments 1 to 11, comprising culturing the host cell of embodiment 13 under conditions that promote the production of the variant polypeptide and recovering the variant polypeptide.

[0101] 15. A method for producing a variant polypeptide, a) providing a parent polypeptide comprising a KEX2 protease cleavage site; b) removing the KEX2 protease cleavage site from the parent polypeptide; c) optionally, substituting one or more additional amino acids of the parent polypeptide; d) preparing the variant polypeptide resulting from steps a) to c); e) optionally, recovering the variant polypeptide and comprising the method.

[0102] 16. A composition comprising a variant polypeptide obtainable by the method according to any one of Embodiments 1 to 11 or Embodiment 15, further comprising one or more compounds selected from the group consisting of milk powder, gluten, granular fat, additional enzymes, amino acids, salts, oxidizing agents, reducing agents, emulsifiers, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids, diacetyl tartaric acid esters of mono- and diglycerides, gums, flavorings, acids, starches, modified starches, humectants, and preservatives.

[0103] 17. Use of the variant polypeptide according to any one of Embodiments 1 to 11 or the composition according to Embodiment 16 in the manufacture of food, preferably in the manufacture of dough and / or baked products.

[0104] 18. The use according to Embodiment 17, wherein the use comprises replacing at least a part of a chemical emulsifier in the manufacture of dough and / or baked products.

[0105] 19. Dough comprising the variant polypeptide according to any one of Embodiments 1 to 11, the variant polypeptide obtainable by the method according to Embodiment 15, or the composition according to Embodiment 16.

[0106] 20. A method for manufacturing dough, comprising the step of mixing an effective amount of the variant polypeptide according to any one of Embodiments 1 to 11, an effective amount of the variant polypeptide obtainable by the method according to Embodiment 15, or an effective amount of the composition according to Embodiment 16 with at least one dough material.

[0107] 21. A method for manufacturing a baked product, comprising baking the dough according to Embodiment 19 or the dough obtained by the method according to Embodiment 20.

[0108] 22. A baked product obtainable by the method according to Embodiment 21.

[0109] 23. The baked product according to embodiment 22, wherein the product is a baked product prepared from bread, cake, or laminated dough.

[0110] 24. The baked product according to embodiment 22 or 23, having an improvement in at least one property selected from the group consisting of an increase in volume, an improvement in flavor, an improvement in the structure of the bread body, an improvement in the softness of the bread body, an improvement in crispiness, a reduction in swelling, and an improvement in anti-aging.

[0111] 25. The use according to embodiment 17 or 18, the dough according to embodiment 19, the method according to embodiment 20 or 21, or the baked product according to embodiment 22, 23 or 24, wherein the dough contains whole grain flour and / or flour having a free fatty acid content between 0.01 and 0.8 w / w%.

[0112] [Examples] Standard genetic techniques such as overexpression of enzymes in host cells, genetic modification of host cells, or hybridization techniques are methods known in the art, for example, as described in Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York (1987). Methods for transformation, genetic modification, etc. of fungal host cells are known, for example, from European Patent No. 0635574, International Publication No. 98 / 46772 pamphlet, International Publication No. 99 / 60102 pamphlet and International Publication No. 00 / 37671 pamphlet, International Publication No. 90 / 14423 pamphlet, European Patent No. 0481008, European Patent No. 0635574, and U.S. Patent No. 6,265,186.

[0113] [Materials and Methods] [Inc.] WT1: This Aspergillus niger strain is used as a wild-type strain. This strain is deposited at the CBS Institute under the deposit number CBS513.88. GBA306: The construction of GBA306 using WT1 as the starting strain is described in detail in WO 2011 / 009700 pamphlet. This GBA306 strain has the following genotype: ΔglaA, ΔpepA, ΔhdfA, adapted BamHI amplicon, ΔamyBII, ΔamyBI, and ΔamyA.

[0114] [Vector] The pGBTOP-16 vector described in WO 15177171A1 pamphlet, WO 16097270A1 pamphlet, and WO 16 / 193292A1 pamphlet was modified to enable Golden Gate cloning (New England Biolabs). Four BsaI sites present in pGBTOP-16 were removed, and two BsaI sites were introduced, one at the 3’ end of the PglaA (promoter) fragment and one at the 5’ end of the 3’glaA (terminator) fragment to enable cloning. As a result, the vector pGBTOP-18 was obtained (Figure 1).

[0115] [Production of the lipase of the present invention in shake flasks] As shown, lipolytic enzymes were produced in shake flasks by growing the A. niger strain. As described in detail in Example 1 of WO 2009 / 106575 pamphlet, spores were inoculated in a preculture of CSL medium, followed by culturing in CSM medium at 34°C for 4 days to obtain broth samples and (cell-free) supernatant samples for subsequent analysis.

[0116] [Protein content (Bradford / SDS-PAGE)] The protein content was determined using the Bradford assay (Bradford, M.M. (1976), “Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding”, Anal. Biochem. 72:248-254) in combination with SDS-PAGE verification, and it was confirmed that the sample showed a purity of over 80% based on band intensity.

[0117] [pH adjustment] The pH adjustment of the phospholipase sample was carried out at the end of fermentation of both the broth sample and the supernatant sample after shake flask fermentation by incubating the sample at the indicated temperature, pH and time. For pH adjustment, the pH of 50 g of broth or 50 g of supernatant obtained from the flask (obtained by collecting the supernatant after 10 minutes of centrifugation) was adjusted to a set point of pH 5.0 ± 0.1 at 30 °C with 4 N NaOH or 3.5 M phosphoric acid. As a result of pH adjustment with alkali / acid, the phospholipase activity was corrected / normalized against the dilution factor.

[0118] [Diacetate treatment] For the diacetate treatment, a 350 g / kg sodium diacetate stock solution was added to the sample containing approximately 50 g of broth or 50 g of supernatant (see “pH treatment” above) until a final sodium diacetate concentration of 15 g / kg was reached. The diacetate treatment was tested at pH 5.0 ± 0.1 at 30 °C for the indicated incubation time. As a result of the addition of the diacetate solution and / or pH adjustment with alkali / acid, the phospholipase activity was corrected / normalized against the dilution factor.

[0119] [Phospholipase enzyme activity assay (PLE)] The PLE phospholipase enzyme activity was measured using the following solutions: - A substrate solution of 0.7% (w / v) L-α-phosphatidylcholine (from egg yolk) in 60 mM acetate buffer pH 5.5 (also containing 1.7% Triton X-100) (Sigma 61755, Zwijndrecht, the Netherlands) - Reagents R1 and R2 prepared according to the instructions described in the attached document of the Wako HR series NEFA-HR(2) diagnostic kit (FUJIFILM / Wako Chemicals).

[0120] The activity analysis was carried out using a programmed Konelab analyzer (Thermo-Fisher) as detailed in the example section (lipolytic enzyme variants) of WO 18114912 pamphlet. The reaction sequence was initiated by equilibrating 40 μL of the substrate solution for 2 minutes and reaching 37 °C. Subsequently, 10 μL of the enzyme solution having an activity between 0.015 - 0.075 U / mL was added and incubated at 37 °C for 20 minutes. Next, to determine the free fatty acid content, 130 μL of NEFA-R1 reagent was added and incubated for 2 minutes, then 65 μL of NEFA-R2 reagent was added and incubated for 3 minutes. At the end of the incubation, the absorbance was measured at 540 nm. The reaction sequence was modified as follows to measure the blank: 130 μL of NEFA-R1 reagent was equilibrated at 37 °C for 65 seconds, then incubated with 10 μL of the enzyme solution for 65 seconds, then incubated with 40 μL of the substrate for 2 minutes, and then incubated with 65 μL of NEFA-R2 reagent for 3 minutes. At the end of the incubation, the absorbance was measured at 540 nm. A 1.0 mM oleic acid standard (NEFA standard 270-77000 / FUJIFILM / Wako Chemicals) was also analyzed in the same way to determine the response coefficient.

[0121] The activity was calculated as follows:

Equation

[0122] 1 U PLE is defined as the amount of enzyme that releases 1 micromole of free fatty acid per minute under the test conditions (pH 5.5 / 37 °C).

[0123] [Example 1: Cloning and expression of lipase variants] [Example 1.1. Cloning and expression] The protein sequence (amino acid sequence) of the reference polypeptide (also called M15) is SEQ ID NO: 1. This polypeptide sequence is composed of a signal peptide 1-15 (bold), an N-terminal propeptide 16-30 (underlined), a mature polypeptide 31-304 with lipolytic activity (plain), and a C-terminal propeptide 307-346 (underlined); the Kex2 protease cleavage site is indicated by bold underlining.

[0124] [SEQ ID NO: 1 (346 amino acids)]

Chemical formula

[0125] The codon-optimized DNA sequences for the expression of lipase proteins (lipase variants and reference polypeptides) in Aspergillus niger were designed to contain additional BsaI type II restriction enzyme sites (bold) to enable subcloning in the Aspergillus expression vector pGBTOP-18. Codon optimization was carried out as described in WO 2008 / 000632 pamphlet. The codon-optimized DNA sequence for the expression of the gene encoding the reference polypeptide of SEQ ID NO: 1 in A. niger is shown in SEQ ID NO: 2.

[0126] [SEQ ID NO: 2]

Chemical formula

[0127] In addition, polypeptide variants named M15_002, M15_004, M15_006, M15_008, M15_055, M15_056, M15_057 and M15_066 were designed to have modifications in the loop (linker) of the KEX2 protease cleavage site and the adjacent C-terminal propeptide, amino acids 305 - 320 of SEQ ID NO: 1. The amino acid RRYRSAESVDKRATMT was replaced with the sequences described in Table 1 to obtain polypeptides having the amino acid sequences shown in SEQ ID NOs: 3 - 10.

[0128]

Table 1

[0129] BsaI type II restriction enzyme sites were introduced into the codon-optimized DNA sequences for the expression of the genes encoding the polypeptide variants named M15_002, M15_004, M15_006, M15_008, M15_055, M15_056, M15_057 and M15_066 in A. niger to enable subcloning in pGBTOP-18 (Figure 1). Following standard procedures, DNA fragments SEQ ID NO: 2 and SEQ ID NOs: 11 - 18 were cloned into pGBTOP-18 through iterative steps of BsaI digestion and ligation (GoldenGate cloning method (New England Biolabs)). As a result, vectors containing the lipase expression cassette under the control of the glucoamylase promoter gave rise to vectors pGBTOP-M15 and pGBTOP-M15_02 - pGBTOPM15_66, respectively.

[0130] Subsequently, using the strains and methods described in WO 2011 / 009700 pamphlet and the references therein, in the co-transformation protocol with pGBAAS-4, A. niger GBA306 was transformed with pGBTOP-M15, pGBTOP-M15_02 to pGBTOP-M15_66, selected in acetamide-containing medium, and colonies were purified according to standard procedures. Transformation and selection were carried out as described in WO 98 / 46772 pamphlet and WO 99 / 32617 pamphlet. Eight transformants expressing the same gene variant were selected as representative transformants, and replica plating was further performed to obtain an inoculation spore suspension of a single strain.

[0131] [Example 2: Fermentation of A. niger Strains Expressing Lipase] Fresh A. niger spores were prepared from the parent strain GBA306 and the GBA306-M15 and GBA306-M15_02 to GBA306-M15_66 expression strains of M15 and M15_02 to M15_66, and using these, sample materials were generated by culturing the strains in 24 deep well plates containing 3 ml of fermentation medium (15% w / v maltose, 6% w / v bacto-soytone, 1.5% w / v (NH4)2SO4, 0.1% w / v NaH2PO4.H2O, 0.1% w / v MgSO4.7H2O, 0.1% w / v L-arginine, 8 per mil w / v Tween-80, 2 per mil w / v Basildon, 2% w / v MES, pH 5.1). After culturing for 6 days at 34 °C, 550 rpm and 80% humidity in a Microton incubator shaker (Infors AG, Bottmingen, Switzerland), 1.5 mL of the sample was taken, the mycelium was separated from the supernatant by centrifugation at 4000 g for 30 minutes, and the supernatant was stored at -20 °C until further analysis. The stored mutant and control supernatant samples were loaded onto the same Coomassie-stained SDS PAGE gel in equal amounts at the same dilution, and then the enzyme expression levels in the supernatant were compared by comparing the band intensities of the 28 - 49 kDa ladder bands with the band of control SEQ ID NO: 1 (Table 2). Thermo Fischer's SeeBlue® Plus2 Pre-Stained Standard was used as the pre-stained ladder in this experiment.

[0132] As can be clearly observed, due to the absence of the parental KEX2 protease cleavage site in the generated lipase polypeptide, surprisingly, in all cases, the yield of the generated polypeptide was significantly increased compared to the reference (M15) polypeptide with the KEX2 protease cleavage site.

[0133]

Table 2

[0134] [Example 3: Cloning and Expression of the Second Enzyme LPV06 and Lipase Variants] The second parent polypeptide (also called LPV06), SEQ ID NO: 28, contains a signal peptide (amino acids 1 - 15), an N-terminal propeptide (amino acids 16 - 30), a mature protein with lipolytic activity and a Kex2 protease cleavage site (amino acids 31 - 304), and a C-terminal propeptide (amino acids 305 - 346). The mature proteins of M15 and LPV06 are 77% identical.

[0135] Polypeptide variants of LPV06 named R3_072 and R3_073 were designed to have modifications in the loop (linker) of the KEX2 protease cleavage site and the adjacent C-terminal propeptide, amino acids 305 - 320 of SEQ ID NO: 28. The amino acids RRYRSAESVDKRATMT (SEQ ID NO: 19) were replaced with the sequences described in Table 3, and polypeptides having the amino acid sequences shown in SEQ ID NOs: 29 - 30 were obtained.

[0136] The designed polypeptides were reverse translated into codon-optimized DNA, and nucleotide SEQ ID NOs: 31, 32, and 33 were obtained. This construct was cloned into pGBTOP-18 and transformed into Aspergillus niger as described in Examples 1 and 2 herein.

[0137]

Table 3

[0138] As described in Example 2, the enzyme-expressing strains were cultured in 24-deep well plates containing 3 ml of fermentation medium. After 6 days of culture, the enzyme expression levels in the supernatant were compared by SDS-PAGE as described in Example 2 above. The control sequence here is SEQ ID NO: 28 (Table 4).

[0139]

Table 4

[0140] As can be clearly observed, due to the absence of the parental KEX2 protease cleavage site in the generated lipase polypeptides (R3_72 and R3_73), surprisingly, in each case, the yield of the generated polypeptide was significantly increased compared to the reference (LPV06) polypeptide with the KEX2 protease cleavage site.

[0141] [Example 4: Lipase Expression as a Result of pH and Diacetate Treatments A. Phospholipase Enzyme (PLE) Activity of Aspergillus niger Strains] The phospholipase enzyme (PLE) activity levels and the amount of protein in the selected broth samples and supernatant samples as described in Examples 2 and 3 were determined for the mutant and control supernatant samples after growth and fermentation on the shake flask scale as described above. Further, pH treatment and diacetate treatment were applied to the selected lipase enzyme mutants in the broth samples and supernatant samples, and then the same protein and phospholipase (PLE) activity assays were performed.

[0142] As can be clearly observed from Tables 2 and 4 (Examples 2 and 3), the productivity of the mutant lipolytic polypeptide obtained from SDS-PAGE analysis was significantly increased due to the absence of the parental KEX2 protease cleavage site. The increase in the protein level of the lipolytic enzyme mutant lacking the KEX2 protease cleavage site corresponded to an increase in the lipase activity level of those mutants in both the broth and the supernatant compared to the M15 reference (data not shown). To enable comparison of the activity and protein levels of each sample over time, each "end of fermentation" sample of each lipolytic enzyme mutant was set to 100% for PLE activity and Bradford protein concentration. Surprisingly, due to the absence of the parental KEX2 protease cleavage site in the lipolytic enzyme mutant, the PLE lipase activity level increased after the application of incubation / diacetate treatment at pH 5.0 (Table 5). The activity level could not be explained by the differences in protein content in different samples. When the reference M15 lost 60% - 70% of the PLE activity in the broth and the supernatant with diacetate treatment, all mutants lacking the parental KEX2 protease cleavage site either lost less PLE activity (M15_002, M15_057), or even increased the PLE activity level (M15_055, M15_066). The same was true for the LPV06 lipase reference, and the mutant enzyme LPV06-R3_72 surprisingly showed an increase in the PLE activity level with diacetate treatment when the reference lost PLE activity with diacetate treatment (Table 5).

[0143] In the next experiment, the M15 reference enzyme with the KEX2 cleavage site was compared with one lipolytic enzyme mutant (M15_055) lacking the KEX2 protease cleavage site in the broth background under various treatments at the end of fermentation. As can be seen from Table 6, due to the absence of the KEX2 protease cleavage site, the PLE lipase activity level increased under various treatments after fermentation, such as incubation at pH 5.0 and diacetate treatment conditions (time variation) as shown in Table 6.

[0144] The pH treatment and diacetate treatment that are carried out are examples of typical conditions and methods that can be applied to generate and purify enzymes on a large scale using broth and / or cell-free supernatant in the sterilization of downstream processes and / or production strains. Thus, the absence of the KEX2 protease cleavage site in the lipolytic enzyme is a means for more efficiently generating more lipase polypeptides and / or generating more active lipase enzymes as measured in an activity assay.

[0145]

Table 5

[0146]

Table 6

Claims

1. A variant of a parent polypeptide, wherein the difference between the variant polypeptide and the parent polypeptide is that the KEX2 protease cleavage site present in the parent polypeptide is absent in the variant polypeptide, and preferably, the KEX2 protease cleavage site consists of amino acids KK, RR, RK, or KR. The parent polypeptide has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, or The parent polypeptide has at least 80% sequence identity with the amino acid sequence described in SEQ ID NO: 34, and the mutant polypeptide further comprises at least one substitution of an amino acid residue at a position corresponding to any of positions 113, 121, 138, 141, 179, 282, 284, 286, or 295 in SEQ ID NO:

34.

2. The mutant polypeptide according to claim 1, wherein the KEX2 protease cleavage site is located immediately before the propeptide in the parent polypeptide, and preferably the propeptide having the KEX2 protease cleavage site immediately before it is located at the C-terminus of the parent polypeptide.

3. The mutant polypeptide according to claim 1 or 2, wherein, when produced under the same conditions as the parent polypeptide, the mutant polypeptide has a higher yield compared to the parent polypeptide, and / or the mutant polypeptide and the parent polypeptide have lipolytic activity, preferably EC3.1.1.3 triacylglycerol lipase.

4. The aforementioned KEX2 protease cleavage site is, (a) Deletion or substitution of at least one amino acid residue at the KEX2 protease cleavage site, (b) Substitution of the KEX2 protease cleavage site with an amino acid motif that does not contain the KEX2 protease cleavage site, optionally with one or more consecutive amino acids adjacent to the KEX2 protease cleavage site. A mutant polypeptide according to claim 1 or 2, which is removed by...

5. The mutant polypeptide according to claim 4, wherein the linker motif adjacent to the C-terminal propeptide is substituted with an amino acid motif selected from the group consisting of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, and 27, and / or the linker motif adjacent to the C-terminal propeptide of the parent polypeptide has the amino acid sequence described in SEQ ID NO:

19.

6. The mutant polypeptide according to claim 1 or 2, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, and 10, and preferably comprising the substitution of at least one amino acid residue at a position corresponding to any of positions 113, 121, 138, 141, 179, 282, 284, 286, and 295.

7. A polynucleotide encoding a mutant polypeptide according to claim 1 or 2, preferably a codon-optimized polynucleotide.

8. Recombinant host cells comprising the polynucleotide described in claim 7.

9. A method for producing a mutant polypeptide according to claim 1 or 2, comprising culturing the host cells according to claim 8 under conditions that promote the production of the mutant polypeptide, and recovering the mutant polypeptide.

10. A method for producing a mature polypeptide having lipolytic activity, comprising: culturing the host cells described in claim 8 under conditions that promote the production of the mutant polypeptide; recovering the mutant polypeptide; and activating the mutant polypeptide to obtain the mature polypeptide having lipolytic activity.

11. A fabric comprising the mutant polypeptide described in claim 1.

12. A method for producing a baked product, comprising baking the dough described in claim 11.

13. The method according to claim 12, wherein the product is a baked product prepared from bread, cake, or layered dough.

14. The dough according to claim 11, wherein the dough comprises whole wheat flour and / or flour containing 0.01 to 0.8 w / w% of free fatty acids.

15. The method according to claim 12 or 13, wherein the dough comprises whole wheat flour and / or flour containing 0.01 to 0.8 w / w% free fatty acids.

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