Modified enzyme variants

Abstract

A polypeptide having an amino acid sequence that, when compared with the amino acid sequence shown in SEQ ID NO:1, contains at least substitutions of amino acid residues corresponding to amino acids at sites 50, 51, 126, 135, and 221, wherein the sites are defined with reference to SEQ ID NO:1.

Description

Technical Field

[0001] This invention relates to a polypeptide with rennet activity. The invention also relates to compositions comprising said polypeptide, the use of said polypeptide or compositions comprising said polypeptide in the preparation of cheese, methods for producing cheese, and the resulting cheese. Background Technology

[0002] Enzymatic coagulation of milk using enzymes such as rennet is an important process in cheese production. The enzymatic coagulation of milk occurs in two stages: in the first stage, proteolytic enzymes act on κ-casein, causing the casein micelle structure to reach a metastable state; in the second stage, the milk subsequently coagulates and forms a curd. Many characteristics of enzymes are crucial for cheese production.

[0003] WO2013 / 164479 explains the proteolytic activity that needs to be modified under cheese-making conditions. In most cheeses, rennet is responsible for "primary" proteolysis, a process that releases peptides, which are then used by lactic acid bacteria for "secondary" proteolysis and generate flavor compounds during maturation. WO2013 / 164479 reports a beneficial mutant rennet polypeptide discovered in this regard, wherein the variant has an amino acid sequence that, when compared with a rennet containing the sequence shown in SEQ ID NO:2, includes at least one substitution of any amino acid residue corresponding to amino acids 2, 22, 40, 48, 50, 51, 53, 61, 62, 76, 88, 98, 99, 109, 112, 117, 125, 126, 135, 144, 160, 161, 163, 187, 189, 194, 200, 201, 202, 203, 221, 223, 240, 242, 244, 254, 267, 271, 273, 278, 280, 284, 289, 292, 294, or 295, the site of which is referenced in SEQ ID NO:2. NO:2 defines a variant as having one or more modified properties compared to a reference polypeptide with rennet activity. WO2013 / 164479 describes up to 115 specific mutant polypeptides in the examples. Variants #71, #74, #95, #98, #103, #104, and #106 are described as exhibiting reduced stimulation of proteolytic activity at lower pH values ​​compared to Maxiren (bovine rennet) and Chymax M (camel rennet). Examples also describe thermostable variants (#95, #98, and #104) containing the S135T and A126G mutations. Furthermore, variants with additional negative surface charges, such as variants #12, #107, #111, and #112, are described as exhibiting significantly higher production efficiency than wild-type rennet. According to WO2013 / 164479, the above variants have multiple alterations in the amino acid sequence of rennet, thereby introducing additional negative charges (aspartic acid and glutamic acid) into the calf rennet sequence.

[0004] WO2013 / 164481 explains that an ideal coagulant for the industrial production of early-maturing cheese not only extends the shelf life of the cheese but also achieves rapid processing / shredding performance in the early stages of maturation, eliminating the need for long-term storage. However, rapid processing requires high proteolytic activity, while long shelf life requires low proteolytic activity against the casein matrix. How to achieve both of these properties in a single coagulant remains unclear. WO2013 / 164481 then reports a beneficial discovery: a polypeptide with rennet activity that: (a) can hydrolyze bovine αS1-casein at F23F24 sites, thereby generating the αS1-I casein fragment (f24-199) faster than camel rennet; and (b) has a higher C / P ratio than bovine rennet. WO2013 / 164481 mentions in its experiments that the mutant combinations at sites A51 and K221 (variants #71, #74, #98, #103, and #110) have a synergistic effect on increasing the C / P ratio. WO2013 / 164481 further mentions in its experiments that all variants containing amino acid changes at sites A51 and / or K221 of mature calf rennet B show a clear hydrolysis of the first cleavage site of α-S1-casein, thereby rapidly generating the α-S1-I casein fragment.

[0005] WO2023 / 194285 (Chr. Hansen) relates to polypeptides encoded by specific DNA sequences. In the examples, these specific DNA sequences were integrated into an expression host of *Aspergilus*. The resulting partial enzyme had a higher C / P ratio than bovine chymotrypsin and exhibited greater degradation of intact α-S1-casein than camel chymotrypsin.

[0006] Kaminogawa et al., in their article titled " 钙不敏感性及其他特性 αS1 - I酪蛋白的 The article published in the Journal of Dairy Science, vol. 63 (1980), pp. 223-227, explains that the α-S1-I casein fragment is a large peptide derived from α-S1-casein through the hydrolysis of the peptide bond between residues 23 and 24 by rennet, and is believed to be related to texture.

[0007] However, as Exterkate et al. in their article entitled " 凝乳酶作用的选择性 在奶酪中溶液中αS1 - 和β - 酪蛋白上的凝乳酶作用的选择性受到调节As demonstrated in the article published in the International Dairy Journal, vol. 7 (1997), pp. 47-54, cleavage of alphaS1 (αS1) casein does not necessarily occur between residues 23 and 24, and cleavage of alphaS1 (αS1) casein alone does not necessarily generate the target α-S1-I casein fragment. Table 1 of Exterkate et al. indicates that this fragment may actually neither be generated nor maintained.

[0008] Although the enzymes WO2013 / 164479 and WO2013 / 164481 have been found to exhibit excellent performance, there is still a need for further improvement in the industry.

[0009] Wild-type camel rennet has higher thermal stability than wild-type bovine rennet. Because rennet is expected to exhibit some thermal instability, causing it to degrade during the heating process in cheese making, cheese manufacturers prefer to use bovine rennet.

[0010] When using bovine rennet, cheese producers require an enzyme with an improved C / P ratio, especially under low pH conditions. As an alternative or supplement, the improved cleavage and / or faster coagulation of alpha S1 (αS1)S1 casein are beneficial.

[0011] Unfortunately, due to the interactions of amino acids within an enzyme, multiple mutations within the enzyme can produce strong mutual effects. For example, the individual effect of one mutation may be canceled out by combining with another mutation. Furthermore, the fact that milk or milk bases can contain different types of casein, including alpha S1 (αS1) casein and kappa (κ) casein, as well as further alpha S2 (αS2) and / or beta (β) casein, further increases the complexity of developing novel enzymes. These casein proteins interact with the enzyme independently and differently; a positive effect on one casein may be accompanied by a negative effect on another.

[0012] If a novel, preferred bovine enzyme and / or cheese production method can be provided, capable of achieving improved proteolytic activity and / or improved C / P ratio under cheese-making conditions and / or rapid processing / shredding performance as mentioned in WO2013 / 164481 and / or improved cleavage and / or selectivity of alpha S1 (αS1) casein. In addition, or as an alternative, shorter production times and / or lower dosage regimens are also desirable. Summary of the Invention

[0013] Advantageously, new coagulant peptides suitable for cattle have been identified, which can achieve an improved C / P ratio, preferably an improved C / P ratio at low pH, such as pH 6.1. Alternatively or supplemented, these new peptides provide improved proteolytic activity under cheese-making conditions as mentioned in WO2013 / 164479, and / or improved processing / shredding properties as mentioned in WO2013 / 164481, and / or improved cleavage and / or selectivity of alpha S1 (αS1) casein, and / or improved production time and / or low-dosage regimens. Therefore, the peptides of the present invention are superior coagulants compared to prior art peptides.

[0014] Therefore, in a first aspect, the present invention provides a polypeptide having rennet activity, wherein the polypeptide has an amino acid sequence that, when compared with the amino acid sequence shown in SEQ ID NO:1, contains at least substitutions of amino acid residues corresponding to amino acids at sites 50, 51, 126, 135, and 221, as defined in SEQ ID NO:1. SEQ ID NO:1 is suitable for representing the amino acid sequence of bovine rennet.

[0015] In a second aspect, the present invention provides a composition comprising the polypeptide described in the first aspect.

[0016] In a third aspect, the present invention provides: (i) a nucleic acid sequence encoding the polypeptide of the first aspect; (ii) a nucleic acid construct comprising the nucleic acid sequence, the nucleic acid sequence being operatively linked to one or more regulatory sequences capable of directing the expression of the polypeptide in a host cell; and / or (iii) a recombinant expression vector comprising the nucleic acid sequence or the nucleic acid construct.

[0017] In a fourth aspect, the present invention provides a recombinant host cell comprising the nucleic acid sequence, nucleic acid construct and / or recombinant expression vector described in the third aspect.

[0018] In a fifth aspect, the present invention provides a method for generating the polypeptide described in the first aspect, the method comprising expressing the nucleic acid sequence and / or nucleic acid construct described in the third aspect in a recombinant host cell described in the fourth aspect.

[0019] In a sixth aspect, the present invention provides the use of the polypeptide described in the first aspect or the composition described in the second aspect in the preparation of cheese.

[0020] In a seventh aspect, the present invention provides a method for producing cheese, wherein the method comprises contacting a certain amount of the polypeptide described in the first aspect or the composition described in the second aspect with a milk base.

[0021] In an eighth aspect, the present invention provides a cheese, wherein the cheese comprises a degraded polypeptide as described in the first aspect, and / or is obtained or available by the method described in the seventh aspect or by the use described in the sixth aspect.

[0022] The above aspects of the present invention can advantageously achieve the modified proteolytic activity under the cheese-making conditions mentioned in WO2013 / 164479, and / or the rapid processing / shredding performance mentioned in WO2013 / 164481, and / or shortened production time; and / or lower dosage regimens.

[0023] Brief description of the attached figures The invention is illustrated in conjunction with the following figures: Figure 1 Regarding the gel results from the optical density scanning analysis performed in Example 4.

[0024] A brief explanation of sequence listings This application contains a list of sequences in a computer-readable form, which is incorporated herein by reference. Table 1 below provides an overview of the sequences.

[0025] Table 1: Sequence List Overview Detailed Implementation

[0026] Definitions Unless otherwise defined or the context clearly indicates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0027] In this specification and the appended claims, the terms “comprise”, “include”, “have”, and variations thereof such as “comprises”, “comprising”, “includes”, and “including” are to be interpreted in an open-ended manner. That is, where the context permits, the above terms are intended to indicate that other components or the whole not expressly stated may also be included.

[0028] In this article, the articles “a” and “an” are used to indicate that the grammatical object modified by the article is one or more (i.e., one or at least one). For example, “an element” can mean one element or more elements.

[0029] Unless otherwise expressly stated, the various embodiments of the invention described herein can be combined with each other.

[0030] The term "milk" is intended to encompass mammalian milk, plant-derived milk, microbial-derived milk, and / or mixtures thereof. Preferably, the milk is mammalian-derived milk. Mammal milk sources include, but are not limited to, cow's milk, sheep's milk, goat's milk, buffalo milk, camel's milk, llama milk, horse's milk, or reindeer milk. In one embodiment, the milk is derived from mammals selected from the group consisting of cows, sheep, goats, buffalo, camels, llama, horses, and deer, and combinations thereof. Plant-derived milk includes, but is not limited to, milk extracted from soybeans, peas, peanuts, barley, rice, oats, quinoa, almonds, cashews, coconuts, hazelnuts, hemp seeds, sesame seeds, and sunflower seeds. Microbial-derived milk includes milk and milk proteins produced in a laboratory by recombinant microorganisms (also known as "laboratory-cultured milk") or milk produced in a bioreactor. Preferably, the milk is non-recombinant, naturally occurring, and / or naturally produced milk. The most preferred milk source is cow's milk. Cow's milk is the most preferred. Furthermore, the term "milk" refers not only to whole milk but also to skim milk or any liquid component or reconstituted milk derived therefrom.

[0031] The term "milk base" refers to a base composition that contains, is composed of, or is derived from milk or milk components. The milk base can be used as a raw material for the fermentation production of cheese. For example, the milk base may contain skim milk or non-skim milk, or reconstituted milk, or be composed of skim milk or non-skim milk, or reconstituted milk. Optionally, the milk base may be concentrated, in powder form, or can be obtained from it. Reconstituted milk is understood herein as a liquid milk obtained by adding a liquid (such as water) to skim milk powder, skim milk concentrate, whole milk powder, or whole milk concentrate. Furthermore, the milk base may or may not have undergone a heat treatment process that achieves at least pasteurization. Preferably, the milk base is derived from bovine sources.

[0032] Any reference to %w / v in this document, such as to 12%w / v econstituted skim milk (RSM), refers to the weight in grams per 100 ml volume of solution; for example, 12%w / v econstituted skim milk corresponds to 12 grams of skim milk powder dissolved in 100 ml of water.

[0033] Rennet and pepsin, both lactucinating enzymes of the mammalian stomach, belong to the aspartic protease class and are classified as peptidases. Therefore, the term "rennet" in this document refers to aspartic proteases, preferably aspartic proteases conforming to group EC3.4.23.4 of the International Union of Biochemistry and Molecular Biology (IUBMB) 1992 Enzyme Nomenclature. In this document, the terms "polypeptide with rennet activity," "protein with rennet activity," "enzyme with rennet activity," "rennet protein," "rennet peptide," "rennet," and the abbreviation "rennet" are used interchangeably. In this document, "rennet activity" preferably refers to aspartic protease activity, and more preferably to the catalytic activity defined by group EC3.4.23.4. In this document, the term rennet refers to both naturally occurring rennet and non-naturally occurring rennet (e.g., rennet produced in the laboratory). Wild-type natural rennets are naturally produced by the chief cells of the stomach of young mammals. Rennets are considered the main enzymatically active component of abomasal enzymes. For example, calf abomasal enzymes can be obtained from the mucosa of the abomasum (the fourth stomach, i.e., the last gastric compartment) of unweaned young calves. Non-naturally produced rennets can be produced via microbial methods using recombinant microorganisms such as yeast or bacteria. These rennets are also referred to herein as "microbial rennets".

[0034] In this article, pro-rennethionein should be understood as the precursor protein or zymogen of rennet. Pro-rennethionein may have a leader sequence (zymogen fragment) at the N-terminus of rennet, which is believed to be cleaved during rennethionein activation. Furthermore, pro-rennethionein should be understood as a rennethionein protein with a hydrophobic leader sequence appended to the N-terminus of rennet. This leader sequence, also known as a secretion signal peptide or precursor fragment, can be cleaved before or during protein secretion. Rennet is initially synthesized intracellularly as pro-rennethionein (e.g., Harris et al., Nucleic acid Research 1982, April 10, pp. 2177-2187, in the article entitled " 小牛前凝乳酶原的cDNA编码的分子克隆和核苷酸序列 针对小牛前凝乳酶原 (As stated in the article).

[0035] Genes or cDNAs encoding rennet or prorenin can be cloned and overexpressed in host organisms. Suitable host organisms for rennet overexpression include Aspergillus species (…). 曲霉属 Kluyveromyces ( ) 克鲁维酵母属 种 Trichoderma ( ) 木霉属 ), Escherichia coli (大肠杆菌 Pichia pastoris ( ) 毕赤酵母属 种 ), yeast ( 酿酒酵母属 ), Yersinia lipolytica ( 解脂耶氏酵母属 Neurospora ( 脉孢菌属 sp.) or Bacillus spp. ( 芽孢杆菌属 ).

[0036] As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules that include an open reading frame encoding the polypeptide described herein. Genes may include coding sequences, non-coding sequences, introns, and regulatory sequences. That is, as used herein, "gene" may refer to an isolated nucleic acid molecule as defined herein. Therefore, in this application, the term "gene" does not refer only to naturally occurring sequences.

[0037] As used herein, the terms “polynucleotide,” “nucleic acid sequence,” or “nucleic acid molecule” are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), and DNA or RNA analogs prepared using nucleotide analogs. Nucleic acid molecules may be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphate-thioester nucleotides). Such oligonucleotides can be used to prepare nucleic acids, for example, with altered base-pairing capabilities or enhanced resistance to nucleases.

[0038] The terms “peptide” and “oligopeptide” as used herein are considered synonyms (as are known in the art) and may be used interchangeably depending on the context, both referring to a chain composed of at least two amino acids linked by peptide bonds. The term “polypeptide” is used herein to refer to a chain containing seven or more amino acid residues. All molecular formulas or sequences of oligopeptides and polypeptides in this document are written from left to right, from the amino terminus to the carboxyl terminus. The single-letter codes for amino acids used herein are well-known in the art and can be found in Berg, Tymoczko, and Stryer. 生物化学 , 6 th edition, chapter 2, WH Freeman and Company, New York, 2007.

[0039] The terms "homology" and "identity percentage" used herein are interchangeable. For the purposes of this invention, they are defined as follows: To determine the identity percentage of two amino acid sequences or two nucleic acid sequences, the sequences are optimally aligned (e.g., a gap may be introduced in the first amino acid or nucleic acid sequence to achieve optimal alignment with the second amino acid or nucleic acid sequence). Then, amino acid residues or nucleotide residues at corresponding amino acid or nucleotide sites are compared. If a site in the first sequence has the same amino acid or nucleotide residue as the corresponding site in the second sequence, then the molecule is identical at that site. The identity percentage between two sequences is a function of the number of shared sites (i.e., identity % = number of shared sites / total number of sites (i.e., overlapping sites) × 100). Preferably, the two sequences are of the same length.

[0040] Sequence alignment can be performed on the full length of the two sequences to be aligned, or on fragments of the two sequences. Suitablely, alignment can be performed on the full length of the two sequences to be aligned. However, sequence identity calculations can also be performed on regions of, for example, 20, 50, 100 or more consecutive amino acid residues.

[0041] Those skilled in the art will understand that various computer programs can be used to determine the homology of two sequences. For example, sequence alignment and determination of the percentage of identity between two sequences can be achieved through mathematical algorithms. In a preferred embodiment, the percentage of identity between two amino acid or nucleic acid sequences is determined using the Needleman algorithm and the Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)), which is integrated into the GAP program of the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using a Blosum62 matrix or a PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. Those skilled in the art will understand that using different parameters will yield slightly different results, but the overall percentage of identity between the two sequences will not change significantly when using different algorithms.

[0042] The protein or nucleic acid sequences of this invention can also be searched in public databases as "query sequences," for example, to identify other family members or related sequences. Such searches can be performed using the BLASTN and BLASTP (version 2.0) programs described in Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed using the BLASTP program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of this invention. To obtain nicked alignment sequences for alignment purposes, the nicked BLAST program described in Altschul et al. (1997) Nucleic Acids Res. 25(17): 3389-3402 can be used. When using BLAST and nicked BLAST programs, the default parameters of the respective programs (e.g., BLASTP and BLASTN) can be used. See the homepage of the National Center for Biotechnology Information (NCBI) at http: / / www.ncbi.nlm.nih.gov / .

[0043] In this article, the term "IMCU" is understood to refer to the International Milk Clotting Unit. One IMCU is equal to approximately 0.126 nmol of bovine rennet B (e.g., Maxiren™ or CHY-MAX™). The potency of a milk-coagulating enzyme (such as rennet) can be determined as the milk-coagulating activity (per milliliter or per gram of IMCU) according to the standard set by the International Dairy Federation (IDF) in ISO 11815|IDF Standard 157A:1997, which was developed by ISO / TC34 Technical Committee on Food Products SC5, Subcommittee on Milk and Dairy Products, and the International Dairy Federation (IDF).

[0044] Upon addition of a diluted coagulant to a standard emulsion substrate, the emulsion flocculates. The coagulation time is the period from the addition of the coagulant to the formation of visible flocs or clumps in the emulsion substrate. The effectiveness of the coagulant sample can be determined by comparing the sample's coagulation time with that of a normal reference standard. This method is further described in detail in IDF standard 157A:1997. Based on the IMCU principle, the time required for visible flocculation to occur after treatment with abomasal enzymes was determined in a standard emulsion substrate with 0.05% calcium chloride and a pH of 6.5. The IMCU / mL of the sample was determined by comparing its coagulation time with a standard having known coagulation activity and the same enzyme composition as the sample.

[0045] The term "C / P" or "C / P ratio" refers to the clotting activity (C) of a specific enzyme sample divided by its proteolytic activity (P). C / P is an evaluation indicator of coagulant specificity. Methods for determining both activities and calculating C / P are described herein (see Examples). The method for determining clotting activity (C) is used to quantify the efficiency of an enzyme sample in hydrolyzing κ-casein (k-CN) at a specific site FM between the amino acids phenylalanine (Phe, F) and methionine (Met, M). Proteolytic activity (P) is used to quantify the ability of a coagulant to hydrolyze casein into small (TCA-soluble) peptide fragments and amino acids.

[0046] Polypeptide In a first aspect, the present invention provides a polypeptide having an amino acid sequence, which, when compared with the amino acid sequence shown in SEQ ID NO:1, contains at least substitutions of amino acid residues corresponding to amino acids at sites 50, 51, 126, 135, and 221, wherein the sites are defined with reference to SEQ ID NO:1. SEQ ID NO:1 is suitable to represent the amino acid sequence of bovine chymotrypsin.

[0047] Preferably, the polypeptide has rennet activity, and more preferably, the polypeptide has α-S1-I casein fragment forming activity, wherein the preferred embodiments are described below.

[0048] Preferably, the polypeptide is a bovine chymotrypsin mutant. More preferably, the polypeptide is a mutant of the polypeptide having the amino acid sequence of SEQ ID NO:1. Such mutant polypeptides are also referred to herein as variant polypeptides. Therefore, the polypeptide is preferably a variant polypeptide of the polypeptide having the amino acid sequence of SEQ ID NO:1.

[0049] More preferably, the polypeptide comprises: - At least mutate N50D, A51V, A126G, and S135T; and - Mutations selected from at least K221M and K221V mutations, The site mentioned therein is defined with reference to SEQ ID NO:1.

[0050] The polypeptide comprises substitutions at amino acid residues corresponding to amino acids at sites 201, 243, 164, or 292, wherein said sites are defined with reference to SEQ ID NO:1; preferably, the polypeptide further comprises substitutions at amino acid residues corresponding to amino acid site 201, wherein said sites are defined with reference to SEQ ID NO:1. The polypeptide preferably comprises at least mutant S201D, Y243E, S164G, or H292D, wherein said sites are defined with reference to SEQ ID NO:1; more preferably mutant S201D, wherein said sites are defined with reference to SEQ ID NO:1.

[0051] Even more preferably, the amino acid sequence of the polypeptide, when compared with the amino acid shown in SEQ ID NO:1, includes: (i) Mutate at least N50D, A51V, A126G, S135T, K221M, and Y243E; or (ii) Mutate at least N50D, A51V, A126G, S135T, S201D, and K221V; or (iii) Mutate at least N50D, A51V, A126G, S135T, S201D, and K221M; or (iv) Mutate at least N50D, A51V, A126G, S135T, S164G, K221M and H292D; The site mentioned therein is defined with reference to SEQ ID NO:1.

[0052] Customers and regulatory agencies typically prefer enzymes with as few mutations as possible compared to the wild type. Therefore, the polypeptide of the present invention is preferably a polypeptide wherein, when compared with the amino acid sequence of SEQ ID NO:1, the amino acid sequence of the polypeptide contains 10 or fewer mutations, more preferably 8 or fewer, further preferably 7 or fewer, and most preferably up to 6 mutations.

[0053] Preferably, the polypeptide has an amino acid sequence that has equal or greater than 90%, more preferably equal or greater than 95%, and most preferably equal or greater than 98% sequence identity with the amino acid sequence of SEQ ID NO:1. Most preferably, the present invention provides a polypeptide wherein, when compared with the amino acid sequence of SEQ ID NO:1, the amino acid sequence of the polypeptide includes at least substitutions at amino acid residues at corresponding sites 50, 51, 126, 135, 201, and 221, wherein said sites are defined with reference to SEQ ID NO:1; wherein the polypeptide has an amino acid sequence that has equal or greater than 90%, more preferably equal or greater than 95%, and most preferably equal or greater than 98% sequence identity with the amino acid sequence of SEQ ID NO:1; and wherein said polypeptide includes at least the mutants N50D, A51V, A126G, S135T, S201D, and K221V.

[0054] Most preferably, the polypeptide is: - A polypeptide having the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; or - Homologs of the polypeptide having the amino acid sequence of SEQ ID NO:5, having an amino acid sequence with greater than 99% sequence identity to SEQ ID NO:5; or - Homologs of the polypeptide having the amino acid sequence of SEQ ID NO:6, having an amino acid sequence with greater than 98% sequence identity to SEQ ID NO:6; or - Homologs of the polypeptide having the amino acid sequence of SEQ ID NO:7, having an amino acid sequence with greater than 98% sequence identity to SEQ ID NO:7; or - Homologous to a polypeptide having the amino acid sequence of SEQ ID NO:8, having an amino acid sequence having greater than 98% sequence identity with SEQ ID NO:8.

[0055] Even more preferably, the polypeptide: -A polypeptide having the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; or - An amino acid sequence having greater than 98% sequence identity with SEQ ID NO:5; or - An amino acid sequence having greater than 98% sequence identity with SEQ ID NO:6; or - Its amino acid sequence is more than 98% identical to that of SEQ ID NO:7; or - An amino acid sequence having greater than 98% sequence identity with SEQ ID NO:8.

[0056] Preferably, the polypeptide of the present invention further comprises: - Rennet activity; and / or -α-S1 casein hydrolytic activity; and / or -α-S1-I casein fragment formation activity.

[0057] More preferably, the polypeptide comprises: a) Rennet activity, wherein the C / P ratio of coagulation activity (C) to proteolytic activity (P) is equal to or greater than that of a polypeptide having the amino acid sequence SEQ ID NO:2; and / or b) α-S1-I casein fragment generation activity, which is equal to or greater than the α-S1-I casein fragment generation activity of a polypeptide having the amino acid sequence SEQ ID NO:2; and / or c) The coagulation time at pH 6.1, which is equal to or less than the coagulation time of the polypeptide having the amino acid sequence SEQ ID NO:2.

[0058] Even more preferably, the polypeptide: The polypeptide is capable of hydrolyzing bovine αs1-casein more rapidly than polypeptides having the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20; or more specifically, it is capable of hydrolyzing bovine αs1-casein more rapidly at the F23F24 site at the amino acid sites of the polypeptide, thereby generating the αs1-I casein fragment (f24-199); or it is expressed as follows: the α-S1-I casein fragment forming activity of the polypeptide is equal to or higher than that of polypeptides having the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20. α-S1-I casein fragment formation activity of polypeptides with amino acid sequences of NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20; or In addition to the first feature mentioned above, the polypeptide preferably contains rennet activity, wherein the C / P ratio of its coagulation activity (C) to its proteolytic activity (P) is equal to or higher than the C / P ratio of polypeptides having the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20; or the α-S1-I casein fragment forming activity of the polypeptide is equal to or greater than the α-S1-I casein fragment forming activity of polypeptides having the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

[0059] In this document, rennet activity is preferably understood as the activity of rennet, more preferably as the activity of rennet classified as EC3.4.23.4. The polypeptide of the present invention is preferably an enzyme classified as EC3.4.23.4. A more detailed description of such rennets can be found in the "Definitions" section above. More preferably, the polypeptide comprises rennet activity, wherein the rennet activity comprises or consists of selective κ-casein hydrolysis activity. Most preferably, the polypeptide of the present invention is an enzyme that cleaves κ-casein at least at the peptide bond between the amino acid residues phenylalanine (F) and methionine (M). For example, when the κ-casein used for this assay is mature bovine κ-casein having the amino acid sequence shown in SEQ ID NO:9, the polypeptide of the present invention is suitable for selectively hydrolyzing (i.e., "cleaving") peptide bonds F105-M106.

[0060] The complete sequence of bovine κ-casein is described in UNIPROT accession number P02668. The sequence of mature bovine κ-casein (i.e., the signal peptide without the 21 amino acids) is shown in SEQ ID NO:9. Positions F105-M106 refer to phenylalanine at position 105 and methionine at position 106 in the sequence of SEQ ID NO:9, respectively. Several variants of this bovine protein exist with slightly different amino acid sequences, but those skilled in the art can readily determine the amino acids corresponding to F105 and M106 in SEQ ID NO:9. The C / P ratio of the polypeptide is preferably equal to or greater than 15, more preferably equal to or greater than 20, further preferably equal to or greater than 30, and most preferably equal to or greater than 40. There is no explicit upper limit to this ratio; in practical applications, the C / P ratio can be equal to or less than 1000, or equal to or less than 500. Preferably, the C / P ratio of the polypeptide of the present invention is equal to or greater than 1.1 times that of the polypeptide having the amino acid sequence SEQ ID NO:2, more preferably equal to or greater than 1.5 times, further preferably equal to or greater than 2 times, even more preferably equal to or greater than 2.5 times, and most preferably equal to or greater than 3 times. That is, the C / P ratio of the polypeptide (determined according to the method described in the examples below) is preferably equal to or greater than 2 times that of the polypeptide having the amino acid sequence shown in SEQ ID NO:2, more preferably equal to or greater than 3 times. It is worth noting that the polypeptide having the amino acid sequence SEQ ID NO:2 is the polypeptide variant numbered #71 in the prior art WO2013 / 164479 and WO2013 / 164481. Preferably, the polypeptide has a C / P ratio that is equal to or greater than 1.5 times that of the polypeptide having the amino acid sequences SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

[0061] The C / P ratio can be suitably obtained by measuring coagulation activity (C) and proteolytic activity (P) and subsequently calculating the ratio of coagulation activity (C) to proteolytic activity (P). The term "C / P ratio" as used herein is preferably further defined by the C and P value determination methods described in the examples below.

[0062] Preferably, the polypeptide also possesses α-S1-casein hydrolytic activity. Hereinafter, "α-S1-casein hydrolytic activity" is preferably understood as the activity of hydrolyzing α-S1-casein. α-S1-casein is also referred to herein as "α-S1-casein" or abbreviated as "αS1 CN". More preferably, the polypeptide of the present invention is an enzyme capable of selectively hydrolyzing α-S1-casein into a first fragment and a second α-S1-I casein fragment. Further preferably, the polypeptide comprises α-S1-casein hydrolytic activity, wherein this α-S1-casein hydrolytic activity comprises or consists of α-S1-casein hydrolytic activity at the peptide bond between the first phenylalanine (F) and the second phenylalanine (F). For example, when the α-S1-casein used for this assay is mature bovine α-S1-casein having the amino acid sequence SEQ ID NO:10, the polypeptide is suitable for catalyzing the hydrolysis of peptide bonds F23-F24.

[0063] Most preferably, the α-S1-casein hydrolytic activity is the activity of hydrolyzing α-S1-casein, preferably the activity of hydrolyzing α-S1-casein between phenylalanine 23 and phenylalanine 24, and releasing the α-S1-I casein fragment. Preferred embodiments of such α-S1-I casein fragments are described below.

[0064] Mature bovine α-S1-casein is also abbreviated herein as mature bovine "αS1-CN". The complete sequence of bovine α-S1-casein is described in UNIPROT accession number P02662. The sequence of mature bovine α-S1-casein (excluding the 15-amino acid signal peptide) is shown in SEQ ID NO:10. The F23-F24 sites refer to the two phenylalanine residues at positions 23 and 24 in the sequence, for example, in SEQ ID NO:10. Several variants of the protein exist with slightly different amino acid sequences, but those skilled in the art can readily identify the amino acid sites corresponding to F23 and F24 in SEQ ID NO:10. When mature bovine α-S1-casein (as shown in SEQ ID NO:10) is hydrolyzed (i.e., "cleaved") at the F23-F24 sites, a first fragment (also referred to herein as fragment "f1-23") and a second fragment (also referred to herein as fragment "f24-199") are generated. The first segment (f1-23) contains the first 23 amino acids of mature bovine α-S1-casein, and the second segment (f24-199) contains the remaining amino acids 24-199 of mature bovine α-S1-casein.

[0065] For example, Exterkate et al., in their title " 凝乳酶对α 溶液中S1 - 和β - 酪蛋白的作用选择性在奶酪中受到调节As explained in the article published in the International Dairy Journal, vol. 7 (1997), pp. 47-54, cleavage of α-S1-casein does not necessarily (only) occur between residues 23 and 24, and simple cleavage of α-S1-casein does not necessarily generate the target α-S1-I casein fragment. Table 1 of Exterkate et al. shows that, under the conditions illustrated in the article, this fragment may actually neither be generated nor maintained.

[0066] In this document, the α-S1-I casein fragment generating activity is preferably understood as the generation and maintenance of the α-S1-I casein fragment activity for at least 24 hours. The α-S1-I casein fragment is also abbreviated herein as "αS1-I-CN". Preferably, the α-S1-I casein fragment is a hydrolysis product, corresponding to the second fragment obtained after hydrolysis of α-S1-casein. That is, preferably, the term "α-S1-I casein fragment" is understood herein to refer to the longest fragment generated by hydrolyzing α-S1-casein at the FF peptide bond between the first phenylalanine (F) and the second phenylalanine (F). More preferably, the α-S1-casein is mature bovine α-S1-casein, which is hydrolyzed at the F23-F24 site to generate the α-S1-I casein fragment f24-199. That is, the α-S1-I casein fragment is preferably the bovine α-S1-I casein f24-199 fragment. An example of this bovine α-S1-I casein f24-199 fragment is provided in SEQ ID NO:11. Several variants of this protein fragment exist with slightly different amino acid sequences, but those skilled in the art can readily determine the amino acids corresponding to SEQ ID NO:11.

[0067] Unwilling to be bound by any theory, the inventors believe that the α-S1-I fragment, due to its reduced hydrophobicity compared to α S1 casein, can play an important role in the early coagulation and building up of cheese, endowing cheese with the textural properties required for rapid processing.

[0068] The hydrolysis of α-S1-casein over a 24-hour period can be represented by the αS1-CN hydrolysis rate, where the αS1-CN hydrolysis rate is... t=24h It was calculated as follows: α-S1-casein (“…”) at time (t) = 0 hours and time (t) = 24 hours. α The difference in α-S1-casein ("αS1-CN") concentration is divided by the concentration of α-S1-casein ("αS1-CN") at time (t) = 0 hours. Therefore, the calculation is as follows: αS1 - CN hydrolysis rate t=24h = (αS1 - CN t=0h αS1 - CN t=24h ) / αS1 - CN t=0h In this study, a higher αS1-CN hydrolysis rate indicates a greater decrease in αS1-CN concentration, and thus indicates an increased degree of hydrolysis.

[0069] Preferably, the polypeptide of the present invention α S1-CN hydrolysis rate t=24h It is equal to or greater than 0.5, more preferably equal to or greater than 0.6, further preferably equal to or greater than 0.7, and most preferably equal to or greater than 0.8.

[0070] Preferably, the polypeptide of the present invention has α S1-CN hydrolysis rate t=24h , wherein α S1-CN hydrolysis rate t=24h It is a polypeptide having the amino acid sequence SEQ ID NO:2 or having the amino acid sequences SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20. α S1-CN hydrolysis rate t=24h The α-S1-casein used in this assay is equal to or greater than 1.1 times, more preferably equal to or greater than 1.2 times, even more preferably equal to or greater than 1.3 times, even more preferably equal to or greater than 1.4 times, and most preferably equal to or greater than 1.5 times. α S1-CN) is preferably α-S1-casein (“bovine milk”) derived from bovine milk. α S1-CN”).

[0071] More preferably, the polypeptide of the present invention is an enzyme capable of hydrolyzing mature bovine α-S1-casein, wherein the hydrolysis of the mature bovine α-S1-casein at sites F23-F24 generates an α-S1-I casein fragment (f24-199), which is a polypeptide having the amino acid sequence SEQ ID NO:2 or having the amino acid sequences SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20, equal to or greater than 1.1 times, more preferably equal to or greater than 1.2 times, further preferably equal to or greater than 1.3 times, even more preferably equal to or greater than 1.4 times, and most preferably equal to or greater than 1.5 times. In this assay, mature bovine α-S1-casein preferably has the amino acid sequence of SEQ ID NO:10, and α-S1-I casein fragment preferably has the amino acid sequence of SEQ ID NO:11.

[0072] The hydrolysis of the F23-F24 bond in α-S1-casein can be determined in various ways. The hydrolysis speed or rate can be compared, for example, in terms of the amount of enzyme (e.g., mg of protein) or equivalent International Cough Units (IMCUs). A higher hydrolysis rate compared to a reference indicates that the peptide of the present invention can hydrolyze α-S1-casein at the F23-F24 site to generate… α S1-I CN (f24-199) is faster than this reference.

[0073] For specific peptides, α-S1-I casein fragments are generated and maintained over a 24-hour period (“ α S1-I CN) can be compared with the reference, and with α S1-I CN generation rate is represented by, where, α S1-I CN generation rate t=24h The calculation is as follows: at time (t) = 24 hours, the α-S1-I casein fragment of a specific polypeptide (“ α The ratio of the S1-I CN concentration to the reference concentration. Therefore, the calculation is as follows: 相对于参考A的αS1 - I CN生成率 t=24h = (αS1 - I CN 变体，t=24h / αS1 - I CN 参考A，t=24h ) Concentration can be expressed as a weight percentage of the α-S1-I casein fragment based on total protein weight, or as a pixel percentage of the α-S1-I casein fragment based on total protein pixels as described below. Preferably, the peptide of the present invention generates and maintains the α-S1-I casein fragment (“…”) over a 24-hour period. α "S1-I CN" is a polypeptide having the amino acid sequence SEQ ID NO:2 or having the amino acid sequences SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20, and is equal to or greater than 1.1 times, more preferably equal to or greater than 1.2 times, and most preferably equal to or greater than 1.3 times. That is, the polypeptide of the present invention... α S1-I CN generation rate t=24h Preferably, the polypeptide having the amino acid sequence SEQ ID NO:2 or having the amino acid sequences SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20 has a content equal to or greater than 1.1 times, more preferably equal to or greater than 1.2 times, and most preferably equal to or greater than 1.3 times. In this assay, the α-S1-I casein fragment (“ α S1-I CN) is preferably an α-S1-I casein fragment derived from bovine milk (“bovine milk”). α S1-I CN”).

[0074] The hydrolysis of α-S1-casein over the 24-hour period mentioned above, as mentioned above α S1-I CN hydrolysis rate t=24h The generation and maintenance of the α-S1-I casein fragment mentioned above, and the above-mentioned α S1-I CN generation rate t=24h All can be appropriately measured according to the methods described in the embodiments.

[0075] The concentrations of α-S1-casein and / or α-S1-I casein fragments are preferably analyzed using PAGE, protein band staining, identification of different bands, and scanning and densitometric analysis of each hydrolysis product. Such analytical methods for quantifying α-S1-casein and α-S1-I casein fragments obtained using different coagulants have been previously documented (see, for example, the article by Bansal et al. entitled "..."). 重组骆驼(单峰驼)凝乳酶作为切达干酪凝固剂的适用性 ”Published in the International Dairy Journal, vol. 19, (2009), pp. 510-517. The examples describe a similar densitometric analysis method for quantifying α-S1-casein and α-S1-I casein fragments.

[0076] The polypeptide of the present invention is preferably a polypeptide, wherein the polypeptide has the following α-S1-I casein fragment generating activity, wherein the percentage of α-S1-I casein fragments measured by optical density after 24 hours ("@24 hours") is equal to or greater than 10%, more preferably equal to or greater than 20%, further preferably equal to or greater than 30%, and most preferably equal to or greater than 40%, based on the total protein content after 24 hours ("@24 hours"). For this optical density percentage, the percentage can be suitable as a "pixel" percentage after optical density analysis, wherein the percentage of pixels in the optical density band representing the α-S1-I casein fragment is calculated based on the total number of pixels in the optical density bands representing all proteins.

[0077] Alternatively, the hydrolysis of the F23-F24 bond in mature bovine α-S1-casein and the formation of its hydrolysis products can be tracked by RP-HPLC and quantified using known techniques (see, for example, Carles and Dumas, entitled "..."). 凝乳酶(凝乳素)对牛α -酪蛋白肽键作用的动力学 s1 :该底物与β - 和κ -酪蛋白行为的比较 o 通过比较肽图谱研究骆驼和牛凝乳酶对牛αS1 - 和β - 酪蛋白的水解作用 (This is from an article published in FEBS Letters vol. 185(2), (1985), pp. 282-286). This technique can be used to conveniently monitor the occurrence rate of α-S1-I fragments and the disappearance rate of intact α-S1-casein. Those skilled in the art will understand that this type of RP-HPLC can also be used to quantify α-S1-casein and its degradation products.

[0078] Another method for quantifying the formation rate of α-S1-I casein fragments is also described in ller et al. titled " 比较肽图谱 Compositions containing polypeptides The article, published in J. Agric. Food Chem. Vol. 60, (2012), pp. 11421-11432, states that...

[0079] Any of the above methods can be used to determine the hydrolysis of bovine α-S1-casein by rennet peptides at F23-F24 sites to generate α-S1-I casein fragments (“ α The rate of S1-I CN, for example, f24-199).

[0080] The rate and selectivity of hydrolysis can be expressed as described in the examples.

[0081] Therefore, the preferred polypeptide of the present invention is: -Suitably, within a 24-hour time period, mature bovine α-S1-casein can be hydrolyzed at sites F23-F24 in an amount higher than that of a reference polypeptide having the amino acid sequence SEQ ID NO:2 or having the amino acid sequences SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20 to generate bovine α-S1-I casein fragments (“ α S1-I CN”, f24-199); - It can be appropriately defined as an enzyme that produces the following properties: when cultured with α-S1 casein at 11°C for 6 hours, the α-S1-I casein fragment ( α S1-I CN) and intact α-S1-casein ( α The ratio of S1-I casein (CN) is preferably equal to or greater than 0.6, more preferably equal to or greater than 0.7, and most preferably equal to or greater than 1.0; or when cultured with α-S1-casein at 11°C for 24 hours, the ratio of α-S1-I casein fragment ... α S1-I CN) and intact α-S1-casein ( α The ratio of S1-I CN is preferably equal to or greater than 1.5, more preferably equal to or greater than 2.0, even more preferably equal to or greater than 3.0, even more preferably equal to or greater than 5.0, even more preferably equal to or greater than 8.0, and most preferably equal to or greater than 10.

[0082] In this document, "α-S1-I fragment generation activity" is preferably understood as the ability to generate α-S1-I casein fragments and retain the α-S1-I casein fragments for at least 24 hours after generation. More preferably, the α-S1-I casein fragment generation activity comprises or consists of the cleavage activity of the peptide on α-S1-casein, wherein within the first 24 hours after the peptide contacts α-S1-casein: - Preferably, 50% (w / w), more preferably 60% (w / w), and even more preferably 70% (w / w) of α-S1-casein is hydrolyzed (i.e., "cleaved") at the site between phenylalanine 23 and phenylalanine 24 to generate α-S1-I casein fragments; and / or - Preferably, 50% (w / w), more preferably 60% (w / w), and even more preferably 70% (w / w) of the generated α-S1-I casein fragments are further hydrolyzed (i.e. "cleaved") at other sites.

[0083] The α-S1-I casein fragment generation activity can be appropriately determined according to the methods shown in the examples or described above.

[0084] Preferably, the polypeptide has a coagulation time ratio (coagulation ratio) of 6.6 to 6.1. pH 6.6 / pH 6.1 The condensation ratio is equal to or greater than 1.10, preferably equal to or greater than 1.20. pH 6.6 / pH 6.1 The measurement can be appropriately performed using a rheological laser as illustrated in the example. The emulsion coagulation process is tracked visually in real time. The moment of coagulation onset is considered the coagulation time point.

[0085] Most preferably, the polypeptide of the present invention is a polypeptide comprising: a) Rennet activity, wherein the C / P ratio of coagulation activity (C) to proteolytic activity (P) is equal to or greater than the C / P ratio of the polypeptide having the amino acid sequence SEQ ID NO:2; and b) α-S1-I casein fragment generation activity, which is equal to or greater than the α-S1-I casein fragment generation activity of a polypeptide having the amino acid sequence SEQ ID NO:2; and c) The coagulation time at pH 6.1 is equal to or less than the coagulation time of the polypeptide having the amino acid sequence SEQ ID NO:2.

[0086] Other preferred options are as described above.

[0087] Without being bound by any theory, the inventors believe that the aforementioned dual selectivity can give cheese advantages in texture, rapid processing performance, and reduced cutting losses in the form of fine pieces. Advantageously, the peptides of the present invention can also enable the rapid production of cheese.

[0088] The polypeptides described in this invention can be isolated naturally occurring polypeptides or non-naturally occurring polypeptides. In this document, "non-naturally occurring polypeptide" is understood to be a polypeptide not naturally produced by any mammal. Preferably, the polypeptide is a non-naturally occurring polypeptide.

[0089] In this document, "isolated" polypeptides or proteins are understood to be polypeptides or proteins removed from their native environment. For example, recombinant polypeptides and proteins expressed in host cells are considered isolated in the sense of the purposes of this invention; similarly, recombinant polypeptides substantially purified by any suitable method are also considered isolated. The polypeptide variants described in this invention can be recovered and purified from recombinant cell cultures using methods known in the art.

[0090] Nucleic acid sequences encoding polypeptides In a second aspect, the present invention provides a composition comprising the polypeptide described in the first aspect.

[0091] More preferably, the pH of the composition is equal to or less than pH 6.6, preferably equal to or less than pH 6.1, and more preferably equal to or less than pH 5.8.

[0092] The composition may optionally contain other components, such as additives and / or other enzymes.

[0093] If other enzymes are present, the preferred enzymes present in the composition are pepsin, more preferably pepsin classified under group EC3.4.23.1.

[0094] Preferably, the composition of the present invention may further contain one or more pH buffers and / or other additives, such as glycerol and / or sodium chloride.

[0095] The composition may be in a liquid, frozen, or lyophilized state. If it is in a liquid state, the composition is preferably a solution, suspension, or emulsion.

[0096] 牛 In a third aspect, the present invention provides: (i) a nucleic acid sequence encoding the polypeptide of the first aspect; (ii) a nucleic acid construct comprising the nucleic acid sequence, the nucleic acid sequence being operatively linked to one or more regulatory sequences capable of directing the expression of the polypeptide in a host cell; and / or (iii) a recombinant expression vector comprising the nucleic acid sequence or the nucleic acid construct.

[0097] The polypeptide and / or nucleic acid sequences described in this invention can be prepared using the methods and techniques described in the examples, and / or using the methods described in the specifications and examples of WO2013 / 164479 and WO2013 / 164481, which are incorporated herein by reference.

[0098] The nucleic acid sequences of the present invention can be conveniently prepared using standard molecular biology techniques well known to those skilled in the art, in conjunction with the sequence information provided herein.

[0099] For example, the desired nucleic acid sequence can be synthesized de novo using standard synthesis techniques. Such synthesis methods are typically automated.

[0100] Alternatively, the nucleic acid sequence described in this invention can be prepared by site-directed mutagenesis of existing nucleic acid sequences (e.g., nucleic acid sequences encoding wild-type rennet, such as the nucleic acid sequence shown in SEQ ID NO:12). SEQ ID NO:12 shows a sequence derived from domestic cattle (…). 乳酸克鲁维酵母The nucleic acid sequence of the wild-type pro-rennet B gene of *Kluyveromyces lactis* has been obtained, and the sequence has been codon-adapted to *Kluyveromyces lactis*. 曲霉 It expresses and carries a linker that can be cloned into pKLAC1. Site-directed mutagenesis can be carried out using a variety of conventional techniques known to those skilled in the art.

[0101] In one such method, mentioned only as an example, PCR amplification is performed on a plasmid template using oligonucleotide "primers" encoding the target substitution. Since the primers are the ends of newly synthesized strands, if a mismatch occurs when binding to the template DNA strand in the first cycle, the primer-based strand (containing the mutation) will be roughly equivalent to the original template concentration after the first cycle. After several consecutive cycles, the mutant strand will amplify exponentially; after 25 cycles, its quantity can reach approximately 8,000,000:1 compared to the original unmutated strand, ultimately yielding a nearly homogeneous solution of the mutant amplified fragment. The template DNA can then be removed by enzymatic digestion, for example, using restriction endonucleases that only cleave methylated DNA (such as DpnI). This template, derived from an alkaline lysis plasmid preparation, is methylated and will be degraded in this step, while the mutant plasmid, being synthesized in vitro and unmethylated, is retained. In this method, a single PCR reaction can introduce more than one mutation (encoding the substitution described herein) into a nucleic acid sequence, for example, by using one or more oligonucleotides, each containing one or more mismatches. Alternatively, one or more mutations can be introduced into the nucleic acid sequence by performing more than one PCR reaction, with each reaction introducing one or more mutations, thereby introducing the modified nucleic acid into the target nucleic acid in a continuous and iterative manner.

[0102] The nucleic acid of this invention can be prepared using cDNA, mRNA, or genomic DNA as a template according to the above-described site-directed mutagenesis technique with suitable mismatched oligonucleotide primers. The resulting nucleic acid sequence can be cloned into a suitable vector and characterized by DNA sequencing.

[0103] The nucleic acid sequence described in this invention may contain one or more deletions (i.e., gaps) compared to the nucleic acid sequence encoding wild-type rennet (such as rennet having the amino acid sequence of SEQ ID NO:1). Such deletions / gaps can also be prepared using site-directed mutagenesis techniques with suitable oligonucleotides. Methods for constructing such deletions are well known to those skilled in the art.

[0104] Furthermore, oligonucleotides corresponding to or capable of hybridizing with the nucleotide sequence described in this invention can be prepared using standard synthetic techniques (e.g., using an automated DNA synthesizer).

[0105] The present invention also includes complementary nucleic acid sequences. A nucleic acid sequence complementary to another nucleotide sequence is a nucleic acid sequence that is sufficiently complementary to another sequence to hybridize with another nucleic acid sequence to generate a stable double strand.

[0106] The present invention also relates to a nucleic acid sequence encoding at least one functional domain of a polypeptide variant described herein. Suitably, the functional domain may comprise one or more of the substitutions described herein.

[0107] The gene or cDNA encoding the polypeptide of the present invention can be cloned and overexpressed in a host organism. Suitable host organisms include Aspergillus spp. ( 克鲁维酵母 Kluyveromyces ( ) 木霉 Trichoderma ( ) 大肠杆菌 ), Escherichia coli ( 毕赤酵母 Pichia pastoris ( ) 酿酒酵母 ), yeast ( ​ ), Yersinia lipolytica ( Yarrow Neurospora ( Neurospora ), Bacillus spp. ( Bacillus Fusarium ( ) Fusarium ), Hansenula genus ( Little Hansen ), genus Aureospora ( Chrysosporium ) or Candida genus ( White Suitable examples of bacterial host organisms are Gram-positive bacteria, such as Bacillus species (Bacillus). Bacillaceae ), including Bacillus subtilis ( Bacillus subtilis ), Bacillus licheniformis ( Bacillus licheniformis ), Bacillus tarda ( Bacillus lentus ), Bacillus brevis ( Bacillus brevis ), thermophilic fat bacillus ( Bacillus stearothermophilus ), Alkaliophilic Bacillus ( Bacillus alkalophilic ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens Bacillus coagulans ( Bacillus coagulans ), Bacillus circularis ( Circulating Bacillus Lactobacillus ( Bacillus laevis ), Bacillus megaterium ( Bacillus megalith ) and Bacillus thuringiensis ( Bacillus thuringiensis Streptomyces ( Streptomyces ), such as Streptomyces griseus ( Streptomyces murineus ); Lactic acid bacteria, including Lactococcus spp. ( Lactococcus spp. ), such as Lactococcus lactis ( Lactococcus lactis ); Lactobacillus ( Lactobacillus spp. ), including Lactobacillus reuteri ( Lactobacillus reuteri Leuconostoc ( ) Leuconostoc spp. ) and Streptococcus spp. Streptococcus spp.Alternatively, Gram-negative bacterial strains can be selected as the host organism, such as Enterobacteriaceae (…). Enterobacteriaceae (including Escherichia coli) E. coli )) or Pseudomonas ( Pseudomonadaceae ) strains.

[0108] Suitable yeast host organisms can be advantageously selected from: the genus *Saccharomyces* (Yeast). Saccharomyces ), including brewer's yeast ( Saccharomyces cerevisiae ), or belong to the genus *Schizosaccharomyces* ( Schizosaccharomyces Other available yeast hosts include the genus Pichia pastoris (Pichia pastoris). Pichia spp. ), such as the methylotrophic strains, including Pichia pastoris ( Shepherd's pie ); and Kluyveromyces ( Kluyveromyces spp. ), including Kluyveromycin (lactic acid yeast) Kluyveromyces lactis ).

[0109] Suitable host organisms among filamentous fungi include: *Cladosporium* ( Acremonium Aspergillus ( ) Aspergillus Fusarium ( ) Fusarium ), genus *Pythium* Humicola Mucor ( ) I am sick. ), Thermophilic filamentous fungi ( Mycelium Neurospora ( Neurospora ), Penicillium ( Penicillium ), genus *Clostridium* ( Thielavia ), genus *Morchella* ( Tolypocladium ) or Trichoderma ( Trichoderma ) species, such as Aspergillus echinosporum ( Aspergillus aculeatus ), Aspergillus awamori ( Aspergillus awamori Aspergillus smut () Aspergillus stinking ), Aspergillus japonicum ( Aspergillus japonicus Aspergillus oryzae ( ) Aspergillus oryzae Aspergillus nidus ( ) Aspergillus nidulans ) or Aspergillus niger ( Aspergillus niger ), including Aspergillus niger var. pamoate ( Aspergillus niger var.awamori ); Fusarium spores ( Fusarium bactridioides Fusarium graminearum ( ), Fusarium graminearum Cereal fusarium ), Crookwell Fusarium ( Fusarium crookwellensis ), Fusarium oxysporum ( Fusarium wilt Fusarium graminearum ( ), Fusarium gramineae Fusarium graminearum ( ), Fusarium grass Fusarium heterosporum ( ), Fusarium heterosporum ), Nuniddi Fusarium ( Fusarium negundi Fusarium oxysporum ( ), Fusarium oxysporum Fusarium reticulata ( ) Fusarium reticulatum ), pink Fusarium ( Fusarium roseum ), Fusarium elderberry ( Fusarium sambucinum ), flesh-colored Fusarium ( Fusarium sarcochroum Fusarium solani ( ), Fusarium solani Fusarium sporotrichioides ), Fusarium sulfonata ( Fusarium sulphureum ), Fusarium moniliforme ( Fusarium torulosum ), Fusarium moniliforme ( Fusarium trichothecioides Fusarium tumefaciens ( ), Fusarium venenatum ); sparse cottony humic mold ( Humicola insolens ), Humicola langinosa Mucor (Milk Black Mold) Mucor miehei Thermophilic filamentous fungi ( ); Thermophilic filamentous fungi ( Myceliophthora thermophila ); Neurospora crassa ( Neurospora crassa ); Penicillium chrysogenum ( Penicillium chrysogenu m), Penicillium carmenereum ( Penicillium camembertii ), Penicillium purpureum ( Penicillium purpurogenum ); Rhizopus oryzae ( Rhizomucor miehei ); Earth-colored Clostridial shell ( Thielavia terrestris Trichoderma harzianum ( ); Tricho-derma harzianum Corning Trichoderma ( ); Trichoderma koningii Trichoderma longifolia ( ); Trichoderma longibrachiatum Trichoderma reesei ( Trichoderma reesei ) or green Trichoderma ( Trichoderma viride ).

[0110] In this invention, the polypeptides of this invention may initially be provided in the form of prorenin, reninogen, or (mature) rennet. Corresponding nucleic acid sequences may also be provided, for example, polynucleotides encoding prorenin, reninogen, or (mature) rennet. The nucleic acid sequences encoding the aforementioned prorenin, reninogen, or (mature) rennet may be optimized for expression in target host cells.

[0111] Therefore, the present invention also provides a nucleic acid construct comprising the above-described nucleic acid sequence and operatively linked to one or more regulatory sequences capable of guiding the expression of the polypeptide in a host cell.

[0112] Suitable host cells include cells from the host organisms exemplified above. Preferably, the polypeptides of the present invention can be produced or generated therein by host cells selected from the group consisting of bacterial cells, fungal cells, and yeast cells. More preferably, the polypeptides of the present invention can be produced by host cells selected from the genus Aspergillus (…). Aspergillus Kluyveromyces ( ) Kluyveromyces Trichoderma ( ) Trichoderma ), Escherichia coli ( Escherichia coli Pichia pastoris ( ) Pichia ), yeast ( Saccharomyces ), Yersinia lipolytica ( YarrowiaNeurospora ( Neurospora ) or Bacillus spp. ( Bacillus The polypeptide is produced or generated in a host cell comprising a group of Kluyveromyces species. Most preferably, the polypeptide is produced by Kluyveromyces genus, more preferably Kluyveromyces lactis. Kluyveromyces lactis ) or Aspergillus, more preferably Aspergillus niger Awamori var. Aspergillus niger var. awamori ) is generated or is generated within it.

[0113] The term "operable link" is intended to refer to a target nucleotide sequence linked to a regulatory sequence in a manner that allows the nucleotide sequence to be expressed (e.g., in an in vitro transcription / translation system, or intracellularly when a vector is introduced into a host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (such as polyadenylation signals).

[0114] The polypeptides of the present invention can be prepared in prokaryotic or eukaryotic host cells using recombinant technology, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending on the host used in the recombinant preparation process, the polypeptides of the present invention may be glycosylated or deglycosylated. Furthermore, the polypeptides of the present invention may also contain initiating methionine residues, which in some cases result from host-mediated processing.

[0115] The present invention also provides a recombinant expression vector comprising the above-described nucleic acid sequence and / or the above-described nucleic acid construct.

[0116] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is the "plasmid," which is a circular double-stranded DNA loop into which an additional DNA fragment can be ligated. Another type is the viral vector, in which an additional DNA fragment can be ligated into the viral genome. Some vectors can replicate autonomously within host cells after introduction (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (such as non-free mammalian vectors) integrate into the host cell genome after introduction, thus replicating along with the host genome. Furthermore, some vectors can direct the expression of genes to which they are operatively linked. These vectors are referred to herein as "expression vectors." Generally, expression vectors commonly used in recombinant DNA technology are in plasmid form. The terms "plasmid" and "vector" are used interchangeably herein because plasmids are the most commonly used vector form. However, this invention also includes other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which can perform equivalent functions.

[0117] The recombinant expression vector of the present invention preferably comprises the nucleic acid sequence of the present invention in a form suitable for expression (preferably constitutive expression) of the nucleic acid sequence in a host cell. This means that the recombinant expression vector may include one or more regulatory sequences selected according to the host cell used for expression, which are operatively linked to the nucleic acid sequence to be expressed.

[0118] The polypeptide and / or nucleic acid sequences of the present invention can be prepared using the methods and techniques described in the examples, and / or using the methods described in the specifications and examples of WO2013 / 164479 and WO2013 / 164481, which are incorporated herein by reference.

[0119] In a fourth aspect, the present invention provides a recombinant host cell comprising the nucleic acid sequence of the third aspect, the nucleic acid construct, and / or the recombinant expression vector. In a fifth aspect, the present invention provides a method for preparing the polypeptide described in the first aspect, comprising expressing the nucleic acid sequence of the third aspect and / or the nucleic acid construct in the recombinant host cell described in the fourth aspect.

[0120] The recombinant host cells used in the above aspects are preferably the host cells described above. Other preferred options are as described above.

[0121] Uses of peptides In a sixth aspect, the present invention provides the use of the polypeptide described in the first aspect or the composition described in the second aspect in the preparation of cheese.

[0122] More preferably, this use includes adding the polypeptides or compositions of the present invention to an emulsion base. Preferred emulsion bases are as described in the context.

[0123] Using the polypeptides of the present invention, coagulants for cheese production can be advantageously obtained, with a short production cycle and processing that can be carried out early in the maturation process without the need for long-term storage.

[0124] As shown in the experimental section of this paper, the polypeptides of the present invention exhibit advantages when used in cheese production. The present invention provides the use of the polypeptide described in the first aspect (preferred variant #N11) or the composition described in the second aspect for improving the ability of cheese to retain moisture and fat during cheese heating. The present invention also provides the use of the polypeptide described in the first aspect (preferred variant #N11) for improving the flowability (free-flowing capacity) of grated cheese (e.g., but not limited to mozzarella). Furthermore, the present invention provides the use of the polypeptide described in the first aspect (preferred variant #N11) for improving the water-holding capacity of cheese.

[0125] method In a seventh aspect, the present invention provides a method for producing cheese, wherein the method comprises contacting the polypeptide described in the first aspect or the composition described in the second aspect with a milk base. More preferably, the polypeptide or composition is added to or supplemented into the milk base.

[0126] Preferably, the present invention provides a method for producing cheese, the method comprising adding an effective amount of the present invention's polypeptide or composition to a milk base or contacting an effective amount of the present invention's polypeptide or composition with a milk base, and performing appropriate subsequent cheese production steps.

[0127] In the context of the method described, the milk base can be derived from plant, microbial, or mammalian sources. Preferably, the milk base is derived from mammalian sources, such as cow's milk, sheep's milk, goat's milk, buffalo milk, camel's milk, rhea milk, horse's milk, or deer milk, or any combination thereof. Preferably, the milk can be selected from the group consisting of cow's milk, camel's milk, buffalo's milk, goat's milk, sheep's milk, and any mixture thereof. More preferably, the milk base is derived from bovine sources. Suitable plant milk sources include soybean, pea, peanut, barley, rice, oat, quinoa, almond, cashew, and coconut milk. Preferred plant milks are soy milk, oat milk, and almond milk. Cow's milk is most preferred. Preferably, the milk base is milk. Preferably, the milk is derived from mammalian sources. Most preferably, the milk is cow's milk.

[0128] The milk base material may suitably comprise, or consist of, fresh skim milk, non-skim milk, or reconstituted milk. Optionally, the milk base material may be obtained by concentration, in powder form, or reconstituted therefrom. Reconstituted milk, as understood herein, is a liquid milk obtained by adding a liquid (such as water) to skim milk powder, skim milk concentrate, whole milk powder, or whole milk concentrate. Furthermore, the milk base material may or may not undergo heat treatment (pretreatment), such as pasteurization or sterilization. In a preferred embodiment, the milk base material has undergone heat treatment such as pasteurization or sterilization.

[0129] Preferably, the polypeptide is added to or brought into contact with the milk base in a certain amount to obtain a concentration range of: equal to or greater than 1 IMCU / L milk base, more preferably equal to or greater than 5 IMCU / L milk base, further preferably equal to or greater than 10 IMCU / L milk base, even more preferably equal to or greater than 15 IMCU / L milk base, even more preferably equal to or greater than 20 IMCU / L milk base, still more preferably equal to or greater than 25 IMCU / L milk base, most preferably equal to or greater than 30 IMCU / L milk base to equal to or less than 100 IMCU / L milk base, more preferably equal to or less than 80 IMCU / L milk base, further preferably not equal to or less than 75 IMCU / L milk base, even more preferably equal to or less than 70 IMCU / L milk base, still more preferably equal to or less than 65 IMCU / L milk base, most preferably equal to or less than 60 IMCU / L milk base.

[0130] More preferably, the present invention provides a method for producing cheese, comprising: (i) adding a variant or composition of the rennet of the present invention to milk to coagulate the milk, thereby obtaining a curd; and (ii) processing the curd into cheese. Herein, curd is preferably understood as a coagulated component of the milk base.

[0131] The preferred amounts of peptides, emulsion base, and peptides added to or in contact with the emulsion base are as described above. Other preferred amounts are described below.

[0132] More preferably, the present invention provides a method for producing cheese, comprising: (a) Coagulating the emulsion base in the presence of the polypeptide of the present invention, wherein the coagulation step is performed before, simultaneously with or after the following operations: (i) fermentation in the presence of bacterial cultures; and / or (ii) Acidification with food-grade organic and / or inorganic acids. To obtain a coagulated emulsion base; and (b) Processing coagulated milk base into cheese.

[0133] The preferred embodiments of the peptides, the emulsion base, and the amounts of peptides added to or in contact with the emulsion base are as described above.

[0134] In step (a), the bacterial culture preferably comprises one or more strains of Streptococcus thermophilus or is composed of one or more strains of Streptococcus thermophilus, and preferably comprises one or more other bacterial strains, more preferably one or more strains of Lactobacillus and / or Lactococcus, and most preferably one or more strains of Lactococcus fat and / or Lactococcus lactis or composed of the like. Preferably, the temperature for step (a) is in the range of 28°C or higher, more preferably 30°C or higher, most preferably 32°C or higher to 47°C or lower, more preferably 45°C or lower, further preferably 42°C or lower, and most preferably 40°C or lower. Step (a) is preferably carried out in a fermenter.

[0135] The execution time of step (a) can vary within a wide range. However, preferably, the duration of step (a) is in the range of 10 minutes or more, more preferably 20 minutes or more, most preferably 30 minutes or more, up to 24 hours or less, more preferably 12 hours or less, further preferably 360 minutes or less, even more preferably 120 minutes or less, still more preferably 90 minutes or less, and most preferably 60 minutes or less. Shorter times facilitate faster production processes, which is desirable for cheese producers. The peptides of this invention can effectively shorten coagulation time, enabling a faster production process.

[0136] Preferably, step (a) involves fermenting the milk substrate in the presence of a bacterial culture until the pH reaches 6.0 or lower, more preferably 5.8 or lower, further preferably 5.5 or lower, and most preferably 5.3 or lower. For practical operational purposes, the pH during step (a) preferably varies within the following ranges: from 6.8 or lower, possibly from 6.7 or lower, or from 6.4 or lower to 3.0 or higher, more preferably to 3.5 or higher, further preferably to 4.0 or higher, and most preferably to 4.2 or higher. More preferably, the pH range during step (a) is from 6.8 or lower, possibly from 6.7 or lower, or from 6.4 or lower to 4.0 or higher, more preferably 4.5 or higher, further preferably to 5.0 or higher, and most preferably higher than 5.1.

[0137] Step (a) may also be referred to herein as the “curdling” step, which suitably yields a coagulated fermented milk base. The coagulated fermented milk base obtained by or that can be obtained by step (a) may suitably comprise curds and whey.

[0138] Step (b) may be suitably performed after step (a), which includes processing the coagulated fermented milk base into cheese. Processing the coagulated fermented milk base into cheese may suitably include cutting, stirring, and / or cooking steps, after which the whey and curd may be separated. The curd may be ground and may or may not be salted.

[0139] Therefore, the method may preferably include one or more separation steps, wherein a portion (preferably curd) of the coagulated fermented milk base is separated from another portion (preferably whey). Herein, curd is preferably understood as the coagulated component of milk. This coagulated component of milk preferably contains aggregated milk proteins, more preferably aggregated casein.

[0140] Preferably, the above method further includes a cheese harvesting step. After harvesting the cheese, it is preferable to allow the cheese to mature. That is, the above method preferably includes an additional cheese maturation step. In this document, "ripened" and "matured" are used interchangeably. The "matured" process is also referred to as the "maturing" process. Preferably, the maturation step of the fermented milk product includes maturation within the following time ranges: from 24 hours or more, more preferably from 48 hours or more, further preferably from 5 days or more, even more preferably from 7 days or more, still more preferably from 2 weeks or more, even more preferably from 1 month or more, most preferably from 3 months or more to 5 years or less, more preferably from 3 years or less, further preferably from 2 years or less, even more preferably from 1 year or less, and most preferably from 10 months or less. The temperature at which the maturation step of the fermented milk product is carried out is preferably in the range of 3°C or more, more preferably from 5°C or more to 20°C or less, and more preferably from 15°C or less.

[0141] The fermented dairy product obtained by the above method is preferably cheese, more preferably mature cheese. This method advantageously enables rapid cheese production, and the flavor and texture of the resulting cheese are improved compared to cheese prepared using rennets from existing technologies. The present invention also relates to cheese obtainable by the above method.

[0142] cottage cheese In an eighth aspect, the present invention provides a cheese, wherein the cheese comprises a degraded polypeptide as described in the first aspect, and / or is prepared or can be prepared by the method described in the seventh aspect or the use described in the sixth aspect.

[0143] Preferably, the cheese matures within the following time ranges: from 1 month or more, more preferably from 3 months or more, and from 24 months or less, more preferably up to 12 months, and preferably matures within the following temperature ranges: preferably from 3°C or more, more preferably from 5°C or more to 20°C or less, more preferably up to 15°C or less.

[0144] Preferably, the mature fermented dairy product (preferably mature cheese) is processed into slices, blocks or shreds within 1-30 days after production.

[0145] Compared with cheese prepared using conventional rennet, the polypeptides, compositions, preparation methods, and fermented dairy products described in this invention can advantageously reduce cheese particle loss; due to the short coagulation time, a rapid production process can be achieved; due to the rapid initial cleavage of α-S1-casein, the cheese matures faster in the early stages; at the same time, the higher C / P ratio can also lead to the possibility of extending the shelf life of the cheese.

[0146] The invention will be further illustrated by the following non-limiting embodiments.

[0147] Example Materials and Methods Culture medium composition YEP2D medium: yeast extract 10 g / L, bacterial peptone 20 g / L, glucose 40 g / L. Adjust pH to 6.7 with 4N NaOH. Incubate at 110°C by autoclaving for 30 minutes.

[0148] YEP2D / MES medium: yeast extract 10 g / L, bacterial peptone 20 g / L, glucose 40 g / L, MES 20 g / L. Adjust pH to 6.7 with 4N NaOH. Incubate at 110°C by autoclaving for 30 minutes.

[0149] YEP2D plates: YEP2D medium containing 1.8%-2% agar. After autoclaving at 110°C for 30 minutes, pour the culture medium into petri dishes.

[0150] strains Kluyveromycin (lactic acid yeast) K. lactis strain GG799: This *Kluyveromyces lactis* ( Kluyveromyces lactis The strain was used as the wild-type strain. This strain was purchased from New England Biolabs, Ipswich, Massachusetts, USA.

[0151] Molecular biology techniques Employing molecular biology techniques well known to those skilled in the art (see Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed. (CSHL Press, Cold SpringHarbor, NY, 2001). Examples of routine design, transformation methods, markers, and selective culture media for gene overexpression can be found in WO2007060247, WO2010102982, US4943529, and the references cited therein.

[0152] Coagulation activity (C) The coagulation activity was determined according to the international standard NEN-ISO 11815:2007 entitled "Milk - Determination of total milk-clotting activity of bovine rennets", published by the Dutch "Nederlands Normalisatie-instituut" (NEN) on July 1, 2007.

[0153] An emulsion solution was prepared by adding 11 g of Nilac milk powder (NIZO Food Science, Ede, the Netherlands) to 100 mL of 4.5 mL MCaCl2 solution (final pH 6.6). The solution was stirred for 30 minutes and then allowed to stand in the dark for another 30 minutes. The emulsion was then ready and used within half an hour. Subsequently, 5 mL of the emulsion solution was added to a test tube and pre-incubated in a 32°C water bath for 5 minutes. 100 μL of enzyme solution was added to the emulsion solution to initiate the reaction. The coagulation of the emulsion was then observed visually in real time. The moment when coagulation began was defined as the coagulation time point. A reference curve for activity determination was obtained using different amounts of diluted liquid rennet reference standard (Chr. Hansen, Denmark). A series of coagulation assays were performed at different concentrations to determine the coagulation time for each dilution and to determine the relationship between the coagulation time and the unit quantity in the test. The coagulation time found in the test sample was calculated back to the IMCU activity of the original rennet standard stock solution determined in this assay, expressed as IMCU / mL. The emulsion coagulation activity of the test samples was used for C / P ratio calculation, as illustrated in Example 3 below.

[0154] Total protein hydrolysis activity (P) Protein hydrolysis activity (P) is based on the work of Kappeler et al. entitled " Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milkThe standard method described in Biochemical and Biophysical Research Communications vol.342 (2006), pp.647–654.

[0155] Proteolytic activity was determined using bovine milk-derived sodium casein (Sigma, C8654) as the substrate. The reaction mixture (750 μL) contained 730 μL of substrate (0.5% sodium casein in 33 mM MES, pH 5.8) and 20 μL of the rennet sample, with a final dose of 70 IMCU / mL. The reaction mixture was incubated at 32 °C for 120 min, and the reaction was terminated by adding 250 μL of 12% (w / w) TCA and vortexing vigorously. The control group contained the same substrate and was treated for the same incubation time; the reaction was terminated first by adding TCA, followed by adding 20 μL of enzyme solution and vortexing. After centrifugation at 12000 rpm for 10 min, the OD280 of the supernatant was measured. The difference (ΔOD) between OD280 (t=120 min) and OD280 (blank) was calculated. This difference was used as a measure of the protein hydrolysis activity of the test sample at pH 5.8 and was used in the calculation of the C / P ratio illustrated in Example 3.

[0156] Example 1: DNA constructs and transformation The synthetic DNA construct was designed to be Xho The sequence begins with a restriction site (encoding amino acids L and E), followed by a frame containing DNA encoding a Kex-protein cleavage site (containing amino acids K and R), and then a frame containing a gene encoding a bovine pro-rennet B variant, which begins with the amino acids alanine, glutamic acid, isoleucine, and threonine, and terminates immediately after a stop codon at the PacI restriction site. As an example, the DNA fragment encoding the wild-type pro-rennet B sequence is shown in SEQ ID NO: 1. Codons were adapted using the method described in patent application US090286280. All variants were designed in the same manner and cloned into the vector pKLAC1 (New England Biolabs, Ipswich, Massachusetts, USA) as an XhoI PacI fragment.

[0157] The resulting open reading frame was prepared using Kluyveromycin (lactic acid yeast) K.lactis It begins with the leader sequence of the α-mating factor, extends through the Kex processing site to the bovine prochymotrypsin B variant.

[0158] The design followed the principles of synthetic DNA constructs. Amino acid alterations were introduced into 133 variants. The selection of new variants and their mutations is shown in Table 2. The altered sites are labeled in contrast to the mature wild-type bovine rennet B sequence (SEQ ID NO:1). All variants contain multiple mutations in the amino acid sequence that introduce the rennet protein.

[0159] The wild-type gene encoding unmutated pro-renin and pro-renin variants containing double coding mutations (A51V and K221V) were used simultaneously for gene cloning and transformation.

[0160] The mutant variant polypeptides listed in WO2013 / 164479 and WO2013 / 164481, designated as polypeptide #71, contain such mutations (A51V and K221V) and have the amino acid sequence SEQ ID NO:2, and are hereinafter referred to as reference polypeptide "Reference A". Reference A is used for comparison with the enzymes of the present invention prepared using the variant genes.

[0161] Additionally, the coagulant CHY-MAX™ Supreme, purchased from Chr. Hansen A / S, was used as reference peptide "Reference B". The coagulant CHY-MAX™ Supreme was considered to have the amino acid sequence SEQ ID NO:4.

[0162] Table 2: Selection of new variants Example 2: Cultivation, Activation, Purification and Concentration Kluyveromyces lactis carrying the mutant bovine chymosinogen gene ( Kluyveromyceslactis The strain was placed on YEP2D agar plates and cultured at 30°C for 48 hours. Yeast cells were picked from the plates and inoculated into 100 mL Erlenmeyer flasks containing 20 mL of YEP2D medium to prepare a preculture. The culture was incubated in a shaker at 30°C and 250 rpm for 24 hours. The amount of preculture medium inoculated into a new 500 mL Erlenmeyer flask containing 100 mL of YEP2D / MES medium was calculated to obtain a final OD600 of 0.01. The main culture was then incubated in a shaker at 30°C and 250 rpm for 65 hours.

[0163] Upon receiving the Erlenmeyer flasks containing the fermentation broth of the given variant, downstream processing begins. The fermentation broth from each Erlenmeyer flask is collected and centrifuged. The supernatant (light phase) containing the target enzyme activity is transferred to the clarification step. The filtrate is collected and transferred to the chemical activation step. This step is performed for 90 minutes at pH 2.35–2.40 and 30°C. The pH is adjusted back to pH 6.05 ± 0.05 to terminate chemical activation. The liquid containing the activated enzyme is then initially concentrated using a 1 kDa MWCO PES spiral wound ultrafiltration system. The concentrate is then transferred to an AMICON 1 kDa MWCO stirred ultrafiltration tank. The salinity of the concentrate is adjusted to 45–50 mS / cm, and the pH is adjusted to pH 4.1. This adjustment is performed before final chromatographic concentration and purification, which is carried out using a hydrophobic interaction medium. The target activity is collected from the fractions eluted from the column.

[0164] Example 3: C / P ratio The specificity (C / P) of rennet is an important indicator of the functional characteristics of enzymes in cheese production. The specificity of a single rennet sample is calculated by dividing the milk coagulation activity (C) by the total proteolytic activity (P). The methods for determining C and P activities are detailed in the "Materials and Methods" section. As shown in Table 3, the C / P ratios of several variants are higher than those of reference A and also higher than those of reference B.

[0165] Table 3: [Table 3: ...] C / P ratios were calculated for different rennet variants, where C and P were determined using the method described above. Example 4: In vitro hydrolysis of α-casein using SDS-PAGE A sterile suspension of 2.78 mg / mL α-casein (Merck, C6780) from bovine milk was prepared in 100 mM potassium buffer containing 2% (w / w) NaCl, pH 6.8. The dissolved casein solution was back-titrated to pH 5.5–5.6 with 1 M HCl. After filtration sterilization, the substrate was aliquoted into 900 μL portions and incubated in 1.5 mL tubes at 11 °C using an Eppendorf thermostat. 100 μL of a coagulant buffer containing 0.36 IMCU / mL in pH 5.5 was added to the α-casein substrate suspension. The casein-coagulant suspension was then incubated at 11 °C and 600 rpm for 2 days. Samples (70 μL each) were taken at 0, 3, 6, and 24 hours for protein profiling analysis and immediately flash-frozen in liquid nitrogen, then stored at -80 °C. These samples were inactivated by adding a 1:1 sample buffer (62mM Tris / 8.1M urea / 0.1M DTT, pH 7.6) and stored at -20°C for subsequent SDS-PAGE analysis. A control group containing 100 μL of buffer was also included to verify substrate quality and serve as a reference for protein mapping. For SDS analysis, 20 μL of sample was mixed with 55 μL of dilution buffer (62mM Tris / 0.1M DTT, pH 7.6) and 25 μL of 4×NuPage LDS loading buffer (Life Technologies), and incubated at 70°C for 5 minutes. Then, 10 μL of the mixture was loaded onto a 10% Bis-Tris NuPage gel. The gels were run in MES buffer with NuPage antioxidant (Life Technologies) in the inner tank and electrophoresed at 15 mA (10 min), 20 mA (20 min), and 30 mA (180 min), followed by staining with Coomassie Instant Blue (Expedeon). The gels were washed with deionized water and then scanned for densitometric analysis of the separated bands using a Typhoon FLA 9500 laser scanner and ImageQuant software.

[0166] Densitometric analysis of the stained bands was performed to quantify the hydrolysis products of α-casein and determine the proteolytic specificity of different variants and the reference enzyme. As previously described, the intensity of all separated bands was quantified using a Typhoon laser scanner and ImageQuant software. Subsequently, the total intensity of all bands in a sample (i.e., a single lane) was determined and used to calculate the relative intensity of α-S1-casein and the α-S1-I fragment. Therefore, the relative intensity of a band is expressed as a percentage of the total intensity in its lane / sample. This allows for better comparison between samples / bands compared to the absolute intensity of a single band, as the relative intensity corrects for potential differences in the total sample protein.

[0167] Density analysis data reflect the relative presence of the α-S1-casein band (relative to the total protein band in the sample), as shown in Table 4 and... Figure 1 As shown, it is clear that variant #N11 hydrolyzes α-S1-casein at the fastest rate.

[0168] Table 4 and Figure 1 It also shows the generation of the α-S1-I casein fragment over time. When α-S1 casein is specifically hydrolyzed between phenylalanine 23 and phenylalanine 24, it releases small peptides (α-S1 amino acid residues 1-23) and the α-S1-I fragment.

[0169] Unwilling to be bound by any theory, the inventors believe that the α-S1-I casein fragment, due to its lower hydrophobicity than α-S1 casein, provides cheese with textural properties suitable for rapid processing and makes an important contribution to the early maturation of cheese.

[0170] The data in Table 4 show that almost no α-S1-I casein fragments were produced when α-casein was cultured using reference B (Chymax Supreme). As shown, the levels of α-S1-I compounds were similar to those in the group without the enzyme in experiments using Chymax Supreme. All other rennets produced α-S1-I compounds, with the highest percentage produced in cultures using variant #N11.

[0171] The detection limit of this method is approximately 2%.

[0172] Table 4: As described in Example 4, after culturing in α-casein solution for 24 hours, the α-S1 hydrolysis rate and the percentage of relative α-S1-I content in the total protein were calculated (the percentages were calculated by optical density analysis of SDS gel). Example 5: Milk coagulation activity in heterogeneous whole milk at different pH levels Following the instrument's instruction manual, the milk coagulation activity of different rennet variants was determined using the Rheolaser Master (Formulaction). Heterogeneous whole milk was preheated in a 40°C water bath for at least 20 minutes, then CaCl2 was added to a final concentration of 4 mM. After incubation at 40°C for another 20 minutes, 1.0 M acetic acid or 0.5 M sodium hydroxide solution was added dropwise to adjust the milk pH to 6.1 or 6.6, respectively. The Rheolaser Master was set to 34°C, and 20 mL of milk was transferred to each of the six Rheolaser tubes. The milk was incubated in the Rheolaser instrument at 34°C for at least 15 minutes. The rennet sample was added to initiate the milk coagulation experiment. For the experiment at pH 6.1, the enzyme dosage was 16 IMCU / L milk; for the experiment at pH 6.6, the dosage was 30 IMCU / L milk. In each Rheolaser run, six milk samples were analyzed per run, including the peptide reference SEQ ID NO:2. The determined coagulation times for each rennet sample at both pH values ​​are shown in Table 5. All values ​​are reported relative to the reference sample of SEQ ID NO:2 peptide in the same batch. Coagulation times were provided by Rheolaser software according to the manufacturer's instructions. Data showed that several variants coagulated faster at pH 6.1 (values ​​below 1).

[0173] Table 5: The relative coagulation times of different rennet variants in whole milk relative to the polypeptide of SEQ ID NO:2 were determined using Rheolaser at pH 6.1 and pH 6.6.

[0174] Example 6: Preparation of 200-liter cheddar cheese Cheddar cheese was prepared using standard American processes. After pasteurization (73°C, 15 seconds), the milk was inoculated with dsm-firmenich Delvo Cheese CH-121 starting culture (1 unit per 1000 liters of cheese milk, batch GT00044164). This allowed the milk to pre-mature for 60 minutes. The rennet dosage for variant #N11 and reference A (i.e., variant peptide #71 in WO2013 / 164479 and WO2013 / 164481) was 40 IMCU / L. This dosage was based on laboratory experiments in Rheolaser, using the same milk samples and conditions as the cheddar experiments (pH 6.6, 50 ml of 33% w / w CaCl2 solution per 200 liters of milk) to ensure consistent cutting times for all cheddar cheese. After approximately 30 minutes, the curd was firm enough to be cut. Cooking began after 10 minutes and continued until 38°C. The pH is monitored regularly. When the pH drops below 6.2, the whey is drained, and the composting process in cheese production begins. The curd is turned regularly, and when the pH reaches 5.3, it is ground and salted. The ground curd with salt is allowed to mature for about 15 minutes, then molded and pressurized overnight. The cheese is unmolded the next morning (Day 1), samples are taken for component analysis, vacuum-packed, and matured at 11°C.

[0175] Example 7: Hydrolysis of α-casein in cheese using SDS-PAGE To monitor the hydrolysis of α-S1-casein in the cheddar cheese prepared in Example 6, 50 g samples were taken from the cheese at appropriate times on day 1 and day 17. For each time point, 8-10 30 mg aliquots were cut from different parts of the 50 g cheese sample and freeze-dried for at least 24 hours. Next, the 50 mg freeze-dried cheese sample was dissolved in 0.5 ml of sample buffer (62 mM Tris / 8.1 M urea / 0.1 M DTT, pH 7.6). For SDS analysis, 20 µl of the dissolved cheese sample was mixed with 180 µl of dilution buffer (62 mM Tris / 0.1 M DTT, pH 7.6). Next, 65 µl of the diluent was mixed with 25 µl of 4× NuPage LDS loading buffer (Life Technologies) and 10 µl of 10× sample reducing agent (Thermo Fisher Scientific), and incubated at 70 °C for 5 min. Then, 5 µl of the mixture was loaded onto a 10% Bis-Tris NuPage gel. The gel was run using MES buffer with NuPage antioxidant (Life Technologies) added to the inner tank, and electrophoresis was performed at 15 mA (10 min), 20 mA (20 min), and 30 mA (180 min). The gel was stained with Coomassie Brilliant Blue Rapid Stain (Expedeon). After washing with deionized water, the gel was scanned for densitometric analysis of the separated bands using a Typhoon FLA 9500 laser scanner and ImageQuant software.

[0176] Densitometric analysis of the stained bands was performed to quantify the hydrolysis products of α-casein and determine the proteolytic specificity of different variants and the reference enzyme. As previously described, the intensity of all separated bands was quantified using a Typhoon laser scanner and ImageQuant software. Subsequently, the total intensity of all bands in a sample (i.e., a single lane) was determined and used to calculate the relative intensity of α-S1-casein and the α-S1-I fragment. Therefore, the relative intensity of a band is expressed as a percentage of the total intensity in its lane / sample. This allows for better comparison between samples / bands compared to the absolute intensity of a single band, as the relative intensity corrects for potential differences in the total sample protein. Densitometric analysis data for the relative presence of the α-S1-casein band are shown in Table 6. It is clearly shown that the α-S1-casein hydrolysis rate was fastest in the cheese sample prepared using variant #N11.

[0177] Table 7 shows the generation of the α-S1-I casein fragment over time. Unlike in vitro α-casein culture systems, it is not possible to detect individual SDS-Page bands of the α-S1-I casein fragment in cheese samples as in in vitro α-S1-I fragment cultures, because these bands overlap with the bands of β-casein present in cheese on SDS gels. However, it is still possible to monitor the generation of the α-S1-I casein fragment in cheese samples by measuring the increase in intensity of the band containing both the α-S1-I fragment and β-casein on the SDS-gel. The results are reported in Table 7. In cheese samples produced with variant #N11 and reference A, the presence of the α-S1-I casein fragment appears to be increased, as the intensity of the combined band containing both the α-S1-I casein fragment and β-casein increases significantly. The fastest increase was observed for peptide variant #N11.

[0178] Table 6 Numerical values ​​indicate the percentage of α-S1-casein in the total protein content of the sample (the percentage was calculated after optical density analysis of the SDS gel). Table 7 Numerical values ​​indicate the percentage of the total protein in the sample relative to the combined content of α-S1-I casein + β-casein (the percentage was calculated after optical density analysis of the SDS gel). Example 8: Comparison of coagulation activity / proteolytic activity of the variants with the eight variants described in WO2013 / 164479 (C / P) The C / P values ​​of the variants described in this patent application (Example 3, Table 3) are compared with the 'relative C / P values' of the eight variants described in WO2013 / 164479. WO2013 / 164479 reports relative C / P values; the measured C / P value of the variant is divided by the C / P measured for the control sample Maxiren. Since reference A in this patent is equivalent to variant #71 in WO2013 / 164479, the data from the two patent applications can be compared. A coefficient is obtained by dividing the C / P ratio described in the variants of this patent by the C / P value of reference A (Table 8). By multiplying the relative C / P ratio of variant #71 in WO2013 / 164479 (value 16.8, see Table 3 of WO2013 / 164479) by the obtained coefficient, we calculated the corrected C / P ratio of the variant in this patent application, which can be directly compared with the C / P value listed in WO2013 / 164479, resulting in Table 9. This value can be corrected via this overlapping enzyme for accurate comparison because the methods used to determine coagulation activity and proteolytic activity are the same (see the "Materials and Methods" section).

[0179] The C / P values ​​(Table 8, last column) of all variants described in this patent application are higher than the previously determined C / P values ​​(Table 9) of the eight variants reported in WO2013 / 164479, except for variant #99, which has a C / P of 43.8, higher than that of variant #N11, which has a C / P of 41.4.

[0180] Table 8 Coefficient calculation: Variant's C / P divided by reference A's C / P Table 9 Relative C / P values ​​of the eight variants reported in WO2013 / 164479 Example 9: In vitro α- peptidoglycan analysis of variant #N11 and eight variants described in WO2013 / 164479 using SDS-PAGE Casein hydrolysis Following the guidelines of WO2013 / 164479, strains of *Kluyveromyces lactis* expressing the eight variants (#95, #96, #97, #98, #99, #100, #109, and #110) described in WO2013 / 164479, as well as a strain expressing variant #N11, were cultured. The supernatant was collected and concentrated 15-fold using a 10 kDa filter (Amicon® Ultra-15, a centrifugal filter unit with a 10 kDa cutoff). The concentrated supernatant was incubated at pH 2 for 90 minutes to activate rennet, followed by pH restoration to pH 6.1. The coagulation activity of the samples was determined using an adjusted coagulation activity assay. In this assay, 40 μL of the activated supernatant was incubated with 200 μL of 1.2% skim milk at pH 6.1, and the absorbance was measured at 600 nm for 60 minutes. The coagulation activity of the concentrated supernatant was calculated using a standard curve of Maxiren® with known IMCU / mL activity (determined using the international standard coagulation assay as described in this patent). Because the conditions of this adjusted assay differ from those used in the international standard assay, the sample coagulation activity is expressed in MCU / mL rather than IMCU / mL. Subsequently, the in vitro α-casein hydrolytic activity of these supernatants was tested as described in Example 4, except that the coagulant was derived from a stock solution containing 0.36 MCU / mL instead of 0.36 IMCU / mL. Densitometric analysis data from SDS-PAGE gels, reflecting the relative presence of α-S1-casein bands (relative to the total protein bands in the sample), are shown in Table 10 and clearly demonstrate that the hydrolysis of α-S1-casein occurred most rapidly in variant #N11. Table 10After culturing with α-casein solution for 24 hours as described in Example 4, the hydrolysis rate of α-S1-casein and the percentage of relative α-S1-I content in the total protein were calculated (the percentages were calculated by optical density analysis of SDS-PAGE gel). * For αS1 t=0, the values ​​of 'enzyme-free sample' t=24 hrs are used because no degradation was detected in this sample.

[0181] Example 10: Comparison of the ability of variant #N11 and reference B to retain moisture and fat during cheese heating Pasta Filata / mozzarella cheese was prepared using variant #N11 and reference B. The resulting cheese was then shredded, and equal portions of the shredded cheese were placed in separate containers. The cheese was heated to obtain completely melted cheese. The release of moisture and fat during heating was measured by pouring the liquid off the solid and subsequently weighing each fraction. The results showed that the cheese prepared using variant #N11 exhibited improved moisture and fat retention: compared to reference B, variant #N11 showed more than 30% less moisture and fat release.

[0182] Example 11: Comparison of the ability of variant #N11 and reference B to retain moisture in cheese Pastafilata / mozzarella cheese was prepared using variant #N11 and reference B. The resulting cheese was then subjected to a water separation method to determine its water-holding capacity. Cheese samples were taken and ground immediately before weighing. 12 grams of cheese were weighed into a small centrifuge tube. This process was repeated twice for each cheese. The tubes were centrifuged at 12,500 rpm for 60 minutes at room temperature. Afterward, the centrifuge tubes were removed from the centrifuge. The volume of liquid in the tube was weighed. This is referred to as the extractable whey. The whey was transferred to small Nalgene tubes and frozen for subsequent analysis. The water extraction yield was calculated using the following formula: Watering off (%) = (whey weight / 12g cheese) × 100 The percentage of total moisture (%) = (water separation rate % / moisture content in cheese) × 100.

[0183] Table 11 The percentage of water separated from cheese out of the total water content of the cheese. Therefore, it can be concluded that variant #N11 exhibits better water-holding capacity compared to reference B. Furthermore, variant #N11 increases total water content without causing significant loss of free water.

[0184] Example 12: Comparison of the flowability of shredded cheese between variant #N11 and reference B Mozzarella cheese was prepared using variant #N11 and reference B, respectively. The resulting cheeses were shredded four months after production, and the flowability of the shredded portions was compared. The comparison showed that the shredded mozzarella cheese produced using variant #N11 had less stickiness and therefore improved free-flowing properties compared to the shredded mozzarella cheese produced using reference B.

Claims

1. A polypeptide having an amino acid sequence, wherein the amino acid sequence, when compared with the amino acid sequence shown in SEQ ID NO:1, contains at least substitutions of amino acid residues corresponding to amino acids at sites 50, 51, 126, 135 and 221, wherein the sites are defined with reference to SEQ ID NO:

1.

2. The polypeptide according to claim 1, comprising at least the mutants N50D, A51V, A126G, and S135T, and at least the mutants K221M or K221V, wherein the site is defined with reference to SEQ ID NO:

1.

3. The polypeptide according to any one of the preceding claims, wherein the polypeptide further comprises a substitution of an amino acid residue corresponding to an amino acid at site 201, site 243, site 164, or site 292, wherein the site is defined with reference to SEQ ID NO:1, preferably, wherein the polypeptide further comprises a substitution of an amino acid residue corresponding to an amino acid at site 201, wherein the site is defined with reference to SEQ ID NO:

1.

4. The polypeptide according to claim 3, comprising at least the mutant S201D, Y243E, S164G, or H292D, wherein the site is defined with reference to SEQ ID NO:1, preferably the mutant S201D, wherein the site is defined with reference to SEQ ID NO:

1.

5. The polypeptide according to any one of the preceding claims, wherein the polypeptide has an amino acid sequence, said amino acid sequence, when compared with the amino acid shown in SEQ ID NO:1, comprising: (i) Mutate at least N50D, A51V, A126G, S135T, K221M, and Y243E; or (ii) Mutate at least N50D, A51V, A126G, S135T, S201D, and K221V; or (iii) At least N50D, A51V, A126G, S135T, S201D, and K221M; or (iv) Mutate at least N50D, A51V, A126G, S135T, S164G, K221M and H292D; The site mentioned therein is defined with reference to SEQ ID NO:

1.

6. The polypeptide according to any one of the preceding claims, wherein the polypeptide has an amino acid sequence having equal or greater than 90%, more preferably equal or greater than 95%, and most preferably equal or greater than 98% sequence identity with SEQ ID NO:

1.

7. The polypeptide according to any one of the preceding claims, wherein the polypeptide is: - A polypeptide having the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; or - Homologs of the polypeptide having the amino acid sequence of SEQ ID NO:5, having an amino acid sequence with greater than 99% sequence identity to SEQ ID NO:5; or - Homologs of the polypeptide having the amino acid sequence of SEQ ID NO:6, having an amino acid sequence with greater than 98% sequence identity to SEQ ID NO:6; or - Homologs of the polypeptide having the amino acid sequence of SEQ ID NO:7, having an amino acid sequence with greater than 98% sequence identity to SEQ ID NO:7; or - Homologous to a polypeptide having the amino acid sequence of SEQ ID NO:8, having an amino acid sequence having greater than 98% sequence identity with SEQ ID NO:

8.

8. The polypeptide according to any one of the preceding claims, wherein the polypeptide is a non-naturally occurring polypeptide.

9. A composition comprising a polypeptide according to any one of claims 1-8.

10. The composition according to claim 9, wherein the pH of the composition is equal to or less than pH 6.6, preferably equal to or less than pH 6.1, and more preferably equal to or less than pH 5.

8.

11. A nucleic acid sequence encoding a polypeptide according to any one of claims 1-8.

12. A nucleic acid construct comprising the nucleic acid sequence of claim 11, said nucleic acid sequence being operatively linked to one or more regulatory sequences capable of directing the expression of the polypeptide of any one of claims 1-8 in a host cell.

13. A recombinant expression vector comprising the nucleic acid sequence according to claim 11 and / or the nucleic acid construct according to claim 12.

14. A recombinant host cell comprising the nucleic acid sequence according to claim 11, the nucleic acid construct according to claim 12, and / or the recombinant expression vector according to claim 13.

15. A method for generating a polypeptide according to any one of claims 1-8, the method comprising expressing the nucleic acid sequence according to claim 11 and / or the nucleic acid construct according to claim 12 in a recombinant host cell according to claim 14.

16. Use of the polypeptide according to any one of claims 1-8 or the composition according to any one of claims 9-10 in the preparation of cheese.

17. A method for producing cheese, wherein the method comprises contacting a polypeptide according to any one of claims 1-8 or a composition according to any one of claims 9-10 with a milk base.

18. The method according to claim 17, wherein, The pH of the emulsion base is equal to or less than pH 6.6, preferably equal to or less than pH 6.1, and more preferably equal to or less than pH 5.

8.

19. A cheese, wherein the cheese comprises a degraded polypeptide according to any one of claims 1-8, and / or the cheese is prepared or can be prepared by the method according to any one of claims 17-18 and / or by use according to claim 16.

20. A cheese, wherein the pH of the cheese is between 5.0 and 6.1, and wherein the cheese contains a polypeptide according to any one of claims 1-8 or a composition according to any one of claims 9-10.

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