WO2025099224A1 - Improved enzyme variants - Google Patents

Abstract

A polypeptide comprising: a) a chymosin activity with a C/P ratio of clotting activity (C) to proteolytic activity (P) equal to or higher than the C/P ratio of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and b) an alpha-S1-I casein fragment formation activity equal to or higher than the alpha-S1- I casein fragment formation activity of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and/or c) a clotting time at pH 6.1 of equal to or less than the clotting time of a polypeptide having an amino acid sequence of SEQ ID NO: 2.

Description

IMPROVED ENZYME VARIANTS

Field of the invention

[001] The invention relates to a polypeptide having chymosin activity. The invention further relates to a composition comprising such polypeptide, to use of such polypeptide or polypeptide- containing composition in the preparation of a cheese, to a process for the production of a cheese and to the resulting cheese.

Background of the invention

[002] Enzymatic coagulation of milk by milk clotting enzymes, such as chymosin, is an important process in the manufacture of cheeses. Enzymatic milk coagulation is a two-phase process: a first phase where a proteolytic enzyme attacks kappa-casein, resulting in a metastable state of the casein micelle structure and a second phase, where the milk subsequently coagulates and forms a coagulum. For the manufacture of cheeses a variety of enzyme characteristics are important.

[003] WO2013/164479 explains the need for modified proteolytic activity under cheese-making conditions. In most cheeses, chymosin is responsible for “primary” proteolysis, which leads to the release of peptides that are later used by lactic acid bacteria for “secondary” proteolysis and flavour formation during ripening. WO2013/164479 reports on the finding of an advantageous variant chymosin polypeptide in this respect, wherein the variant has an amino acid sequence which, when aligned with the chymosin comprising the sequence set out in SEQ ID NO: 2, comprises at least one substitution of an amino acid residue corresponding to any of amino acids 2, 22, 40, 48, 50, 51 , 53, 61 , 62, 76, 88, 98, 99, 109, 1 12, 1 17, 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 said positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having chymosin activity. In its examples, WO2013/164479 describes a broad range of 115 specific variant polypeptides. Variants #71 , #74, #95, #98, #103, #104 and #106 are stated to show a reduced stimulation of the proteolytic activity with lower pH compared to Maxiren (bovine chymosin) and Chymax M (camel chymosin). The examples further mention thermolabile variants (#95, #98 and #104) containing the mutations S135T and A126G. In addition, it is stated that variants that contained additional negative surface-charge, like variant #12, and #107, #111 and #112 had a clearly higher productivity than wild-type chymosin. According to WO2013/164479 these variants had multiple changes in the amino acid sequence of chymosin which has led to the introduction of extra negative charges (aspartate and glutamate) in the amino acid sequence of calf chymosin.

[004] WO2013/164481 explains that the ideal coagulant for the industrial production of young cheese is one that leads not only to a prolonged storage, but also to fast processibility/ shreddability early in the ripening, without extensive storage. However, since fast processibility requires high proteolytic activity but prolonged storage requires low proteolytic activity on the casein matrix, it was not clear how these two properties could be combined in a single coagulant. Subsequently WO2013/164481 reports on the advantageous finding of a polypeptide having chymosin activity which (a) is capable of hydrolysing bovine alpha s1-casein at position F23F24 so as to form as1-l casein fragment (f24-199) more rapidly than camel chymosin; and (b) has a C/P ratio higher than the C/P ratio of bovine chymosin. In its experiments, WO2013/164481 mentions that a combination of mutations at positions A51 and K221 (variants #71 , #74, #98, #103 and #110) gave a synergistic effect on the increase in C/P. In the experiments it is further mentioned that all variants that contain changes in amino acids at position A51 and/or K221 of the mature calf chymosin B, show a clear hydrolysis of the first cut in alpha-s1 -casein, leading to a rapid formation of fragment alpha-s1-l casein.

[005] WO2023/194285 (Chr. Hansen) refers to polypeptides encoded by specific DNA sequences. In the examples these specific DNA sequences were integrated into Aspergilus expression hosts. Some of the enzymes so produced were stated to provide a higher C/P ratio than bovine chymosin and a higher degradation of intact alphaSI -casein than camel chymosin.

[006] Kaminogawa et al., in their article titled “Calcium Insensitivity and Other Properties of aS1 -I Casein", published in the Journal of Dairy Science, vol. 63 (1980), pages 223-227, explain that alpha S1-I casein fragment is a large peptide, derived from alpha S1 -casein by chymosin hydrolysis of the bond between residues of 23 and 24, that is thought to have a relationship to texture.

[007] However, as illustrated by Exterkate et al., in their article titled “The Selectivity of Chymosin Action on aS1- and /3-caseins in Solution is Modulated in Cheese", published in International Dairy Journal, vol. 7 (1997), pages 47-54, cleavage of the alpha S1 (aS1) casein does not necessarily occur between residues 23 and 24 and the mere cleavage of an alpha S1 (aS1) casein does not necessary lead to the formation of the desired alpha S1-I casein fragment. Table 1 of Exterkate et al. illustrates that in fact, such fragment may be neither formed nor maintained.

[008] Although the enzymes of WO2013/164479 and WO2013/164481 have been found to work very well, industry is calling for further improvements.

[009] Cheese manufacturers are in need of an enzyme that has a combination of optimum enzyme characteristics.

[010] Unfortunately, due to the interactions between amino acids within the enzyme, multiple mutations within the enzyme can strongly influence each other. The individual effect of one mutation may for example be cancelled by a combination with another mutation. The development of new enzymes is further complicated by the fact that a milk or milk-base may comprises different types of casein proteins, including the alpha S1 (aS1) casein and kappa (K) casein, and further alpha S2 (aS2) casein and/or beta (p) casein. These casein proteins interact each individually and differently with the enzyme and a positive effect in respect of one casein protein may be accompanied by a negative effect in respect of another casein protein. [011] It would be an advancement in the art to provide a new enzyme and/or a new process for production of cheese that allows for modified proteolytic activity under cheese-making conditions as mentioned in WO2013/164479, but also fast processibility/shreddability as mentioned in WO2013/164481 . Additionally or in the alternative, a short production time and/or a low applied dosage scheme is/are desired.

Summary of the invention

[012] Advantageously a coagulant polypeptide has now been identified which allows for modified proteolytic activity under cheese-making conditions as mentioned in WO2013/164479 and fast processibility/shreddability as mentioned in WO2013/164481 . In addition, the polypeptide of the invention can be used to shorten production time of the cheese. The polypeptide of the invention is therefore a superior coagulant compared to the polypeptides of the prior art.

[013] Surprisingly, the new enzyme has a double selectivity. The new enzyme has a high and selective clotting and proteolysis activity in respect of kappa-casein, whilst simultaneously it has a high and selective proteolysis activity in respect of alpha S1 (aS1) casein. Advantageously, the desired aS1-l casein fragment is stably formed and not immediately cleaved further. The enzyme is further able to function at normal and acidified (low) pH conditions. In addition, the new enzyme allows cheese manufacturers to reduce production time and/or to decrease enzyme dosage.

[014] Accordingly, in a first aspect, the present invention provides a polypeptide comprising: a) a chymosin activity with a C/P ratio of clotting activity (C) to proteolytic activity (P) equal to or higher than the C/P ratio of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and b) an alpha-S1 -I casein fragment formation activity equal to or higher than the alpha-S1 -I casein fragment formation activity of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and/or c) a clotting time at pH 6.1 of equal to or less than the clotting time of a polypeptide having an amino acid sequence of SEQ ID NO: 2.

[015] In a second aspect, the invention provides a composition comprising the polypeptide of the first aspect.

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

[017] In a fourth aspect, the invention provides a recombinant host cell comprising the nucleic acid sequence, the nucleic acid construct and/or the recombinant expression vector of the third aspect. [018] In a fifth aspect, the invention provides a method for producing the polypeptide of the first aspect, comprising expressing the nucleic acid sequence and/or the nucleic acid construct of the third aspect in the recombinant host cell of the fourth aspect.

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

[020] In a seventh aspect, the invention provides a process for the production of a cheese, wherein the process comprises contacting an amount of the polypeptide of the first aspect or a composition of the second aspect with a milk base.

[021] In an eighth aspect, the invention provides a cheese, wherein the cheese comprises an deteriorated polypeptide of the first aspect and/or is obtained or obtainable by the process of the seventh aspect or by the use of the sixth aspect.

[022] The above aspects according to the invention advantageously allow for modified proteolytic activity under cheese-making conditions as mentioned in WO2013/164479, and/or fast processibility/shreddability as mentioned in WO2013/164481 and/or a short production time and/or a low applied dosage scheme.

Brief description of the drawings

[023] The invention is illustrated by the following figures:

[024] Figure 1 : Gel results in respect of the densitometric analysis carried out in Example 4.

Brief description of the sequence listing

[025] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. An overview of the sequences is provided by Table 1 below.

Table 1 : Overview of sequence listings:

Detailed description of the invention

Definitions

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

[027] Throughout the present specification and the accompanying claims, the words "comprise”, "include" and “having” and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows. [028] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

[029] Unless explicitly indicated otherwise, the various embodiments of the invention described herein can be cross-combined.

[030] The term "milk" is intended to encompass milks from mammals, milks from plant sources, milks from microbial sources and/or mixtures thereof. Preferably, the milk is from a mammal source. Mammal sources of milk include, but are not limited to cow milk, sheep milk, goat milk, buffalo milk, camel milk, llama milk, horse milk or reindeer milk. In an embodiment, the milk is from a mammal selected from the group consisting of cow, sheep, goat, buffalo, camel, llama, horse and deer, and combinations thereof. Plant sources of milk include, but are not limited to, milk extracted from soy bean, pea, peanut, barley, rice, oat, quinoa, almond, cashew, coconut, hazelnut, hemp, sesame seed and sunflower seed. Microbial sources of milk include milk and milk proteins produced by recombinant micro-organisms in a laboratory (also referred to as “lab-grown milk”) or bioreactor. Preferably the milk is a non-recombinant, naturally occurring and/or naturally produced, milk. Cows are most preferred as a source for milk. Bovine milk is most preferred. In addition, the term "milk" refers to not only whole milk, but also skim milk or any liquid component derived thereof or reconstituted milk.

[031] The term "milk-base" refers to a base composition, comprising or consisting of milk or milk ingredients, or derived from milk or milk ingredients. The milk-base can be used as a raw material for the fermentation to produce a cheese. The milk-base may for example comprise or consist of skimmed or non-skimmed milk, or reconstituted milk. Optionally the milk-base may be concentrated or in the form of powder or may be reconstituted from such. By reconstituted milk is herein understood liquid milk obtained by adding liquid, such as water, to a skim milk powder, skim milk concentrate, whole milk powder or whole milk concentrate. Furthermore, the milk-base may or may not have been subjected to a thermal processing operation which is at least as efficient as pasteurization. Preferably the milk-base is from a bovine source.

[032] Any references to %w/v herein, such as for example references to 12% w/v reconstituted skim milk (RSM), refer to weight in grams present per volume of 100 ml solution, for example 12% w/v RSM corresponds to 12 grams of skim milk powder dissolved per 100 ml water.

[033] Chymosin and pepsin, the milk clotting enzymes of the mammalian stomach, are aspartic proteases belonging to a broad class of peptidases. That is, the term “chymosin” suitably refers herein to an aspartic protease, preferably an aspartic protease of EC group 3.4.23.4, in line with the Enzyme Nomenclature, 1992 of the International Union of Biochemistry and Molecular Biology, IUBMB. The terms “polypeptide having chymosin activity”, “protein having chymosin activity”, “enzyme having chymosin activity”, “chymosin protein”, “chymosin peptide”, “chymosin enzyme” and simply “chymosin” are used interchangeably herein. By “chymosin activity” is herein preferably understood an aspartic protease activity, preferably a catalytic activity as defined for enzyme classification EC group 3.4.23.4. The term chymosin refers herein to naturally occurring chymosin and non-naturally occurring chymosin, for example produced in a laboratory. Wild-type, naturally occurring, chymosin can be naturally produced by gastric chief cells in juvenile mammals. Chymosin is thought to be the main enzymatic component in rennet. Calf rennet can for example be obtained from the lining of the abomasum (the fourth and final, chamber of the stomach) of young, unweaned calves. Non-naturally produced chymosin can be produced microbially by recombinant micro-organisms, such as yeasts or bacteria. Such chymosin is herein also referred to as “microbial chymosin”.

[034] Pro-chymosin is in the present context to be understood as the precursor protein or proenzyme of chymosin. Pro-chymosin may possess a leader sequence (pro-part) on the N- terminal side of chymosin and said leader sequence is believed to be cleaved off during activation of the pro-chymosin. Furthermore in this context, pre-pro-chymosin is to be understood as a protein of pro-chymosin, to which is added on the N-terminal end of pro-chymosin a hydrophobic leader sequence. This leader sequence, also called secretion signal or pre-part, can be cleaved off before secretion or when the protein is secreted. Chymosin may in the cell be initially synthesised as pre- pro-chymosin (as for example described by Harris et al., Nucleic acid Research 1982, April 10, pages 2177-2187, in the article titled “Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosiri”).

[035] A gene or cDNA coding for chymosin or pro-chymosin may be cloned and over-expressed in a host organism. Suitable host organisms for chymosin over-expression include Aspergillus sp., Kluyveromyces sp., Trichoderma sp., Escherichia coll, Pichia sp., Saccharomyces sp., Yarrowia sp., Neurospora sp. Or Bacillus sp..

[036] As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a polypeptide as described herein. A gene may include coding sequences, non-coding sequences, introns and regulatory sequences. That is to say, a “gene”, as used herein, may refer to an isolated nucleic acid molecule as defined herein. Accordingly, the term “gene”, in the context of the present application, does not refer only to naturally-occurring sequences.

[037] 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) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases. [038] As used herein, the terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkages. The word "polypeptide" is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. The one-letter code of amino acids used herein is commonly known in the art and can be found in Berg, Tymoczko and Stryer, Biochemistry, 6^th edition, chapter 2, W.H Freeman and Company, New York, 2007.

[039] The terms “homology” and “percent identity” are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid for optimal alignment with a second amino or nucleic acid sequence). The amino acid or nucleotide residues at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions (i.e. overlapping positions) x 100). Preferably, the two sequences are the same length.

[040] A sequence comparison may be carried out over the entire lengths of the two sequences being compared or over fragment of the two sequences. Suitably the comparison can be carried out over the full length of the two sequences being compared. However, sequence identity may be carried out over a region of, for example, twenty, fifty, one hundred or more contiguous amino acid residues.

[041] The skilled person will be aware of the fact that several different computer programs are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

[042] The protein sequences or nucleic acid sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTN and BLASTP programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10. BLAST protein searches can be performed with the BLASTP program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped 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 at http://www.ncbi.nlm.nih.gov/.

[043] Herein, the term “IMCU” is understood to refer to International Milk Clotting Units. One IMCU equals about 0.126 nmol of bovine chymosin B (e.g. Maxiren™ or CHY-MAX™). The strength of a milk clotting enzyme (such as chymosin enzyme) can be determined as the milk clotting activity (IMCU per ml or per gram) in accordance with the standard as set by the International Dairy Federation (IDF) in ISO 11815|IDF standard 157A:1997 as prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF).

[044] Following the addition of diluted coagulant to a standard milk substrate, the milk will flocculate. The milk clotting time is the time period from addition of the coagulant until formation of visible flocks or flakes in the milk substrate. The strength of a coagulant sample can be found by comparing the milk clotting time for the sample to that of a reference standard, a normal. This is also further explained in IDF standard 157A:1997. In line with the IMCU principle the determination of the time needed for visible flocculation of renneted standard milk substrate with 0.05% calcium chloride, pH6.5. IMCU/ml of a sample is determined by comparison of the clotting time to that of a standard having known milk clotting activity and having the same enzyme composition of the sample.

[045] The term “C/P” or “C/P ratio” refers to the clotting activity (C) divided by the proteolytic activity (P) of a specific enzyme sample. The C/P is a measurement of the specificity of the coagulant. The methods to measure both activities and calculate the C/P is described herein (see the Examples). The method to measure the clotting activity (C) will quantify the efficiency of the enzyme sample to hydrolyse kappa-casein (k-CN) at the specific position F-M, between amino acids phenylalanine (Phe, F) and methionine (Met, M). The proteolytic activity (P) will quantify the ability of the coagulant to hydrolyse casein into small (TCA-soluble) peptide fragments and amino acids.

The polypeptide

[046] In a first aspect, the invention provides a polypeptide comprising: a) a chymosin activity with a C/P ratio of clotting activity (C) to proteolytic activity (P) equal to or higher than the C/P ratio of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and b) an alpha-S1 -I casein fragment formation activity equal to or higher than the alpha-S1 -I casein fragment formation activity of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and/or c) a clotting time at pH 6.1 of equal to or less than the clotting time of a polypeptide having an amino acid sequence of SEQ ID NO: 2.

[047] Preferably, the polypeptide of the first aspect is a polypeptide comprising: a) a chymosin activity with a C/P ratio of clotting activity (C) to proteolytic activity (P) higher than the C/P ratio of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and b) an alpha-S1-l casein fragment formation activity higher than the alpha-S1-l casein fragment formation activity of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and/or c) a clotting time at pH 6.1 of less than the clotting time of a polypeptide having an amino acid sequence of SEQ ID NO: 2.

[048] By chymosin activity is herein preferably understood the activity of a chymosin enzyme, more preferably a chymosin enzyme of enzyme class EC 3.4.23.4. Preferably the polypeptide of the invention is an enzyme in enzyme class EC 3.4.23.4. A more detailed description of such a chymosin enzyme is provided above under definitions. More preferably the polypeptide comprises a chymosin activity, wherein such chymosin activity comprises or consists of a selective kappacasein hydrolyzing activity. Most preferably the polypeptide of the invention is an enzyme that cleaves the kappa-casein at least at the peptide bonding between amino acid residues phenylalanine (F) and methionine (M). For example, where the kappa-casein for this determination is a mature bovine kappa-casein having the amino acid sequence set out in SEQ ID NO: 9, the polypeptide of the invention suitably selectively hydrolyses (that is “cleaves”) the peptide bonding F105-M106.

[049] The complete sequence of a bovine kappa-casein is reported in UNIPROT accession number P02668. The sequence of a mature bovine kappa-casein (i.e. without the 21 amino acid signal peptide) is set out in SEQ ID NO: 9. Position F105-M106 refers to the phenylalanine and the methionine at respectively positions 105 and 106 in a sequence such as SEQ ID NO:9. There are several alternatives for this bovine protein with slightly different amino acid sequences, but the skilled person will be able to easily identify the amino acids corresponding to F105 and M106 in SEQ ID NO: 9.

[050] Wild type camel chymosin has a higher thermostability than wild type bovine chymosin. As it is desirable that the chymosin enzyme has a certain thermolability, causing it to disintegrate during heat applying steps in a cheese making process, cheese manufacturers have a preference for bovine chymosin. Hence preferably the polypeptide of the invention is a bovine chymosin or a mutant of bovine chymosin. Preferably the polypeptide is a mutant of bovine chymosin. More preferably the polypeptide is a mutant of the polypeptide having amino acid sequence SEQ ID NO:

1 . Such a mutant polypeptide is sometimes also referred to as a variant polypeptide. Hence, the polypeptide is preferably a variant polypeptide of the polypeptide having the amino acid sequence of SEQ ID NO:1.

[051] When applying bovine chymosin, cheese manufacturers are in need of enzymes that have an improved C/P ratio. More preferably, when applying bovine chymosin, cheese manufacturers are in need of enzymes that have an improved C/P ratio at low pH, for example at a pH of 6.1 or even lower.

[052] The polypeptide preferably has a C/P ratio of equal to or more than 15, more preferably equal to or more than 20, even more preferably equal to or more than 30, and most preferably equal to or more than 40. There is no upper limit, but in practice the C/P ratio may be equal to or less than 1000, and possibly equal to or less than 500. Preferably the C/P ratio of the polypeptide of the invention is equal to or more than 1.1 , more preferably equal to or more than 1.5, even more preferably equal to or more than 2, still more preferably equal to or more than 2.5, and most preferably equal to or more than 3, times higher than the C/P ratio of the polypeptide having amino acid sequence SEQ ID NO: 2. That is, the polypeptide preferably has a C/P ratio (as determined according to the below Examples) that is equal to or more than double, preferably equal to or more than triple, the C/P ratio of a polypeptide having the amino acid sequence set out in SEQ ID NO:

2. Noticeable, the polypeptide having amino acid sequence SEQ ID NO: 2 is the polypeptide variant numbered #71 of prior art WO2013/164479 and WO2013/164481 .

[053] The C/P ratio can suitably be determined by determining a Clotting activity (C) and a Proteolytic activity (P) and subsequently calculating the ratio of Clotting activity (C) to Proteolytic activity (P). As used herein, the term "C/P ratio" is preferably further defined by the methods for determining a C-value and a P-value, respectively, as described in the below Examples.

[054] Preferably the polypeptide also has alpha-S1 -casein hydrolyzing activity. By an alpha-S1- casein hydrolyzing activity is herein preferably understood the activity to hydrolyze alpha-S1 -casein protein. The alpha-S1 -casein protein is herein also referred to as simply “alpha-S1 -casein” or abbreviated as “aS1 CN”. More preferably the polypeptide of the invention is an enzyme that selectively hydrolyzes alpha-S1 -casein into a first fragment and a second alpha S1-I casein fragment. Even more preferably the polypeptide comprises alpha-S1 -casein hydrolyzing activity, wherein such alpha-S1 -casein hydrolyzing activity comprises or consists of an alpha-S1 -casein hydrolyzing activity at the peptide bonding between a first phenylalanine (F) and a second phenylalanine (F). For example, where the alpha-S1 -casein for this determination is a mature bovine alpha-S1 -casein protein having the amino acid sequence set out in SEQ ID NO: 10, the polypeptide suitably catalyses hydrolysis of the peptide bonding F23-F24.

[055] Most preferably the alpha-S1 -casein hydrolyzing activity is an activity to hydrolyse alpha- S1-casein, preferably between phenylalanine 23 and phenylalanine 24, under the release of an alpha-S1 -I casein fragment. Preferences for such an alpha-S1 -I casein fragment are described below.

[056] The mature bovine alpha-S1 -casein is herein also abbreviated as mature bovine “aS1-CN”. The complete sequence of a bovine alpha-S1 -casein is reported in UNIPROT accession number P02662. The sequence of a mature bovine alpha s1 -casein (i.e. without the 15 amino acid signal peptide) is set out in SEQ ID NO: 10. Position F23-F24 refers to the two phenylalanine residues at positions 23 and 24 in a sequence such as SEQ ID NO: 10. There are several alternatives for this protein with slightly different amino acid sequences, but the skilled person will be able to easily identify the amino acids corresponding to F23 and F24 in SEQ ID NO: 10. When a mature bovine alpha-S1 -casein, such as for example the alpha-S1 -casein of SEQ ID NO: 10, is hydrolyzed (i.e. “cleaved”) at the position F23-F24, a first fragment is formed (herein also referred to as fragment “f1 -23”) and a second fragment is formed (herein also referred to as fragment “f24-199”). Such first fragment (“f1 -23”) comprises the first 23 amino acids of the mature bovine alpha-S1 -casein protein, and the second fragment (“f24-199”) comprises the remaining amino acids 24-199 of the mature bovine alpha-S1 -casein protein.

[057] As explained by Exterkate et al., in their article titled “The Selectivity of Chymosin Action on aS1- and /3-caseins in Solution is Modulated in Cheese “, published in International Dairy Journal, vol. 7 (1997), pages 47-54, cleavage of the alpha-S1 (aS1) casein does not necessarily occur (only) between residues 23 and 24 and the mere cleavage of an alpha-S1 -casein does not necessary lead to the formation of the desired alpha-S1 -I casein fragment. Table 1 of Exterkate et al. illustrates that in fact, under the conditions exemplified in the article, such fragment may be neither formed nor maintained.

[058] By alpha-S1 -I casein fragment formation activity is herein preferably understood the activity to form and maintain an alpha-S1 -I casein fragment during at least 24 hours. An alpha-S1 -I casein fragment is herein also abbreviated as “aS1-l-CN”. Preferably, such alpha-S1 -I casein fragment is a hydrolysis product, corresponding to the second fragment obtained after hydrolysis of an alpha- S1-casein. That is, preferably the term “alpha-S1-l casein fragment” is understood herein to refer to the longest fragment formed when hydrolyzing an alpha-S1 -casein protein at the peptide bonding F-F, between the first phenylalanine (F) and the second phenylalanine (F). More preferably the alpha-S1 -casein protein is a mature bovine alpha-S1 -casein that is being hydrolyzed at position F23-F24 so as to form an alpha-S1 -I casein fragment f24-199. That is, preferably the alpha-S1 -I casein fragment is a bovine alpha-S1 -I casein f24-199 fragment. An example of such a bovine alpha-S1-l casein f24-199 fragment is provided in SEQ ID NO: 11. There are several alternatives for this protein fragment with slightly different amino acid sequences, but the skilled person will be able to easily identify the amino acids corresponding to those in SEQ ID NO: 11 .

[059] Without wishing to be bound by any kind of theory, inventors believe this alpha S1-I fragment provides an important contribution in early knitting of the cheese by giving the cheese the desired textural properties for fast processing because of its decreased hydrophobicity compared to the alpha S1 casein.

[060] The hydrolysis of the alpha-S1 -casein protein over a period of 24 hours can be expressed in terms of an aS1-CN hydrolysis ratio, wherein the aS1-CN hydrolysis ratio t=24h is calculated as the difference in concentration of alpha-S1 -casein (“aS1-CN”) at time(t)=0 hours and time (t)=24 hours, divided by the concentration of alpha-S1 -casein (“aS1-CN”) at time(t)=0 hours. Hence: aS1-CN hydrolysis ratio t=24h = (aS1-CN _t=oh - aS1-CN _t=24h) / aS 1-CN t=oh

[061] Herein, a higher aS1-CN hydrolysis ratio is indicative of an increased reduction in aS1-CN concentration and hence indicative of an increased hydrolysis.

[062] Preferably the polypeptide of the invention has an aS1-CN hydrolysis ratio t=24h of equal to or more than 0.5, more preferably equal to or more than 0.6, yet more preferably equal to or more than 0.7 and most preferably equal to or more than 0.8.

[063] Preferably the polypeptide of the invention has an aS1-CN hydrolysis ratio t=24h, wherein such aS1-CN hydrolysis ratio t=24h is equal to or more than 1.1 , more preferably equal to or more than 1 .2, still more preferably equal to or more than 1 .3, even more preferably equal to or more than 1 .4, and most preferably equal to or more than 1.5 times the aS1 -CN hydrolysis ratio t=24h of a polypeptide having the amino acid sequence of SEQ ID NO: 2. Preferably the alpha-S1 -casein (“aS1-CN”) in such a determination is an alpha-S1 -casein derived from bovine milk (“bovine aS1- CN”).

[064] More preferably the polypeptide of the invention is an enzyme capable of hydrolyzing mature bovine alpha-S1 -casein, wherein the hydrolysis of such mature bovine alpha-S1 -casein at position F23-F24, forming alpa-S1-l casein fragment (f24-199), is equal to or more than 1.1 , more preferably equal to or more than 1 .2, still more preferably equal to or more than 1 .3, even more preferably equal to or more than 1 .4, and most preferably equal to or more than 1 .5, times more rapid than the polypeptide having amino acid sequence SEQ ID NO: 2. For this determination, the mature bovine alpha-S1 -casein may preferably have an amino acid sequence of SEQ ID NQ:10 and the alpa-S1-l casein fragment may preferably have an amino acid sequence of SEQ ID NO:11. [065] Hydrolysis of the F23-F24 bond in alpha-S1 -casein can be measured in different ways. Speed or rate of hydrolysis may be compared, for example, in terms of amount of enzyme (for example in terms of mg protein) or in terms of an equivalent amount of IMCUs. A higher rate of hydrolysis compared to a reference is indicative of a polypeptide of the invention that is capable of hydrolysing alpha s1 -casein at position F23-F24 so as to form aS1-l CN (f24-199) more rapidly than such reference.

[066] The formation and maintenance of the alpha-S1-l casein fragment (“aS1-l-CN“) over a period of 24 hours for a specific polypeptide can be compared to a reference and expressed in terms of an aS1-l-CN formation ratio, wherein the aS1-l-CN formation ratio t=24h is calculated as the ratio in concentration of alpha-S1-l-casein fragment (“aS1-l-CN”) at time(t)=24 hours for the specific polypeptide and the reference. Hence: aS1 -I -CN formation ratio t=24h vis-a-vis Reference A = (aS1-l-CN variant, t=24h / aS 1 -I -CN R_Sf A, t=24h) The concentration can be expressed as a weight percentage alpha-S1 -I casein fragment, based on the total weight of proteins or as a pixel percentage alpha-S1 -I casein fragment, based on the total amount of pixels for proteins as explained below.

[067] Preferably the polypeptide of the invention has a formation and maintenance of the alphas'! -I casein fragment (“aS1-l-CN“) over a period of 24 hours that is equal to or more than 1.1 , more preferably equal to or more than 1 .2, and most preferably equal to or more than 1 .3, times the formation and maintenance of the alpha-S1 -I casein fragment over a period of 24 hours by the polypeptide having amino acid sequence SEQ ID NO: 2. That is, preferably the polypeptide of the invention has an aS1-l-CN formation ratio t=24h vis-a-vis the polypeptide having amino acid sequence of SEQ ID NO:2 that is equal to or more than 1.1 , more preferably equal to or more than 1.2, and most preferably equal to or more than 1.3. Preferably the alpha-S1-l casein fragment (“aS1-l-CN”) in such a determination is an alpha-S1 -I casein fragment derived from bovine milk (“bovine aS1-l-CN”).

[068] The above mentioned hydrolysis of alpha-S1 -casein protein over a period of 24 hours, the above mentioned aS1-CN hydrolysis ratio t=24h, the above mentioned formation and maintenance of alpha-S1 -I casein fragment and the above mentioned aS1-l-CN formation ratio t=24h , can each suitably be determined as described in the Examples.

[069] Preferably concentrations of alpha-S1 -casein protein and/or alpha-S1-l casein fragment can be analyzed using PAGE, staining of the protein bands, identification of the different bands, and scanning and densitometric analysis of the different hydrolysis products. Such an analytical method to quantify alpha-S1 -casein and alpha-S1-l casein fragment, obtained with different coagulants, has been described previously in literature (see for example the article of Bansal et al. titled “Suitability of recombinant camel (Camelus dromedarius) chymosin as a coagulant for Cheddar cheese”, published in the International Dairy Journal, vol. 19, (2009), pages 510-517). A comparable densitometric analysis to quantify alpha-S1 casein and alpha-S1 -I casein fragment is described in the Examples.

[070] The polypeptide according to the invention is preferably a polypeptide, wherein the polypeptide has an alpha-S1 -I casein fragment formation activity with a densitometric alpha S1-I casein fragment percentage after 24 hours (“@ 24 hours”) of equal to or more than 10%, more preferably equal to or more than 20%, even more preferably equal to or more than 30% and most preferably equal to or more than 40%, based on the total protein content after 24 hours (“@ 24 hours”). For such a densitometric percentage, the percentage may suitably be a “pixel” percentage following a densitometric analysis, where the percentage of pixels in a densitometric band representing alpha-S1 -I casein fragment is calculated on the basis of the total amount of pixels for all densitometric bands representing proteins.

[071] Alternatively, hydrolysis of the F23-F24 bond in mature bovine alpha-S1 casein and formation of the hydrolysis products can be followed using RP-HPLC and quantified using a described technique (see for example the article by Carles and Dumas, titled “Kinetics of the action of chymosin (rennin) on a peptide bond of bovine a_si-casein: Comparison of the behaviour of this

and K₀-caseins", published in FEBS Letters vol. 185(2), (1985), pages 282- 286). The rate of the appearance of the alpha-S1 I fragment and the disappearance of the intact alpha-S1 casein can be conveniently monitored using this technique. It will be clear to a person skilled in the art that such RP-HPLC can also be used to quantify alpha-S1 casein and its degradation products.

[072] A further method to quantify the rate of formation of alpha-S1 -I casein fragment is also described in the article by M0ller et al. titled “Camel and Bovine Chymosin Hydrolysis of Bovine aS1- and /3-Caseins Studied by Comparative Peptide Mapping”, published in J. Agric. Food Chem. Vol. 60, (2012), pages 11421-11432.

[073] Any of the above methods may be used to determine the rate at which a chymosin polypeptide is capable of hydrolysing bovine alpha-S1 -casein at position F23-F24 so as to form alpha-S1 -I casein fragment (“aS1-l-CN”, e.g. f24-199).

[074] Rate and selectivity of the hydrolysis can be expressed as described in the Examples.

[075] Accordingly, preferably the polypeptide of the invention:

- may suitably be capable of hydrolysing mature bovine alpha-S1 -casein at position F23-F24 so as to form bovine alpha-S1-l casein fragment (“aS1-l-CN”, f24-199) over a period of 24 hours in a higher amount than a reference polypeptide having amino acid sequence SEQ ID NO: 2,

- may suitably be defined as an enzyme that produces: a ratio of alpha-S1-l casein fragment (aS1- l-CN) to intact alpha-S1 casein (aS1-CN) of preferably equal to or more than 0.6, more preferably equal to or more than 0.7, and most preferably equal to or more than 1 .0, when the incubation with alpha S1 casein is performed for 6 hours at 1 1 °C; or a ratio of alpha-S1-l casein fragment (aS1-l- CN) to intact alpha-S1 casein (aS1-CN) of preferably equal to or more than 1.5, more preferably equal to or more than 2.0, even more preferably equal to or more than 3.0, still more preferably equal to or more than 5.0, yet more preferably equal to or more than 8.0, and most preferably equal to or more than 10, when the incubation with alpha-S1 -casein is performed for 24 hours at 11 °C.

[076] By an alpha-S1 -I fragment formation activity is herein preferably understood the capability to form such alpha-S1 -I casein fragment and the maintenance of such alpha-S1 -I casein fragment for at least 24 hours following formation. More preferably the alpha-S1 -I casein fragment formation activity comprises or consists of a cleavage activity by the polypeptide of the alpha-S1 casein protein, wherein within the first 24 hours following the contact of the polypeptide and the alpha-S1 casein protein: - preferably equal to or more than 50% (w/w), more preferably equal to or more than 60% (w/w), even more preferably equal to or more than 70% (w/w), of the alpha S1 casein protein is hydrolysed (i.e. “cleaved”) at the position between phenylalanine 23 and phenylalanine 24, under formation of an alpha-S1 -I casein fragment; and/or

- preferably equal to or less than 50% (w/w), more preferably equal to or less than 60% (w/w), even more preferably equal to or less than 70% (w/w), of the alpha-S1-l casein fragment so formed is hydrolysed (i.e. “cleaved”) further at other positions.

[077] The alpha-S1 -I casein fragment formation activity can suitably be determined as illustrated in the Examples or as described above.

[078] Preferably the polypeptide further has a ratio of clotting time at pH 6.6 to clotting time at pH 6.1 (clotting ratio _PH 6.6/_PH 6.I) of equal to or more than 1 .10, preferably of equal to or more than 1 .20. Such clotting ratio _PH 6.6/ _PH 6.I can suitably be determined by means of a rheolaser as exemplified in the Examples. The milk clotting was here followed visually in time. The moment coagulation starts is considered the point of clotting time.

[079] Most preferably the polypeptide of the invention is a polypeptide comprising: a) a chymosin activity with a C/P ratio of clotting activity (C) to proteolytic activity (P) equal to or higher than the C/P ratio of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and b) an alpha-S1 -I casein fragment formation activity equal to or higher than the alpha-S1 -I casein fragment formation activity of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and c) a clotting time at pH 6.1 of equal to or less than the clotting time of a polypeptide having an amino acid sequence of SEQ ID NO: 2.

Further preferences are as set out above.

[080] Even more preferred, the polypeptide of the invention is a polypeptide comprising: a) a chymosin activity with a C/P ratio of clotting activity (C) to proteolytic activity (P) higher than the C/P ratio of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and b) an alpha-S1 -I casein fragment formation activity higher than the alpha-S1 -I casein fragment formation activity of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and c) a clotting time at pH 6.1 of less than the clotting time of a polypeptide having an amino acid sequence of SEQ ID NO: 2.

Further preferences are as set out above.

[081] Without wishing to be bound by any kind of theory, it is believed that the above double selectivity can allow for advantages in texture, fast processability and reduced cutting losses in the form of fines of cheese. Advantageously, the polypeptide according to the invention further also allows for rapid production of the cheese. [082] Preferably the polypeptide of the invention is a polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 1 , comprises at least a substitution of the amino acid residues corresponding to the amino acids in positions 50, 51 , 126, 135 and 221 , wherein said positions are defined with reference to SEQ ID NO: 1 . Preferably this polypeptide has chymosin activity and preferably this polypeptide has alpha- S1-I casein fragment formation activity, where further preferences are as set out above.

[083] More preferably the polypeptide comprises:

- at least the mutations N50D, A51V, A126G, S135T; and

- at least a mutation selected from the mutations K221 M and K221 V, wherein said positions are defined with reference to SEQ ID NO: 1 .

[084] More preferably, the polypeptide additionally comprises a substitution of the amino acid residue corresponding to the amino acids in position 201 or position 243 or position 164 or position 292, wherein said position is defined with reference to SEQ ID NO: 1 , preferably wherein the polypeptide additionally comprises a substitution of the amino acid residue corresponding to the amino acid in position 201 , wherein said position is defined with reference to SEQ ID NO: 1. Preferably, such a polypeptide comprises at least the mutation S201 D or Y243E or S164G or H292D, wherein said positions are defined with reference to SEQ ID NO: 1 , preferably mutation S201 D, wherein said position is defined with reference to SEQ ID NO: 1.

[085] Even more preferably the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 1 , comprises at least the mutations N50D, A51V, A126G, S135T, S201 D and K221V, wherein said positions are defined with reference to SEQ ID NO: 1.

[086] Customers and regulatory authorities have a preference for a minimum amount of mutations of the enzyme vis-a-vis the wild type. Preferably the polypeptide of the invention is therefore a polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 1 , comprises equal to or less than 10, more preferably equal to or less than 8, even more preferably equal to or less than 7, and most preferably at most 6 mutations.

[087] Preferably the polypeptide has an amino acid sequence with equal to or more than 90%, more preferably equal to or more than 95%, and most preferably equal to or more than 98%, sequence identity with the amino acid sequence of SEQ ID NO: 1.

[088] Preferably the polypeptide has an amino acid sequence with equal to or more than 90%, more preferably equal to or more than 95%, and most preferably equal to or more than 98%, sequence identity with the amino acid sequence of SEQ ID NO: 2.

[089] The invention most preferably provides a polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 1 , comprises at least a substitution of the amino acid residues corresponding to the amino acids in positions 50, 51 , 126, 135, 201 and 221 , wherein said positions are defined with reference to SEQ ID NO: 1 , wherein said polypeptide has an amino acid sequence with equal to or more than 90%, more preferably equal to or more than 95%, and most preferably equal to or more than 98%, sequence identity with the amino acid sequence of SEQ ID NO: 1 and wherein the polypeptide comprises at least the mutations N50D, A51V, A126G, S135T, S201 D and K221V.

[090] Most preferably the polypeptide is a polypeptide that has an amino acid sequence of SEQ ID NO: 6; or is a homologue of a polypeptide having amino acid sequence of SEQ ID NO: 6, wherein this homologue has an amino acid sequence with more than 98%, more preferably equal to or more than 99%, and most preferably equal to or more than 99.5% sequence identity with SEQ ID NO: 6. [091] The above polypeptide(s) may conveniently comprise: a) a chymosin activity with a C/P ratio of clotting activity (C) to proteolytic activity (P) equal to or higher than the C/P ratio of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and b) an alpha-S1 -I casein fragment formation activity equal to or higher than the alpha-S1 -I casein fragment formation activity of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and/or c) a clotting time at pH 6.1 of equal to or less than the clotting time of a polypeptide having an amino acid sequence of SEQ ID NO: 2, or more preferred a) chymosin activity with a C/P ratio of clotting activity (C) to proteolytic activity (P) higher than the C/P ratio of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and b) an alpha-S1-l casein fragment formation activity higher than the alpha-S1-l casein fragment formation activity of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and/or c) a clotting time at pH 6.1 of less than the clotting time of a polypeptide having an amino acid sequence of SEQ ID NO: 2

Further preferences are as mentioned above.

[092] The polypeptide according to the invention can be an isolated naturally occurring polypeptide or a non-naturally occurring polypeptide. By a non-naturally occurring polypeptide is herein understood a polypeptide that is not naturally produced by any mammal. Preferably the polypeptide is a non-naturally occurring polypeptide.

[093] By an "isolated" polypeptide or protein is herein understood a polypeptide or protein removed from its native environment. For example, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention as are recombinant polypeptides which have been substantially purified by any suitable technique. A polypeptide variant according to the invention can be recovered and purified from recombinant cell cultures by methods known in the art.

[094] Preferably, the claimed polypeptide performs better in at least one of the claimed characteristics when compared to, not only SEQ ID NO: 2, but also when compared to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19 or 20. The composition comprising the polypeptide

[095] In a second aspect, the invention provides a composition comprising the polypeptide of the first aspect.

[096] More preferably such a composition has a pH equal to or less than pH 6.6, preferably equal to or less than pH 6.1 , more preferably equal to or less than pH 5.8.

[097] The composition may optionally comprise other ingredients such as additives and/or other enzymes.

[098] If present, a preferred other enzyme that can be present in the composition is a pepsin enzyme, more preferably a pepsin enzyme of EC group EC 3.4.23.1 .

[099] Preferably the composition according to the invention may further comprise one or more pH buffers and/or other additives such as glycerol and/or sodium chloride.

[100] The composition can be liquid, frozen or freeze-dried. If liquid, the composition is preferably a solution, suspension or emulsion.

Nucleic acid sequences encoding the polypeptide

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

[102] The polypeptides and/or nucleic acid sequences of the present invention can be generated using the methods and techniques as described in the Examples and/or as described in the description and Examples of WO2013/164479 and WO2013/164481 , incorporated herein by reference.

[103] Conveniently, the nucleic acid sequence of the invention can be generated using standard molecular biology techniques well known to those skilled in the art taken in combination with the sequence information provided herein.

[104] For example, using standard synthetic techniques, the required nucleic acid sequence may be synthesized de novo. Such a synthetic process will typically be an automated process.

[105] Alternatively, a nucleic acid sequence of the invention may be generated by use of site- directed mutagenesis of an existing nucleic acid sequence, for example a nucleic acid sequence encoding a wild-type chymosin, such as the nucleic acid sequence of SEQ ID NO: 12. SEQ ID NO: 12 sets out the nucleic acid sequence of the wild type pro-chymosin B gene sequence from Bos taurus with codon adaptation for expression in K. lactis and with linkers to allow cloning into pKLACI . Site-directed mutagenesis may be carried out using a number of techniques well known to those skilled in the art. [106] In one such method, mentioned here merely by way of example, PCR is carried out on a plasmid template using oligonucleotide "primers" encoding the desired substitution. As the primers are the ends of newly-synthesized strands, should there be a mis-match during the first cycle in binding the template DNA strand, after that first round, the primer-based strand (containing the mutation) would be at equal concentration to the original template. After successive cycles, it would exponentially grow, and after 25, would outnumber the original, unmutated strand in the region of 8 million: 1 , resulting in a nearly homogeneous solution of mutated amplified fragments. The template DNA may then be eliminated by enzymatic digestion with, for example using a restriction enzyme which cleaves only methylated DNA, such as Dpn1 . The template, which is derived from an alkaline lysis plasmid preparation and therefore is methylated, is destroyed in this step, but the mutated plasmid is preserved because it was generated in vitro and is unmethylated as a result. In such a method more than one mutation (encoding a substitution as described herein) may be introduced into a nucleic acid sequence in a single PCR reaction, for example by using one or more oligonucleotides, each comprising one or more mis-matches. Alternatively, more than one mutation may be introduced into a nucleic acid sequence by carrying out more than one PCR reaction, each reaction introducing one or more mutations, so that altered nucleic acids are introduced into the nucleic acid in a sequential, iterative fashion.

[107] A nucleic acid of the invention can be generated using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate mis-matched oligonucleotide primers according to the site-directed mutagenesis technique described above. A nucleic acid sequence derived in this way can be cloned into an appropriate vector and characterized by DNA sequence analysis.

[108] A nucleic acid sequence of the invention may comprise one or more deletions, i.e. gaps, in comparison to nucleic acid sequence encoding a wild type chymosin, such as the chymosin having amino acid sequence SEQ ID NO: 01. Such deletions/gaps may also be generated using site- directed mutagenesis using appropriate oligonucleotides. Techniques for generating such deletions are well known to those skilled in the art.

[109] Furthermore, oligonucleotides corresponding to or hybridizable to nucleotide sequences according to the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

[1 10] Also, complementary nucleic acid sequences are included in the present invention. A nucleic acid sequence which is complementary to another nucleotide sequence is one which is sufficiently complementary to the other nucleotide sequence such that it can hybridize to the other nucleotide sequence thereby forming a stable duplex.

[1 11] The invention also relates to nucleic acid sequences encoding at least one functional domain of a polypeptide variant of the invention. Suitably, such a domain may comprise one or more of the substitutions described herein. [1 12] A gene or cDNA coding for the polypeptide of the invention may be cloned and overexpressed in a host organism. Suitable host organisms include Aspergillus, Kluyveromyces, Trichoderma, Escherichia coll, Pichia, Saccharomyces, Yarrowia, Neurospora, Bacillus, Fusarium, Hansenula, Chrysosporium or Candida. Examples of suitable bacterial host organisms are gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium and Bacillus thu- ringiensis, Streptomyces species such as Streptomyces murinus, lactic acid bacterial species including Lactococcus spp. such as Lactococcus lactis, Lactobacillus spp. including Lactobacillus reuteri, Leuconostoc spp. and Streptococcus spp. Alternatively, strains of a gram negative bacterial species such as a species belonging to Enterobacteriaceae, including E. coll or to Pseudomonadaceae may be selected as the host organism.

[1 13] A suitable yeast host organism may advantageously be selected from a species of Saccharomyces including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces. Further useful yeast host organisms include Pichia spp. such as methylotrophic species hereof, including Pichia pastoris, and Kluyveromyces spp. including Kluyveromyces lactis.

[1 14] Suitable host organisms among filamentous fungi include species of Acremonium,

Aspergillus, Fusarium, Humicola, Mucor, Myceliophtora, Neurospora, Penicillium, Thielavia, Tolypocladium or Trichoderma, such as e. g. Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus oryzae, Aspergillus nidulans or Aspergillus niger, including Aspergillus nigervar. awamori, Fusarium bactridioides, Fusa- rium cereals, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichiodes, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola langinosa, Mucor miehei,

Myceliophtora thermophila, Neurospora crassa, Penicillium chrysogenum, Penicillium camenbertii, Penicillium purpurogenum, Rhizomucor miehei, Thielavia terestris, Tricho- derma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesii or Trichoderma viride.

[1 15] In the invention, the polypeptide of the invention may originally be provided in the form of pre-prochymosin, prochymosin or (mature) chymosin. A corresponding nucleic acid sequence may also be provided, for example a polynucleotide that encodes a pre-prochymosin, prochymosin or (mature) chymosin is provided. The nucleic acid sequence encoding for such a pre-prochymosin- , prochymosin- or (mature) chymosin-encoding sequence may be optimized for expression in a desired host cell. [1 16] The invention therefore also provides a nucleic acid construct comprising such a nucleic acid sequence operably linked to one or more control sequences capable of directing the expression of the polypeptide in a host cell.

[1 17] Suitable host cells include cells from the above exemplified host organisms. Preferably the polypeptide according to the invention can be produced by or in a host cell, selected from the group consisting of bacteria cells, fungus cells and yeast cells. More preferably the polypeptide according to the invention can be produced by or in a host cell chosen from the group consisting of Aspergillus, Kluyveromyces, Trichoderma, Escherichia coll, Pichia, Saccharomyces, Yarrowia, Neurospora or Bacillus. Most preferably the polypeptide is produced by or in Kluyveromyces, more preferably Kluyveromyces lactis, or Aspergillus, more preferably Aspergillus niger var. awamori.

[1 18] The term “operatively linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signal).

[1 19] Polypeptides of the present invention can for example be produced, with the help of recombinant techniques, in a prokaryotic or eukaryotic host cell, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

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

[121] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms “plasmid” and “vector” can be used interchangeably herein as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[122] The recombinant expression vectors of the invention preferably comprise a nucleic acid sequence of the invention in a form suitable for, preferably constitutive, expression of the nucleic acid sequence in a host cell, which means that the recombinant expression vector may include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.

[123] The polypeptides and/or nucleic acid sequences of the present invention can be generated using the methods and techniques as described in the Examples and/or as described in the description and Examples of WO2013/164479 and WO2013/164481 , incorporated herein by reference.

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

[125] Preferably the recombinant host cell for these aspects is a host cell as described above. Further preferences are also as described above.

Use of the polypeptide

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

[127] More preferably such use comprises the addition of the polypeptide of the invention or the composition of the invention to a milk base. Preferences for such a milk base are as described above and below.

[128] Use of a polypeptide according to the invention may advantageously lead to a coagulant for the production of cheese, that can be produced within a short production time and that can also be processed early in the ripening, without extensive storage.

[129] As disclosed herein within the experimental part, a polypeptide of the invention shows advantages when used in the cheese. The invention provides use of a polypeptide of the first aspect (preferably variant #N11) or the composition of the second aspect for improving (increasing) moisture and fat holding by the cheese during cheese heating. Further provided is use of a polypeptide of the first aspect (preferably variant #N1 1) for improving (increasing) flowability (free- flowing capacity) of grated cheese (for example but not limited to mozzarella cheese). Additionally provide is use of a polypeptide of the first aspect (preferably variant #N11) for improving (increasing) the water-binding capacity of cheese. The process

[130] In a seventh aspect, the invention provides a process for the production of a cheese, wherein the process comprises contacting the polypeptide of the first aspect or a composition of the second aspect with a milk base. More preferably the polypeptide, respectively the composition, is added or supplemented to the milk-base.

[131] Preferably, the invention provides a process for the production of a cheese, which process comprises adding or otherwise contacting a milk clotting effective amount of the polypeptide of the invention or a composition of the invention to/with a milk-base and carrying out appropriate further cheese manufacturing steps.

[132] In the above and below processes, the milk-base can be derived from a plant-based source a microbial source or a mammal source. Preferably, the milk-base is derived from a mammal source, such as cow, sheep, goat, buffalo, camel, llama, horse or deer milk or any combination thereof. Preferably the milk may be selected from the group of cow's milk, camel's milk, buffalo milk, goat's milk, sheep's milk and a mixture of any such milk types. More preferably the milk-base is from a bovine source. Suitable plant-based sources of milk include, soy, pea, peanut, barley, rice, oat, quinoa, almond, cashew, and coconut milk. Preferred plant-based sources are soy milk, oat milk and almond milk. Bovine milk is most preferred. Preferably the milk-base is milk. Preferably the milk is derived from a mammalian source. Most preferably the milk is bovine (“cow”) milk.

[133] The milk-base may suitably comprise or consist of fresh skimmed or non-skimmed milk, or reconstituted milk. Optionally the milk-base may be concentrated or in the form of powder or may be reconstituted from such. By reconstituted milk is herein understood liquid milk obtained by adding liquid, such as water, to a skim milk powder, skim milk concentrate, whole milk powder or whole milk concentrate. Furthermore, the milk-base may or may not have been subjected to thermal (pre-)processing, such as pasteurization or sterilization. In one preferred embodiment the milk-base has been subjected to thermal (pre-) processing, such as pasteurization or sterilization.

[134] Preferably the polypeptide is added to or otherwise contacted with the milk-base in an amount to achieve a concentration in the range from equal to or more than 1 IMCU/L milk-base, more preferably from equal to or more than 5 IMCU/L milk-base, even more preferably from equal to or more than 10 IMCU/L milk-base, yet more preferably from equal to or more than 15 IMCU/L milk-base, still more preferably from equal to or more than 20 IMCU/L, even still more preferably from equal to or more than 25 IMCU/L and most preferably from equal to or more than 30 IMCU/L milk-base, to equal to or less than 100 IMCU/L milk-base, more preferably to equal to or less than 80 IMCU/L milk-base, even more preferably to equal to or less than 75 IMCU/L milk-base, yet more preferably to equal to or less than 70 IMCU/L milk-base, still more preferably to equal to or less than 65 IMCU/L milk-base, and most preferably to equal to or less than 60 IMCU/L milk-base.

[135] More preferably, the invention provides a process forthe production of cheese, comprising, (i) supplementing milk with a chymosin variant or composition according to the invention, to effect coagulation of the milk, wherein a curd is obtained; and (ii) processing the curd into cheese. By a curd is herein preferably understood the coagulated fraction of the milk-base.

[136] Preferences for the polypeptide, the milk-base and the amounts of polypeptide to be added to or otherwise contacted with the milk-base are as described herein above. Further preferences are as described hereinbelow.

[137] Even more preferably the invention provides a process for preparing a cheese, comprising:

(a) coagulating a milk-base in the presence of the polypeptide of the invention, wherein the coagulation is carried out before, simultaneously with, or after:

(i) fermenting in the presence of a bacterial culture; and/or

(ii) acidifying with a food grade organic and/or anorganic acid, to produce a coagulated milk-base; and

(b) processing the coagulated milk-base into a cheese.

[138] Preferences for the polypeptide, the milk-base and the amounts of polypeptide to be added to or otherwise contacted with the milk-base are as described herein above.

[139] In step (a) the bacterial culture preferably comprises or consists of one or more Streptococcus thermophilus strain(s), and preferably one or more other bacterial strains, more preferably one or more Lactobacillus and/or Lactococcus bacterial strains, most preferably one or more Lactococcus cremoris strain(s) and/or Lactococcus lactis strain(s). Preferably step (a) is carried out at temperatures in the range from equal to or more than 28°C, more preferably from equal to or more than 30°C, and most preferably from equal to or more than 32°C, to equal to or less than 47°C, more preferably to equal to or less than 45°C, even more preferably to equal to or less than 42°C and most preferably to equal to or less than 40°C. Preferably step (a) is carried out in a fermentation vessel.

[140] The time period during which step (a) is carried out may vary widely. However, preferably step (a) is carried out during a period in the range from equal to or more than 10 minutes, more preferably from equal to or more than 20 minutes, and most preferably from equal to or more than 30 minutes, to equal to or less than 24 hours, more preferably to equal to or less than 12 hours, still more preferably to equal to or less than 360 minutes, even more preferably to equal to or less than 120 minutes, still even more preferably to equal to or less than 90 minutes and most preferably to equal to or less than 60 minutes. The shorter periods have the advantage that the process becomes a more speedy process which is desired from a cheese producer perspective. The polypeptide according to the invention can advantageously accelerate clotting time and can allow for a more speedy production process.

[141] Preferably step (a) comprises fermenting the milk-base in the presence of the bacterial culture until a pH of equal to or less than 6.0, more preferably equal to or less than 5.8, even more preferably equal to or less than 5.5 and most preferably equal to or less than 5.3 is reached. For practical purposes, the pH during step (a) preferably varies in the range from equal to or less than 6.8, possibly from equal to or less than 6.7 or from equal to or less than 6.4 to equal to or more than 3.0, more preferably equal to or more than 3.5, still more preferably to equal to or more than 4.0 and most preferably equal to or more than 4.2. More preferably, the pH during step (a) more preferably varies in the range from equal to or less than 6.8, possibly from equal to or less than 6.7 or from equal to or less than 6.4 to equal to or more than 4.0, more preferably equal to or more than 4.5, still more preferably to equal to or more than 5.0 and most preferably equal to or more than 5.1 .

[142] Step (a) may also be referred to herein as the “curdling” step, which step may conveniently yield a coagulated fermented milk-base The coagulated fermented milk-base obtained or obtainable from step (a) may suitably comprise curd and whey.

[143] Conveniently step (a) may be followed by a step (b) comprising processing the coagulated fermented milk-base into a cheese. Such processing of the coagulated fermented milk-base into a cheese may conveniently comprise steps of cutting, stirring and/or cooking, whereafter any whey can conveniently be separated from the curd. The curd can advantageously be milled, whereafter it may or may not be subjected to salting.

[144] The process may thus preferably comprise one or more separation steps where one part of a coagulated fermented milk-base, preferably the curd, is separated from another part, preferably the whey. By a curd is herein preferably understood the coagulated fraction of the milk. Such a coagulated fraction of milk preferably comprises aggregated milk proteins, more preferably aggregated casein protein.

[145] Preferably the processes as described above further include a step of retrieving a a cheese. After retrieval of the cheese, the cheese is preferably matured. That is, preferably the processes described above comprise an additional step of maturing of the cheese, respectively cheese. The terms “ripened”, respectively “ripening”, and “matured”, respectively “maturing”, are used interchangeably herein. The “maturation” process is also referred to as “ripening” process. Preferably the step of maturing of the fermented milk product comprises a maturation during a period of time in the range from equal to or more than 24 hours, more preferably from equal to or more than 48 hours, still more preferably from equal to or more than 5 days, yet more preferably from equal to or more than 7 days, even more preferably from equal to or more than 2 weeks, and still even more preferably from equal to or more than 1 month, yet even more preferably from equal to or more than 3 months, to equal to or less than 5 years, more preferably to equal to or less than 3 years, even more preferably to equal to or less than 2 years, still more preferablty to equal to or less than 1 year, yet more preferably to equal to or less than 10 months. Preferably the step of maturing of the fermented milk product is carried out at a temperature in the range from equal to or more than 3°C, more preferably from equal to or more than 5°C to equal to or less than 20°C, more preferably equal to or less than 15°C. [146] The fermented milk product produced in the processes above is preferably a cheese, more preferably a matured cheese. The above process can advantageously allow for a speedy production of a cheese having improved flavor and cheese texture compared to cheeses produced with the chymosin enzyme of the prior art. The invention also relates to a cheese obtainable by the above process.

The cheese

[147] In an eighth aspect, the invention provides a cheese, wherein the cheese comprises an deteriorated polypeptide of the first aspect and/or is obtained or obtainable by the process of the seventh aspect or by the use of the sixth aspect.

[148] Preferably the cheese has matured during a time period in the range from equal to or more than 1 month, more preferably equal to or more than 3 months and equal to or less than 24 months, more preferably equal to or less than 12 months, preferably at a temperature in the range from equal to or more than 3°C, more preferably from equal to or more than 5°C to equal to or less than 20°C, more preferably equal to or less than 15°C.

[149] Preferably the matured fermented milk product, preferably the matured cheese, is processed within 1 to 30 days after production into slices, blocks or shreds.

[150] The polypeptide, composition, process and fermented milk product according to the invention advantageously allow for reduced cheese particle losses compared to cheeses produced with standard chymosin, a fast process, due to the short clotting times, a fast early development in maturation due to the rapid first cut in alpha-S1-casein, whilst in addition leading to the possibility of prolonged storage of the cheese due to the high C/P value.

[151] The present invention is further illustrated by the following, non-limiting, Examples.

Examples

Methods and materials

Medium composition

[152] YEP2D medium: 10 g/l yeast extract, 20 g/l bacto-peptone, 40 g/l glucose. pH was set to pH 6.7 with 4N NaOH. Medium was autoclaved for 30 minutes at 110°C.

[153] YEP2D/MES medium: 10 g/l yeast extract, 20 g/l bacto-peptone, 40 g/l glucose, 20 g/l MES. pH was set to pH 6.7 with 4N NaOH. Medium was autoclaved for 30 minutes at 110°C.

[154] YEP2D plates contain YEP2D medium with 1.8-2% agar. Medium was autoclaved for 30 minutes at 110°C and poured in petridishes.

Strains

[155] K. lactis strain GG799: The Kluyveromyces lactis strain is used as a wild-type strain. The strain is obtained from New England Biolabs, Ipswich, Massachusetts, USA.

Molecular biology techniques

[156] Molecular biology techniques known to the skilled person were used (see: Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, NY, 2001). Examples of the general design of expression vectors for gene over expression, transformation, use of markers and selective media can be found in W02007060247, WO2010102982 and US4943529 and references herein.

Clotting activity (C)

[157] The clotting activity was determined as based on international standard NEN-ISO 11815:2007 titled “Milk - Determination of total milk-clotting activity of bovine rennets” published 1 July 2007, by the Dutch “Nederlands Normalisatie-instituut” (NEN).

[158] A milk solution was prepared by adding 11 gram of Nilac milk powder (NIZO Food Science, Ede, the Netherlands) to 100 ml 4.5 mM CaCI2 (resulting pH 6.6). The solution was stirred for 30 minutes and kept in the dark for another 30 minutes. The milk is then ready and was used within half an hour. Subsequently, 5 ml milk solution was added to a test tube and pre-incubated for 5 minutes in a water bath of 32°C. The reaction was started by adding 100 pl enzyme solution to the milk solution. The milk clotting was followed visually in time. The moment coagulation starts is the point of clotting time. Different amounts of a diluted Liquid Chymosin Reference Standard (Chr. Hansen, Denmark) were used to obtain the reference curve for activity determination. A series of clotting measurements at different concentrations was performed and clotting time for each dilution was determined and the relation between the clotting time and the amount of units in the assay was determined. The clotting time found in the tested samples was calculated back to the IMCU activity of the original chymosin standard stock solution, determined with this assay, and expressed in IMCU/ml. This milk-clotting activity of the tested samples was used in the calculation of the C/P as exemplified below in Example 3.

General proteolytic activity (P)

[159] The proteolytic activity (P) was based on the standard method as described by Kappeler et al., titled “Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk", published in Biochemical and Biophysical Research Communications vol. 342 (2006), pages 647-654.

[160] Proteolytic activity was estimated using casein sodium salt from bovine milk (Sigma, C8654) as substrate. The reaction mix (750 pl) contained: 730 pl substrate (0.5% casein sodium salt in 33 mM MES, pH 5.8) and 20 pl chymosin sample to be tested at a final dosage of 70 IMCU/ml. The reaction mix was incubated for 120 min at 32°C and the reaction was terminated by addition of 250 pl 12% (w/w) TCA with vigorous stirring on a vortex mixer. The blank, containing the same substrate and getting the same incubation time treatment, was also stopped by addition of TCA whereafter 20 pl enzyme solution was added and stirred on a vortex mixer. The OD280 of the supernatants was measured after centrifugation at 12,000 rpm for 10 min. The difference (deltaOD) between OD280 (t=120 min) and OD280 (blank) was calculated and was used as a measure of the proteolytic activity at pH5.8 in the tested samples and used for the calculation of the C/P as exemplified in Example 3.

Example 1 : DNA constructs and transformation

[161] Synthetic DNA constructs were designed to start with a Xhol restriction site, encoding amino acids L and E, followed in frame with DNA encoding a kex-protease cleavage site with amino acids K and R, followed by in frame genes encoding variants of bovine pro-chymosin B starting with amino acids Alanine, Glutamic acid, Isoleucine and Threonine, ending with a Pad restriction site just after the stop codon. As an example, a DNA fragment encoding the wild type pro-chymosin B sequence is listed as SEQ ID NO: 1. Codon usage was adapted according to the method described in patent application US090286280. All variants were designed in a similar fashion and cloned as Xhol Pad fragments in vector pKLACI (New England Biolabs, Ipswich, Massachusetts, USA).

[162] The resulting open reading frames start with the leader sequence of the K. lactis Mating Factor alpha and progresses over the kex processing site to the bovine pro- chymosin B variants.

[163] Following the design of synthetic DNA constructs were designed. Amino acid changes were introduced in the 133 variants. A selection of the new variants and their mutations is depicted in Table 2. Position of the change is indicated in comparison with the mature wild-type bovine chymosin B sequence (SEQ ID NO: 1). All variants have multiple changes introduced into the amino acid sequence of the chymosin protein.

[164] A wild-type gene encoding the unchanged pro-chymosin protein as well as a pro-chymosin variants with two coding mutations (A51V and K221V) was also used in gene cloning and transformation.

[165] The mature variant polypeptide listed in WO2013/164479 and WO2013/164481 as variant polypeptide # 71 , comprising such mutations (A51V and K221V) and having the amino acid sequence of SEQ ID NO: 2, is used below as a reference polypeptide “Reference A”. Reference A was used to compare with the enzymes of the current invention, made with the variant genes. [166] As additional reference polypeptide “Reference B”, coagulant CHY-MAX™ Supreme, commercially available from Chr. Hansen A/S was used. This coagulant CHY-MAX™ Supreme is believed to have the amino acid sequence of SEQ ID NO: 4.

Table 2: Selection of new variants

Example 2: Cultivation, activation, purification and concentration

[167] The Kluyveromyces lactis strains harbouring a mutant bovine pro-chymosin gene were placed on YEP2D agar plate and grown for 48 hours at 30°C. A pre-culture in 20 ml of YEP2D medium in 100 ml Erlenmeyer flasks was inoculated with the yeast cells taken from the plates. The cultures were grown for 24 hours in an incubator shaker at 30°C and 250 rpm. The amount of preculture for inoculation of new 500 ml Erlenmeyer flasks with 100 ml YEP2D/MES medium was calculated to give a final OD600=0.01 . These main cultures were grown for 65 hours in an incubator shaker at 30°C and 250 rpm.

[168] Downstream processing works started upon receiving the Erlenmeyer flasks containing the fermentation broth of a given variant. The broth from each Erlenmeyer flask was harvested and centrifuged. The centrifugation supernatant (light phase), containing the target enzymatic activity, moved to a clarification step. The filtrate was collected and transferred to the chemical activation step. This step was done at a pH 2.35-2.40, Temp 30C °C for 90 min. The chemical activation was quenched by adjusting pH back to pH 6.05 ± 0.05. The liquid, containing the activated enzyme, was concentrated first using a 1 kDa MWCO PES spiral wound ultra-filtration system. The concentrate was then transferred to an AMICON 1 kDa MWCO stirred ultra-filtration cell. The concentrate salt and pH were adjusted to 45-50 mS/cm and pH 4.1 , respectively. This adjustment was done prior to the final chromatography concentration and purification. The latter was done based using a hydrophobic interaction media. The target activity was collected on the fractions from the column elution.

Example 3: C/P ratio

[169] The specificity of chymosin (C/P) is an important parameter for the functional properties of the enzyme in the cheese making process. The specificity of an individual chymosin sample was calculated by dividing the milk clotting activity (C) by the general proteolytic activity (P). The method to measure the C and P activity is described in Materials and Methods section. As can be seen in Table 3, various variants had a C/P ratio higher than Reference A, and higher than Reference B.

Table 3: C/P calculated for different chymosin variants, wherein is that the C and P are determined as above.

Example 4: In vitro alpha casein hydrolysis, using SDS PAGE

[170] A sterile suspension of 2.78 mg/ml a-casein (Merck, C6780) from bovine milk was prepared in 100 mM potassium buffer pH 6.8 containing 2% w/w NaCI. The dissolved casein solution was titrated back to pH 5.5-5.6 using 1 M HCI. This substrate was after filter sterilization aliquoted into 900 pl portions and incubated at 11 °C in a 1.5 ml tubes in an Eppendorf thermo mixer. A volume of 100 pl suspension containing 0.36 IMCU/mL coagulant in buffer pH5.5 was added to the alpha casein substrate suspension. Subsequently, the casein-coagulant suspension was incubated at 11 °C and 600 rpm for 2 days. These incubations were sampled after 0, 3, 6 and 24 hours by taking a volume of 70 pl for protein profile analysis and was put immediately into liquid nitrogen before storage at -80°C. These samples were inactivated by adding 1 :1 sample buffer (62mM Tris/ 8.1 M Urea/ 0.1 M DTT pH7.6) and stored at - 20°C for further analysis on SDS PAGE. The controls containing 10OpI buffer was taken along in order to check the substrate quality and having a reference in protein profiling. For the SDS profiling, 20pl (microliter) sample was mixed with 55pl (microliter) dilution buffer (62mM Tris/ 0.1 M DTT pH7.6) and 25pl 4x NuPage LDS loading buffer (Life Technologies) and incubated for 5 min at 70°C whereafter 10pL was put on a 10% Bis-Tris NuPage gel. The gels were run in MES buffer with NuPage antioxidant (Life Technologies) in the inner chamber and after electrophoresis at 15mA (10min), 20mA (20min) and 30mA (180min), the gels were stained with Coomassie Instant Blue (Expedeon). The gels were washed with demineralized water whereafter the gels were scanned and used for de densitometric analysis on the separated bands using a Typhoon FLA 9500 laser-scanner and Image Quant software.

[171] The densitometric analysis of the stained bands was done to quantify the hydrolytic products from the alpha-casein and to determine the proteolytic specificity of the different variants and reference enzymes. As mentioned, the intensity of all separated bands was quantified using the Typhoon laser and Image Quant software. Subsequently, the total intensity of all bands together in one sample (so per lane) was determined and used to calculate the relative intensity of the alpha- S1 and alpha S1-I fragment. The relative intensity of a band thus represents the intensity of that band as a percentage of the total intensity in that lane/sample. This allows better comparison between samples/lanes compared to absolute intensities of single bands, as the relative representation corrects for potential differences in total loaded protein.

[172] The data of the densitometric analysis indicating the relative presence of alpha-S1 casein bands (relative to the total protein bands in that sample) are shown in table 4 and figure 1 and clearly indicate that alpha-S1 casein hydrolysis occurs fastest in variant #N11 .

[173] Table 4 and figure 1 also show the formation of alpha S1-I casein fragment in time. When alpha S1 casein is specifically hydrolysed between phenylalanine 23 and phenylalanine 24, a small peptide is released (alpha S1 amino acid residues 1-23) and the alpha-S1 -I fragment. [174] Without wishing to be bound by any kind of theory, inventors believe this alpha S1-I casein fragment plays an important contribution in early knitting of the cheese by giving the cheese the desired textural properties for fast processing because of its decreased hydrophobicity compared to the alpha S1 casein. [175] The data in Table 4 shows that the alpha S1-I casein fragment is hardly formed in the alpha casein incubations with Reference B (Chymax Supreme). As illustrated, in the experiment with Chymax Supreme, the levels of alpha S1-I compound are similar to the experiment where no enzyme was used. All other chymosins form the alpha S1-I compound, and the highest percentage is formed in incubations with variant #N11 . [176] The detection limit for this method was about 2%.

Table 4: The alpha S1 hydrolysis ratio and the relative alpha-S1 -I content as percentage of total protein in a sample after 24 hours of incubation with an alpha-casein solution as described in example 4 (the percentage was calculated after densitometric analysis of the SDS gels)

Example 5: Milk clotting activity in non-homogenized full fat milk at varying pH [177] The milk clotting activity of the different chymosin variants was subsequently tested using the Rheolaser Master (Formulaction) according to the instructions of the manufacturer. Nonhomogenized full fat milk was incubated in a water bath at 40°C for at least 20 minutes prior addition of CaCI2 to a final concentration of 4mM. After an additional 20 minutes incubation at 40°C, the milk pH was adjusted to pH 6.1 or 6.6 via dropwise addition of 1.0 M acetic acid or 0.5M sodium hydroxide respectively. The temperature of the Rheolaser Master was set to 34°C and 20 ml of milk was pipetted into each tube of the Rheolaser (six in total). The milk was set to incubate in the Rheolaser instrument for at least 15 minutes at 34°C. The milk clotting experiments were started by addition of the chymosin samples; forthe milk clotting experiments at pH 6.1 the applied enzyme dosage was 16 IMCU per liter of milk, and for the experiments at pH 6.6 a dosage of 30 IMCU per liter of milk was applied. In each run of the Rheolaser, six milk samples were analyzed including a polypeptide of SEQ ID NO: 2 reference for each run. The determined clotting times for the chymosin samples at the two pH values are listed in Table 5 and the values are reported relative to the polypeptide of SEQ ID NO: 2 reference sample from the same run. The clotting time is provided by the Rheolaser software according to the instructions of the manufacturer. The data show that various variants have faster clotting times (values lower than 1) at pH 6.1 .

Table 5: Relative clotting times for different chymosin variants compared to Polypeptide of SEQ ID NO: 2 in full fat milk at pH 6.1 and 6.6 determined with the Rheolaser.

Example 6: preparation Cheddar cheese at 200-liter scale

[178] Cheddar cheese was manufactured using a generic US style recipe. After pasteurisation (15 seconds at 73°C), the milk was inoculated using dsm-firmenich Delvo Cheese CH-121 starter culture (1 units per WOOL cheese milk, batch GT00044164). The milk was allowed to pre ripen for 60 minutes. The rennet was dosed at 40 IMCU per 1 L for variant #N11 and Reference A (variant polypeptide # 71 of WO2013/164479 and WO2013/164481). The dosages were based on lab trials in the Rheolaser using the same milk sample as used for the Cheddar trial and the same conditions (pH 6,6 and 50ml 33% w/w CaCI2 solution per 200 liter milk) in order to have the same cutting time for all Cheddar cheeses. After approximately 30 minutes, the coagulum was firm enough to be cut. Cooking commenced after 10 minutes to a temperature of 38°C. Regular pH checks were made and when the pH had dropped below 6.2, the whey was drained off and the cheddaring part of the manufacturing process started. The curd slabs were turned at regular intervals and when the pH reached 5.3, the slabs were milled and salted. The milled, salted curd was allowed to mellow for approximately 15 minutes after which the curd was moulded and pressed overnight. The following morning (i.e. on day 1) the cheeses were removed from the moulds, sampled for compositional analysis, vacuum packed and ripened at 11 °C.

Example 7: In cheese alpha casein hydrolysis, using SDS PAGE

[179] To monitor the alpha-S1 casein hydrolysis in the Cheddar cheeses prepared in example 6,

50 g (gram) samples were taken from the cheeses in time, at day 1 and 17. For each cheese time point sample, eight to ten 30mg pieces were taken from different parts of the 50g cheese sample and freeze-dried for at least 24 hours. Subsequently, 50mg of the freeze-dried cheese sample was dissolved in 0.5 ml sample buffer (62mM Tris/ 8.1 M Urea/ 0.1 M DTT pH7.6). For the SDS profiling, 20pl dissolved cheese sample was mixed with 180pl dilution buffer (62mM Tris/ 0.1 M DTT pH7.6). Next, 65pl of this dilution was mixed with 25pl 4x NuPage LDS loading buffer (Life Technologies) and 10ul Sample reducing agent 10x (Life Technologies) and incubated for 5 min at 70°C whereafter 5uL was put on a 10% Bis-Tris NuPage gel. The gels were run in MES buffer with NuPage antioxidant (Life Technologies) in the inner chamber and after electrophoresis at 15mA (10min), 20mA (20min) and 30mA (180min), the gels were stained with Coomassie Instant Blue (Expedeon). The gels were washed with demineralized water whereafter the gels were scanned and used for de densitometric analysis on the separated bands using a Typhoon FLA 9500 laserscanner and Image Quant software.

[180] The densitometric analysis of the stained bands was done to quantify the hydrolytic products from the alpha-casein and to determine the proteolytic specificity of the different variants and reference enzymes. As mentioned, the intensity of all separated bands was quantified using the Typhoon laser and Image Quant software. Subsequently, the total intensity of all bands together in one sample (so per lane) was determined and used to calculate the relative intensity of the alpha-

51 casein and alpha S1-I casein fragment. The relative intensity of a band thus represents the intensity of that band as a percentage of the total intensity in that lane/sample. This allows better comparison between samples/lanes compared to absolute intensities of single bands, as the relative representation corrects for potential differences in total loaded protein.

The data of the densitometric analysis of the relative presence of the alpha-S1 casein bands are shown in table 6. It is very clear that the alpha-S1 casein hydrolysis occurred the fastest in the cheese samples produced with the variant #N11 .

Table 7 shows the formation of alpha-S1-l casein fragment in time. In cheese samples however, it is not possible to monitor the individual SDS Page band for alpha-S1 -I casein fragment as done in the in vitro alpha casein incubations, as this band overlaps on SDS gel with the beta casein band that is present in the cheese. However, it was still possible to monitor the alpha-S1 -I casein fragment formation in cheese samples via measuring the increase in intensity of the band on SDS- gel containing both the alpha-S1 -I casein fragment and the beta casein. These data are reported in table 7. In the cheese samples produced with both variant #N11 as well as Reference A the presence of alpha-S1-l casein fragment appears to have increased, as the intensity of the combined band containing alpha-S1 -I casein fragment and beta casein increased significantly. The fastest increase was observed for the polypeptide variant #N11 .

Table 6. Numbers indicate relative alpha-S1 casein content as percentage of total protein in sample (the percentage was calculated after densitometric analysis of the SDS gels)

Table 7. Numbers indicate relative alpha-S1 -I casein + beta-casein combined content as percentage of total protein in sample (the percentage was calculated after densitometric analysis of the SDS gels)

Example 8: Clotting activity over protease activity (C/P) of variants compared to eight variants described in WQ2013/164479

The C/P values of the variants described in the present patent application (example 3, table 3) were compared to the ‘relative C/P values’ of eight variants described in WO2013/164479. WO2013/164479 reports relative C/P values; the measured C/P values of the variants were divided by the C/P measured for the control sample Maxiren. A comparison of the data in the 2 patent applications is possible because Reference A from the present patent application eguals variant #71 from WO2013/164479. By dividing the C/P ratio of the variants described in the present patent application over the C/P value of Reference A, a factor was obtained (table 8). By subseguently multiplying the ‘relative C/P ratio’ of variant #71 from WO2013/164479 (value of 16.8, table 3 in WO2013/164479) with the obtained factor, we calculated ‘a corrected’ C/P ratio for the variants in the present patent application that can be directly compared with the C/P values listed in WO2013/164479 resulting in table 9. The values could be corrected for accurate comparison via this overlapping enzyme because the same methods were used to determine the clotting and the protease activity (described in Materials and Methods section).

The C/P values of all variants described in the present patent application (last column, table 8) are improved compared to the previously determined C/P values of the eight variants reported in WO2013/164479 (table 9) except for variant #99 with a C/P of 43,8 which is higher than the C/P of variant #N11 which is 41 ,4.

Table 8. Calculation of factor: C/P of variant divided over C/P of Reference A.

Table 9. Relative C/P values of eight variants as reported in WO2013/164479.

Example 9: Comparing in vitro alpha casein hydrolysis using SDS PAGE of variant #N11 to eight variants described in WQ2013/164479.

Kluyveromyces lactis strains expressing eight variants described in WO2013/164479 (#95, #96, #97, #98, #99, #100, #109 and #110) and a strain expressing variant #N11 were cultivated as described in WO2013/164479. Supernatants were harvested and 15x concentrated using a 10 kDa cut-off filter (Centrifugal filter units, Amicon® Ultra-15, 10 kD Ultracel-10). Concentrated supernatants were incubated for 90 minutes at pH2 to activate the chymosin and the pH was restored to pH6.1 . An adjusted clotting activity assay was used to determine the clotting activity of the samples. In this assay, 40 pl of activated supernatants was incubated with 200 pl 1 .2% skimmed milk at pH6.1 and the absorbance at 600 nm was measured for 60 minutes. Using a calibration curve of Maxiren® with a known IMCU/ml activity (determined with the international standard clotting assay as described in this patent), the milk clotting activity of the concentrated supernatants was calculated. As the conditions in this adjusted assay are different from the conditions used in the international standard assay, the milk clotting activity of the samples was expressed in MCU/ml instead of IMCU/ml. Next, these supernatants were tested fortheir in vitro alpha casein hydrolysis activity as described in Example 4, except that the coagulant was dosed from a stock solution containing 0.36 MCU/ml instead of 0.36 IMCU/ml. The data of the densitometric analysis of the SDS PAGE gels indicating the relative presence of alpha-S1 casein bands (relative to the total protein bands in that sample) are shown in table 10 and clearly indicate that alpha-S1 casein hydrolysis occurs fastest in variant #N11 .

Table 10. The alpha S1 hydrolysis ratio and the relative alpha-S1 -I content as percentage of total protein in a sample after 24 hours of incubation with an alpha-casein solution as described in example 4 (the percentage was calculated after densitometric analysis of the SDS PAGE gels)

^_as alphaSI t=0 the value of the ‘no enzyme sample’ at t=24hrs was used because no degradation was detected in that sample. Example 10: Comparing variant #N11 and Reference B in cheese for ability to hold moisture and fat during heating

Variant #N11 and Reference B were used in the preparation of Pasta Filata/mozzarella. The produced cheese was subseguently subjected to shredding and egual amounts of shredded cheese were put into separate containers. The cheese was heated to result in melted cheese. The moisture and fat release upon heating was tested by decanting the liguids from the solid and weighing each fraction afterwards and showed that cheese prepared with variant #N11 resulted in improved moisture and fat holding by the cheese: variant N11 showed more than 30% less moisture and fat release of the cheese when compared to Reference B.

Example 11 : Comparing variant #N11 and Reference B in cheese for ability to hold moisture in cheese

Variant #N11 and Reference B were used in the preparation of Pasta Filata/mozzarella. The produced cheese was subseguently subjected to a watering-off method to define moisture retention. A cheese sample was taken and grinded immediately before weighing out. A weight of 12 grams of cheese was put into a small centrifuge tube. This was done for each cheese in duplicate. The tubes were centrifuged at 12,500 rpm for 60 minutes at room temperature. Thereafter, the tubes were removed from the centrifuge. The amount of liguid in the tubes was weighted. This is called the expressible serum. The serum was transferred to small Nalgene tubes and frozen until further analysis. The next formulas were used for calculation to express amount of watering off:

% watering off = (weight of serum/12 g cheese)*100

% of total moisture = (% watering off/amount of moisture in the cheese)*100

Table 11 . Amount of moisture which is separated from the cheese as % of the total moisture in the cheese

It is concluded that variant #N11 shows better water-binding capacity compared to Reference B. Additionally, variant #N11 allows the total moisture to be increased without too much loss of free moisture. Example 12: Comparing variant #N11 and Reference B in cheese for flowability of grated cheese Variant #N11 and Reference B were used in the preparation of mozzarella. The produced cheese was 4 months after production grated and the flowability of the resulting parts were compared. This comparison showed that grated mozzarella produced with variant #N11 sticks les together and hence has an improved free-flowing capacity compared grated mozzarella produced with reference B.

Claims

Claims

1 . A polypeptide comprising: a) a chymosin activity with a C/P ratio of clotting activity (C) to proteolytic activity (P) equal to or higher than the C/P ratio of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and b) an alpha-S1 -I casein fragment formation activity equal to or higher than the alpha-S1- I casein fragment formation activity of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and/or c) a clotting time at pH 6.1 of equal to or less than the clotting time of a polypeptide having an amino acid sequence of SEQ ID NO: 2.

2. The polypeptide according to claim 1 comprising a) chymosin activity with a C/P ratio of clotting activity (C) to proteolytic activity (P) higher than the C/P ratio of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and b) an alpha-S1 -I casein fragment formation activity higher than the alpha-S1 -I casein fragment formation activity of a polypeptide having an amino acid sequence of SEQ ID NO: 2; and/or c) a clotting time at pH 6.1 of less than the clotting time of a polypeptide having an amino acid sequence of SEQ ID NO: 2.

3. The polypeptide according to any of the preceding claims, wherein the polypeptide has a C/P ratio of equal to or more than 15, more preferably 20, preferably equal to or more than 30.

4. The polypeptide according to any one of the preceding claims, wherein the polypeptide has an alpha-S1 -I casein fragment formation activity with a densitometric alpha S1-I casein fragment percentage® 24 hours of equal to or more than 10%, more preferably equal to or more than 20%, even more preferably equal to or more than 30% and most preferably equal to or more than 40%, based on the total protein content @ 24 hours

5. A polypeptide according to any one of the preceding claims, wherein the polypeptide has a clotting ratio _PH 6.6 / _PH 6.I of clotting time at pH 6.6 to clotting time at pH 6.1 (clotting ratio PH 6 6/ _PH 6.1), as preferably determined by rheolaser, of equal to or higher than the clotting ratio _PH 6 6 / pH 6 i of bovine chymosin of SEQ ID NO:2.

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

7. The polypeptide according to any of the preceding claims which has an amino acid sequence with equal to or more than 90%, more preferably equal to or more than 95%, and most preferably equal to or more than 98%, sequence identity with the amino acid sequence of SEQ ID NO: 1 .

8. A composition comprising the polypeptide according to any one of claims 1 to 7.

9. The composition according to claim 8, wherein such composition has a pH equal to or less than pH 6.6, preferably equal to or less than pH 6.1 , more preferably equal to or less than pH 5.8.

10. A nucleic acid sequence encoding a polypeptide according to any one of claims 1 to 7.

11. A nucleic acid construct comprising the nucleic acid sequence according to claim 10 operably linked to one or more control sequences capable of directing the expression of the polypeptide according to any one of claims 1 to 7 in a host cell.

12. A recombinant expression vector comprising the nucleic acid sequence according to claim 10 and/or the nucleic acid construct according to claim 11 .

13. A recombinant host cell comprising the nucleic acid sequence according to claim 10, the nucleic acid construct according to claim 11 , and/or the recombinant expression vector according to claim 12.

14. A method for producing the polypeptide according to any one of claims 1 to 7 comprising expressing the nucleic acid sequence according to claim 10 and/or the nucleic acid construct according to claim 11 in a recombinant host cell according to claim 13.

15. Use of a polypeptide according to any one of claims 1 to 7 or the composition according to any one of claims 8 to 9 in the preparation of a cheese.

16. A process for the production of a cheese, wherein the process comprises contacting a polypeptide according to any one of claims 1 to 7 or a composition according to any one of claims 8 to 9 with a milk base.

17. The process according to claim 11 , wherein the milk base has a pH equal to or less than pH 6.6, preferably equal to or less than pH 6.1 , more preferably equal to or less than pH 5.8.

18. A cheese, wherein the cheese comprises a deteriorated polypeptide according to any one of claims 1 to 7 and/or is obtained or obtainable by the process according to any one of claims 16 to 17 and/or by the use according to claim 15.

19. A cheese, wherein the cheese has a pH of between 5.0 and 6.1 and wherein the cheese contains the polypeptide according to any one of claims 1 to 7 or the composition according to any one of claims 8 to 9.