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WO2025099225A1 - Variants enzymatiques améliorés - Google Patents

Variants enzymatiques améliorés Download PDF

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Publication number
WO2025099225A1
WO2025099225A1 PCT/EP2024/081638 EP2024081638W WO2025099225A1 WO 2025099225 A1 WO2025099225 A1 WO 2025099225A1 EP 2024081638 W EP2024081638 W EP 2024081638W WO 2025099225 A1 WO2025099225 A1 WO 2025099225A1
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WIPO (PCT)
Prior art keywords
polypeptide
seq
acid sequence
amino acid
milk
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PCT/EP2024/081638
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English (en)
Inventor
Johannes Gustaaf Ernst VAN LEEUWEN
Loes Elizabeth Bevers
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DSM IP Assets BV
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DSM IP Assets BV
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Publication of WO2025099225A1 publication Critical patent/WO2025099225A1/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A23C19/0326—Rennet produced by fermentation, e.g. microbial rennet; Rennet produced by genetic engineering
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6478—Aspartic endopeptidases (3.4.23)
    • C12N9/6483—Chymosin (3.4.23.4), i.e. rennin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/23—Aspartic endopeptidases (3.4.23)
    • C12Y304/23004—Chymosin (3.4.23.4), i.e. rennin

Definitions

  • 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.
  • Enzymatic coagulation of milk by milk clotting enzymes 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.
  • a variety of enzyme characteristics are important.
  • WO2013/164479 explains the need for modified proteolytic activity under cheese-making conditions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Wild type camel chymosin has a higher thermostability than wild type bovine chymosin.
  • 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.
  • 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.
  • 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.
  • the new polypeptides have now been identified which allow for an improved C/P ratio, preferably an improved C/P ratio at low pH, such as a pH of 6.1 .
  • the new polypeptides provide improvements in modified proteolytic activity under cheese-making conditions as mentioned in WO2013/164479, and/or improvements in fast processibility/shreddability as mentioned in WO2013/164481 , and/or improvements in cleavage and/or selectivity of an alpha S1 (aS1) casein, and/or improvements in production time and/or a low applied dosage scheme.
  • the polypeptides of the invention are therefore superior coagulants compared to the polypeptides of the prior art.
  • the present invention provides a polypeptide having chymosin activity, 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 .
  • SEQ ID NO: 1 suitably represents the amino acid sequence of a bovine chymosin.
  • the invention provides a composition comprising the polypeptide of the first 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 1 Gel results in respect of the densitometric analysis carried out in Example 4.
  • milk is intended to encompass milks from mammals, milks from plant sources, milks from microbial sources and/or mixtures thereof.
  • 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.
  • 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.
  • 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.
  • milk refers to not only whole milk, but also skim milk or any liquid component derived thereof or reconstituted milk.
  • 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.
  • the milk-base may be concentrated, or in the form of powder, or may be reconstituted from such.
  • 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.
  • the milk-base may or may not have been subjected to a thermal processing operation which is at least as efficient as pasteurization.
  • the milk-base is from a bovine source.
  • 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.
  • Chymosin and pepsin 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.
  • 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”.
  • 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.
  • 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 pre prochymosin’”).
  • 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..
  • 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.
  • 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).
  • oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • 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.
  • 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.
  • 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.
  • % identity number of identical positions/total number of positions (i.e. overlapping positions) x 100).
  • the two sequences are the same length.
  • 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.
  • 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.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402.
  • IMCU International Milk Clotting Units.
  • One IMCU equals about 0.126 nmol of bovine chymosin B (e.g. MaxirenTM or CHY-MAXTM).
  • the strength of a milk clotting 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
  • 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.
  • 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.
  • 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 present invention 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 and 221 , wherein said positions are defined with reference to SEQ ID NO: 1.
  • SEQ ID NO: 1 suitably represents the amino acid sequence of a bovine chymosin.
  • this polypeptide has chymosin activity and preferably this polypeptide has alphas'! -I casein fragment formation activity, where further preferences are as set out below.
  • 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.
  • 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.
  • 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.
  • polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 1 , comprises:
  • 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.
  • 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.
  • 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.
  • polypeptide is:
  • polypeptide that has an amino acid sequence of SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or
  • polypeptide [056] Even more preferably, the polypeptide:
  • - is a polypeptide that has an amino acid sequence of SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or
  • polypeptide of the invention further has:
  • the polypeptide comprises: 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/or 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.
  • the polypeptide • is capable of hydrolysing bovine alpha s1 -casein more rapidly than a polypeptide having an amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19 or 20 or more specifically phrased is capable of hydrolysing bovine alpha s1 -casein at position F23F24 so as to form as1-l casein fragment (f24-199) more rapidly than a polypeptide having an amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19 or 20; alternatively phrased the polypeptide comprises 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: 13, 14, 15, 16, 17, 18, 19 or 20, or
  • the polypeptide preferably comprises 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: 13, 14, 15, 16, 18, 19 or 20, or the polypeptide comprises 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: 13, 14, 15, 16, 17, 18, 19 or 20.
  • C clotting activity
  • P proteolytic activity
  • 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.
  • 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.
  • the polypeptide comprises a chymosin activity, wherein such chymosin activity comprises or consists of a selective kappacasein hydrolyzing activity.
  • 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).
  • the polypeptide of the invention suitably selectively hydrolyses (that is “cleaves”) the peptide bonding F105-M106.
  • 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.
  • 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.
  • 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.
  • the polypeptide having amino acid sequence SEQ ID NO: 2 is the polypeptide variant numbered #71 of prior art WO2013/164479 and WO2013/164481 .
  • the polypeptide has a C/P ratio that is at least equal or more than 1 .5 times the C/P ratio of a polypeptide having amin acid sequence SEQ ID NO: 13, 14, 15, 16, 18, 19 or 20.
  • 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).
  • 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.
  • the polypeptide also has alpha-S1 -casein hydrolyzing activity.
  • 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”.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • a mature bovine alpha-S1 -casein such as for example the alpha-S1 -casein of SEQ ID NO: 10
  • 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”).
  • first fragment (“f1 -23”) comprises the first 23 amino acids of the mature bovine alpha-S1 -casein protein
  • the second fragment (“f24-199”) comprises the remaining amino acids 24-199 of the mature bovine alpha-S1 -casein protein.
  • 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”.
  • alpha-S1 -I casein fragment is a hydrolysis product, corresponding to the second fragment obtained after hydrolysis of an alpha- S1-casein.
  • 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.
  • bovine alpha-S1-l casein f24-199 fragment is provided in SEQ ID NO: 11.
  • SEQ ID NO: 11 An example of such a bovine alpha-S1-l casein f24-199 fragment is provided in SEQ ID NO: 11.
  • 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 .
  • a higher aS1-CN hydrolysis ratio is indicative of an increased reduction in aS1-CN concentration and hence indicative of an increased hydrolysis.
  • the alpha-S1 -casein (“aS1-CN”) in such a determination is an alpha-S1 -casein derived from bovine milk (“bovine aS1-CN”).
  • 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 or having the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19 or 20.
  • the mature bovine alpha-S1 -casein may preferably have an amino acid sequence of SEQ ID NO:10 and the alpa-S1- I casein fragment may preferably have an amino acid sequence of SEQ ID NO:11 .
  • 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.
  • alpha-S1-l casein fragment (“aS1-l-CN“) over a period of 24 hours for a specific polypeptide
  • aS1-l-CN“ alpha-S1-l casein fragment
  • the polypeptide of the invention has a formation and maintenance of the alpha- S1-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 or having the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19 or 20.
  • 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”).
  • 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.
  • 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”).
  • 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.
  • 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 s i-casein: Comparison of the behaviour of this substrate with that of /3- and K 0 -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.
  • 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).
  • Rate and selectivity of the hydrolysis can be expressed as described in the Examples.
  • polypeptide of the invention preferably amino acids:
  • 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 or having the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19 or 20,
  • - 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.
  • 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:
  • 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
  • alpha-S1-I casein fragment formation activity can suitably be determined as illustrated in the Examples or as described above.
  • the polypeptide further has a ratio of clotting time at pH 6.6 to clotting time at pH 6.1 (clotting ratio P H 6.6/ P H 6.I) of equal to or more than 1 .10, preferably of equal to or more than 1 .20.
  • clotting ratio P H 6.6/ P H 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.
  • 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.
  • the polypeptide according to the invention further also allows for rapid production of the cheese.
  • the polypeptide according to the invention can be an isolated naturally occurring polypeptide or a non-naturally occurring polypeptide.
  • a non-naturally occurring polypeptide is herein understood a polypeptide that is not naturally produced by any mammal.
  • the polypeptide is a non-naturally occurring polypeptide.
  • an "isolated" polypeptide or protein is herein understood a polypeptide or protein removed from its native environment.
  • 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.
  • composition comprising the polypeptide
  • the invention provides a composition comprising the polypeptide of the first aspect.
  • 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.
  • the composition may optionally comprise other ingredients such as additives and/or other enzymes.
  • 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 .
  • composition according to the invention may further comprise one or more pH buffers and/or other additives such as glycerol and/or sodium chloride.
  • the composition can be liquid, frozen or freeze-dried. If liquid, the composition is preferably a solution, suspension or emulsion.
  • 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.
  • 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.
  • 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.
  • 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.
  • PCR is carried out on a plasmid template using oligonucleotide "primers" encoding the desired substitution.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention also relates to nucleic acid sequences encoding at least one functional domain of a polypeptide variant of the invention.
  • a domain may comprise one or more of the substitutions described herein.
  • 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.
  • 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.
  • Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Suitable host cells include cells from the above exemplified host organisms.
  • 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 coli, 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.
  • 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).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signal).
  • the invention also provides a recombinant expression vector comprising the above nucleic acid sequence and/or the above nucleic acid construct.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector 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).
  • vectors e.g., non-episomal mammalian vectors
  • Other 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.
  • 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”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably herein as the plasmid is the most commonly used form of vector.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the recombinant host cell for these aspects is a host cell as described above. Further preferences are also as described above.
  • 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.
  • 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.
  • 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.
  • 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 #N11) 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 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.
  • 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.
  • the milk-base can be derived from a plant-based source a microbial source or a mammal source.
  • 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.
  • 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.
  • 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.
  • the milk-base is milk.
  • the milk is derived from a mammalian source. Most preferably the milk is bovine (“cow”) milk.
  • the milk-base may suitably comprise or consist of fresh skimmed or non-skimmed milk, or reconstituted milk.
  • the milk-base may be concentrated or in the form of powder, or may be reconstituted from such.
  • 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.
  • the milk-base may or may not have been subjected to thermal (pre-)processing, such as pasteurization or sterilization.
  • pre- thermal
  • pre- thermal
  • 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-
  • 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.
  • a curd is herein preferably understood the coagulated fraction of the milk-base.
  • 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:
  • 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).
  • 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.
  • step (a) is carried out in a fermentation vessel.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • step (a) may be followed by a step (b) comprising processing the coagulated fermented milk-base into a cheese.
  • 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.
  • 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.
  • 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.
  • the processes as described above further include a step of retrieving a a 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.
  • 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.
  • 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.
  • 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 invention provides a cheese, wherein the cheese comprises a 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.
  • 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.
  • the matured fermented milk product preferably the matured cheese, is processed within 1 to 30 days after production into slices, blocks or shreds.
  • 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.
  • 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.
  • 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.
  • YEP2D plates contain YEP2D medium with 1.8-2% agar. Medium was autoclaved for 30 minutes at 110°C and poured in petridishes.
  • 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.
  • 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).
  • 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 CaCL (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.
  • proteolytic activity 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.
  • 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 OD280 of the supernatants was measured after centrifugation at 12,000 rpm for 10 min.
  • Example 1 DNA constructs and transformation
  • 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.
  • variant polypeptide # 71 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.
  • Reference B As additional reference polypeptide “Reference B”, coagulant CHY-MAXTM Supreme, commercially available from Chr. Hansen A/S was used. This coagulant CHY-MAXTM Supreme is believed to have the amino acid sequence of SEQ ID NO: 4.
  • 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.
  • These main cultures were grown for 65 hours in an incubator shaker at 30°C and 250 rpm.
  • 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.
  • C/P The specificity of chymosin
  • C/P 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.
  • 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.
  • 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.
  • the intensity of all separated bands was quantified using the Typhoon laser and Image Quant software.
  • the total intensity of all bands together in one sample 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.
  • Table 4 and figure 1 also show the formation of alpha S1-I casein fragment in time.
  • 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.
  • 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 increased decreased hydrophobicity compared to the alpha S1 casein.
  • 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-homoqenized full fat milk at varying pH
  • 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.
  • 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.
  • 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
  • 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).
  • 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.
  • Table 7 shows the formation of alpha-S1-l casein fragment in time.
  • Example 8 Clotting activity over protease activity (C/P) of variants compared to eight variants described in WQ2013/164479
  • 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.
  • 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.
  • the milk clotting activity of the concentrated supernatants was calculated.
  • the milk clotting activity of the samples was expressed in MCU/ml instead of IMCU/ml.
  • 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
  • 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.

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Abstract

L'invention concerne un polypeptide, le polypeptide ayant une séquence d'acides aminés qui, lorsqu'elle est alignée avec la séquence d'acides aminés définie dans SEQ ID NO : 1, comprend au moins une substitution des résidus d'acides aminés correspondant aux acides aminés aux positions 50, 51, 126, 135 et 221, lesdites positions étant définies en référence à SEQ ID NO : 1.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4943529A (en) 1982-05-19 1990-07-24 Gist-Brocades Nv Kluyveromyces as a host strain
WO2007060247A2 (fr) 2005-11-28 2007-05-31 Dsm Ip Assets B.V. Préparations d'enzymes au goût agréable
US20090286280A1 (en) 2006-06-29 2009-11-19 Dsm Ip Assets B.V. Method for achieving improved polypeptide expression
WO2010102982A1 (fr) 2009-03-10 2010-09-16 Dsm Ip Assets B.V. Procédé d'amélioration du rendement d'un polypeptide
WO2013164479A2 (fr) 2012-05-03 2013-11-07 Dsm Ip Assets B.V. Variants d'enzymes améliorés
WO2023194285A2 (fr) 2022-04-06 2023-10-12 Chr. Hansen A/S Protéase aspartique, procédés et utilisations associés

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4943529A (en) 1982-05-19 1990-07-24 Gist-Brocades Nv Kluyveromyces as a host strain
WO2007060247A2 (fr) 2005-11-28 2007-05-31 Dsm Ip Assets B.V. Préparations d'enzymes au goût agréable
US20090286280A1 (en) 2006-06-29 2009-11-19 Dsm Ip Assets B.V. Method for achieving improved polypeptide expression
WO2010102982A1 (fr) 2009-03-10 2010-09-16 Dsm Ip Assets B.V. Procédé d'amélioration du rendement d'un polypeptide
WO2013164479A2 (fr) 2012-05-03 2013-11-07 Dsm Ip Assets B.V. Variants d'enzymes améliorés
WO2013164481A1 (fr) 2012-05-03 2013-11-07 Dsm Ip Assets B.V. Variants enzymatiques améliorés
WO2023194285A2 (fr) 2022-04-06 2023-10-12 Chr. Hansen A/S Protéase aspartique, procédés et utilisations associés

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