EA040259B1 - CHymosin variants with improved milk-clotting properties - Google Patents

Description

The present invention relates to variants of chymosin with improved milk-clotting properties.

Prior Art

Chymosin (EC 3.4.23.4) and pepsin (EC 3.4.23.1), mammalian gastric milk-clotting enzymes, are aspartic proteases belonging to a large class of peptidases.

When produced in cells of the gastric mucosa, chymosin and pepsin are formed as an enzymatically inactive precursor of prochymosin and a precursor of pepsinogen, respectively. Upon excretion of chymosin, the N-terminal peptide fragment, the pre-fragment (signal peptide) is cleaved off to obtain prochymosin, including the pro-fragment. Prochymosin is an essentially inactive form of the enzyme, which, however, is activated under acidic conditions to active chymosin via autocatalytic removal of the pro moiety. This activation occurs in vivo in the gastric lumen at a suitable pH or in vitro under acidic conditions.

The structural and functional characteristics of the bovine, i.e. Bos taurus, preprochymosin, prochymosin and chymosin. The pre-moiety of bovine pre-prochymosin contains 16 aa (amino acid) residues and the pro-moiety of the corresponding prochymosin has a length of 42 aa residues. Active bovine chymosin contains 323 aa.

Chymosin is naturally produced in mammalian species such as cattle, camels, goats, buffaloes, sheep, pigs, humans, monkeys, and rats.

Bovine and camel chymosin have been commercially available to the dairy industry for a number of years.

Enzymatic coagulation of milk by means of milk-clotting enzymes such as chymosin and pepsin is one of the most important processes in cheese production. Enzymatic coagulation of milk is a two-phase process: the first phase, where the proteolytic enzyme, chymosin or pepsin, acts on k-casein, resulting in a metastable state of the casein micelle structure, and the second phase, where the milk then coagulates and forms a coagulate (ref. 1).

WO02/36752A2 (Chr. Hansen) describes the recombinant production of camel chymosin.

WO2013/174840A1 (Chr. Hansen) describes bovine and camel chymosin mutants/variants.

WO2013/164479A2 (DSM) describes bovine chymosin mutants.

The references listed immediately below may be considered in the present context as references describing chymosin mutants:

Suzuki et al: Site directed mutagenesis reveals functional contribution of Thr218, Lys220 and Asp304 in chymosin, Protein Engineering, vol. 4, January 1990, pages 69-71;

Suzuki et al: Alteration of catalytic properties of chymosin by site-directed mutagenesis, Protein Engineering, vol. 2, May 1989, pages 563-569;

van den Brink et al: Increased production of chymosin by glycosylation, Journal of biotechnology, vol. 125, September 2006, pages 304-310;

Pitts et al: Expression and characterization of chymosin pH optima mutants produced in Trichoderma reesei, Journal of biotechnology, vol. 28, March 1993, pages 69-83;

M.G. Williams et al: Mutagenesis, biochemical characterization and X-ray structural analysis of point mutants of bovine chymosin, Protein engineering design and selection, vol. 10, September 1997, pages 991-997;

Strop et al: Engineering enzyme subsite specificity: preparation, kinetic characterization, and x-ray analysis at 2.0 ANG resolution of Val111phe site mutated calf chymosin, Biochemistry, vol. 29, October 1990, pages 9863-9871;

Chitpinityol et al: Site-specific mutations of calf chymosin B which influence milk-clotting activity, Food Chemistry, vol. 62, June 1998, pages 133-139;

Zhang et al: Functional implications of disulfide bond, Cys45-Cys50, in recombinant prochymosin, Biochimica et biophysica acta, vol. 1343, December 1997, pages 278-286.

None of the prior art references above directly and unambiguously describe any of the chymosin variants with improved specific clotting activity and increased C/P compared to the parent material from which the variant is derived, as described below.

Brief summary of the invention

The object of the present invention is to provide chymosin variants which, compared to the parent polypeptide, have a specific clotting activity (IMCU International Milk-clotting Unit/mg total protein) that is at least 110% of the specific clotting activity of the parent polypeptide and/or at least 200% C/P ratio of its parent polypeptide as shown here.

Based on intelligent design and comparative analysis of different variants, the present inventors have identified a number of amino acid positions that are important because a change in one or more positions in the parent polypeptide can result in an improved chymosin variant with either increased specific clotting activity or with increased C/P ratios, or with an increase in both properties.

The amino acid numbering used here to define the variant is based on the mature peptide. As is known in the art, various wild-type natural chymosin polypeptide sequences derived from different mammalian species (such as, for example, cows, camels, sheep, pigs, or rats) have relatively high sequence similarity/identity. In FIG. 1 this is represented by the alignment of the respective different chymosin sequences.

Because of this relatively close sequence match, the three-dimensional structures of the various natural wild-type chymosins are also considered to be relatively similar.

In this context, wild-type natural chymosin (such as bovine or camel chymosin) here may be an example of a parental polypeptide, i.e. the parent polypeptide for which modification is made in order to obtain a variant of the present invention.

Without being limited by theory, it is believed that the discussed chymosin amino acid positions are essential for any chymosin enzyme considered here of interest (e.g., bovine, camel, sheep, pig, rat, etc.) chymosins, in the sense that by making a change at one or more of these positions, an improved variant of chymosin as a whole (eg, an improved variant of bovine, camel, ovine, porcine, or rat chymosin) can be obtained.

As discussed herein, the sequence of SEQ ID NO: 2 of mature chymosin of the commonly known Camelius dromedarius is used here as a reference sequence for determining the amino acid position of the parental chymosin polypeptide of interest (eg, camel, sheep, bovine, etc.). Alternatively, it may be referred to here as camel chymosin. The sequence is also shown in Fig. 1.

In the present context, it is considered that a parental chymosin polypeptide (e.g., sheep or rat) that has at least 80% sequence identity with the mature polypeptide of SEQ ID NO: 2 (camel chymosin) can be considered here as sufficiently structurally related, for example, with bovine or camel chymosin to be improved by changing any amino acid position as described here.

Embodiments of the present invention are described below.

Definitions

All definitions of these respective terms are in accordance with what would be understood by a person skilled in the art in relation to the relevant technical context.

The term chymosin refers to an enzyme of class EC 3.4.23.4. Chymosin is highly specific and preferentially curdles milk by cleaving one 104-Ser-Phe-|Met-Ala-107 bond in the κ-chain of casein. As a side activity, chymosin also cleaves α-casein mainly between Phe23 and Phe24 and β-casein mainly between Leu192 and Tyr193 (refs 2, 3). The resulting aS1(l-23) and β(193-209) peptides will be further cleaved with proteases from microbial cultures added to ripened cheese (ref. 4). An alternative name for chymosin used in the art is rennin.

The term chymosin activity refers to the chymosin activity of the enzyme chymosin, as understood by one of skill in the present context.

One skilled in the art knows how to determine the appropriate chymosin activity.

As is known in the art, the corresponding so-called C/P ratio is determined by dividing the specific clotting activity (C) by the proteolytic activity (P).

As is known in the art, a higher C/P ratio generally implies that protein loss during, for example, cheese production due to non-specific protein degradation is reduced, which can result in improved cheese yield.

The term isolated variant means a variant that is modified by human action. In one aspect, the variant is at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, and at least 90% pure, as determined by SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

The term mature polypeptide means the peptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In this context, the corresponding mature chymosin polypeptide can be referred to as the active sequence of the chymosin polypeptide, i.e. without pre-part and/or pro-part. Suitable examples of a mature polypeptide here are, for example, the mature polypeptide of SEQ ID NO: 1 (bovine chymosin), which is located from amino acid position 59 to amino acid position 381 of SEQ ID NO: 1, or the mature polypeptide of SEQ ID NO: 2 (camel chymosin) , which is located from amino acid position 59 to amino acid position 381 of SEQ ID NO: 2.

The term parental or parental polypeptide having chymosin activity means a polypeptide that has been modified to produce variants of the enzyme of the present invention. The parent may be a natural (wild-type) polypeptide or a variant thereof.

The term sequence identity refers to the similarity of two amino acid sequences or two nucleotide sequences.

For the purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet 16: 276-277), version 3.0.0 or later is preferred. Possible parameters used are the gap opening penalty of 10, the gap extension penalty of 0.5, and the EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM62). The Needle result marked as the longest identity (obtained using the nobrief option) is used as the percent identity and is calculated as follows.

(Identical remnants of 100)/(Alignment length - total number of gaps in alignment)

For the purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000 above), preferably version 3.0.0 or later. Possible parameters used are the gap opening penalty of 10, the gap extension penalty of 0.5, and the EDNAFULL replacement matrix (EMBOSS version of NCBI NUC4.4). The Needle result marked as the longest identity (obtained using the -nobrief option) is used as the percent identity and is calculated as follows.

(Identical deoxyribonucleotides 100)/(Length of alignment - total number of breaks in alignment).

The term variant means a peptide having chymosin activity and containing a modification, i.e. substitution, insertion and/or removal at one or more (several) positions. Substitution means the replacement of an amino acid occupying a position with another amino acid; removal means removal of the amino acid occupying the position; and insertion means adding 1-3 amino acids adjacent to the amino acid occupying the position.

The amino acid may be a naturally occurring or non-naturally occurring amino acid, eg substitution, eg, in particular, with D-isomers (or D-forms), eg D-alanine, may be theoretically possible.

The term wild-type peptide refers to a nucleotide sequence or peptide sequence that occurs in nature, i. a nucleotide sequence or a peptide sequence that has not been subjected to targeted mutations by a human.

Brief description of graphic materials

Fig. 1 - alignment of the corresponding different chymosin sequences.

As one of skill in the present context will understand, the respective percentages of sequence identity of a mature chymosin polypeptide, e.g., sheep, bactrian camel, camel, pig, or rat, with the mature polypeptide of SEQ ID NO: 3 (bovine chymosin, i.e. the amino acid sequence from position 59 to 381 SEQ ID NO: 3) are relatively similar to the above percentages of sequence identity.

Fig. 2 - 3D structure of camel chymosin (detail, PDB: 4AA9) with bound casein model shown in green as a rod structure, κ-casein is located in the substrate-binding cleft of chymosin with a cleavable bond between residues 105 and 106. Mutations R242E, Y243E, N249D , G251D, N252D, R254E, S273D, Q280E, F282E are highlighted in blue.

Fig. 3 - 3D structure of bovine chymosin (PDB: 4AA8) with a model of bound κ-casein shown in green as a rod-like structure, κ-casein is placed in the substrate-binding cleft of chymosin with a cleavable bond between residues 105 and 106. Positions H292 and Q294 are highlighted in yellow color.

Fig. 4 - 3D structure of camel chymosin (detailed, PDB: 4AA9). Residues Y11, L12, and D13 of the N-terminus of the protein, as well as the potential interaction of Y11 with partner D290, are highlighted in purple in the form of a rod-shaped structure.

Detailed description of the invention

Determination of the amino acid position of the chymosin of interest.

As discussed above, as a reference sequence for determining the amino acid position of the corresponding chymosin polypeptide of interest (e.g., camelids, sheep, bovines, etc.), the well-known camelid chymosin sequence disclosed as SEQ ID NO: 2 is used here.

The amino acid sequence of another chymosin polypeptide is aligned with the polypeptide disclosed in SEQ ID NO: 2, and based on the alignment, the position number of the amino acid corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 2 is determined,

- 3 040259 using the ClustalW algorithm as described in Working Example 1 in this specification.

Based on the above well-known computer programs, one skilled in the art can easily determine the amino acid position in a chymosin polypeptide of interest here (eg, camels, sheep, bovines, etc.).

In FIG. 1 shows an example of alignment.

As an example: in Fig. 1, it can be seen that here the bullish reference SEQ ID NO: 3 used here has a G at position 50 and Camelus_dromedarius (SEQ ID NO: 2 here) has an A at this position 50.

Designation of options.

When describing embodiments of the present invention, the designations described below are intended to facilitate comparison. Use accepted IUPAC (International Union of Pure and Applied Chemistry) one-letter or three-letter amino acid abbreviations.

The specific variations discussed in this notation section below may not correspond to relevant embodiments of the present invention, i.e. this section of designations is only intended to describe the present associated designation as such.

Replacements. For an amino acid substitution, the following designation is used: original amino acid, position, substituted amino acid. Accordingly, the theoretical substitution of threonine with alanine at position 226 is referred to as Thr226Ala or T226A. Multiple mutations are separated by adding labels (+), for example Gly205Arg+Ser411Phe or G205R+S411F, representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively. A substitution, e.g. designated 226A, refers to a substitution of a parent amino acid (e.g., T, Q, S, or other parent amino acid) with alanine at position 226.

Removal. To remove an amino acid, the following notation is used: original amino acid, position, *. Accordingly, removal of the glycine at position 195 is referred to as Gly195* or G195*. Multiple deletions are separated by adding marks (+), eg Gly195*+Ser411* or G195*+S411*.

Inserts. For an amino acid insert, the following designation is used: original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after the glycine at position 195 is designated Gly195GlyLys or G195GK. Multiple amino acid insertions are designated [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after the glycine at position 195 is indicated as Gly195GlyLysAla or G195GKA.

In such cases, the inserted(e) amino acid(s) residue(s) are numbered by adding lower case letters to the position number of the amino acid residue preceding the inserted(s) amino acid(s) residue(s). In the example above, the sequence would look like this:

Parental: Option: 195 195 195a 195b G G - K - A

Multiple modifications. Variants containing multiple modifications are separated by adding labels (+), for example, Arg170Tyr+Gly195Glu or R170Y+G195E represent the replacement of tyrosine and glutamic acid with arginine and glycine at positions 170 and 195, respectively.

Various replacements. If different substitutions can be introduced at the same position, then these different substitutions are separated by a comma, for example, Arg170Tyr,Glu or R170Y,E represents the substitution of arginine with tyrosine or glutamic acid at position 170. Thus, Tyr167Gly,Ala+Arg170Gly,Ala or Y167G,A +R170G,A stands for the following options:

Tyr167Gly+Arg170Gly, Tyr167Gly+Arg170Ala, Tyr167Ala+Arg170Gly, and Tyr167Ala+Arg170Ala.

Preferred parental polypeptide having chymosin activity.

Preferably, the parent polypeptide has at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with the mature polypeptide of SEQ ID NO: 3 (bovine chymosin) and/or SEQ ID NO: 2 (camel chymosin).

As an example herein, a suitable corresponding parental polypeptide may, for example, be bovine chymosin A, as is known in the art, bovine chymosin A may have only one amino acid difference compared to bovine chymosin B of SEQ ID NO: 3.

In a preferred embodiment, the parent polypeptide has at least 90% sequence identity with the mature polypeptide of SEQ ID NO: 3 (bovine chymosin), more preferably the parent polypeptide has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 3 (bovine chymosin) and even more preferably, the parent polypeptide has at least 97% sequence identity with the mature polypeptide of SEQ ID NO: 3 (bovine chymosin). It may be preferred that the parent polypeptide be the mature polypeptide of SEQ ID NO: 3 (bovine chymosin).

- 4 040259

As one skilled in the art will understand, the respective parental polypeptide having chymosin activity may be, for example, a variant of, for example, the corresponding wild-type chymosin.

For example, a variant of bovine chymosin, e.g., with 5-10 modifications (e.g., substitutions) compared to the mature wild-type bovine chymosin polypeptide of SEQ ID NO: 3, may still be a parent polypeptide that has at least 95% sequence identity with the mature polypeptide SEQ ID NO: 3 (bovine chymosin).

As one skilled in the art would understand, the parent polypeptide may be a polypeptide that shares at least 80% sequence identity with the mature polypeptide of SEQ ID NO: 2 (camel). In a preferred embodiment, the parent polypeptide has at least 92% sequence identity with the mature polypeptide of SEQ ID NO: 2 and/or SEQ ID NO: 3, more preferably the parent polypeptide has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 2 and/or SEQ ID NO: 3, and even more preferably, the parent polypeptide has at least 97% sequence identity with the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3. It may be preferred that the parent polypeptide be the mature polypeptide of SEQ ID NO : 2 (camel chymosin).

In other words, in general, an isolated chymosin polypeptide variant corresponding herein may contain modifications (eg, substitutions) at positions other than those stated herein.

For example, a variant of bovine chymosin, for example with 5-10 modifications (e.g., substitutions) compared to a wild-type camel chymosin polypeptide having SEQ ID NO: 2, may still be a parent polypeptide that has at least 95% sequence identity with the mature a polypeptide of SEQ ID NO: 2.

It may be preferred that an isolated variant of bovine chymosin contain fewer than 30 amino acid modifications (e.g., substitutions) compared to the mature polypeptide of SEQ ID NO: 2 (camel chymosin), or it may be preferred that an isolated variant of camel chymosin contain less than 20 amino acid modifications ( e.g., substitutions) compared to the mature polypeptide of SEQ ID NO: 2, or it may be preferred that the isolated bovine chymosin variant contains less than 10 amino acid modifications (e.g., substitutions) compared to the mature polypeptide of SEQ ID NO: 2, or it may be preferred so that the isolated variant of camel chymosin contained less than 5 amino acid modifications (eg, substitutions) compared to the mature polypeptide of SEQ ID NO: 2 (camel chymosin).

A method for obtaining isolated variants of the chymosin polypeptide.

As discussed above, as is known in the art, one of ordinary skill in the art can prepare and purify chymosin and chymosin variants in a conventional manner.

In other words, if one skilled in the art has a parental polypeptide relevant here having a chymosin activity of interest (e.g., bovine, camel, sheep, pig, or rat) and guidance disclosed herein, obtaining a variant of such parental chymosin of interest is routine work for one skilled in the art.

An example of a suitable method for the production and isolation of chymosin (variant or parent) may be, for example, the well-known technology based on fungal recombinant expression/production, such as described in WO02/36752A2 (Chr. Hansen).

It is also routine work for one skilled in the art to modify one or more positions of the parent polypeptide having chymosin activity, where the modification comprises substitution, deletion or insertion at least one amino acid position as described herein.

As known to the person skilled in the art, this can be obtained, for example, by so-called site-directed mutagenesis and recombinant expression/production technology.

It is also routine work for the skilled artisan to determine the presence or absence of chymosin activity in the parent polypeptide relevant here (eg, wild-type camel or bovine chymosin) and/or the chymosin variant relevant here.

As known in the art, the specificity of chymosin can be determined by the so-called C/P ratio, which is determined by dividing the specific clotting activity (C) by the proteolytic activity (P). As is known in the art, a higher C/P ratio generally implies that protein loss during, for example, cheese production due to reduced non-specific protein degradation is reduced, i. cheese yield improved.

Determination of milk-clotting activity of milk.

Milk-clotting activity can be determined using the REMCAT method, which is a standard method developed by the International Dairy Federation (IDF method).

Milk-clotting activity is determined based on the time required for visible

- 5 040259 flocculation of a standard milk substrate obtained from low-temperature thickened and spray-dried skimmed milk powder with 0.5 g per liter of calcium chloride solution (pH-6.5). The clotting time of the rennet sample is compared with the clotting time of a reference standard having a known milk-clotting activity and having the same enzyme composition according to IDF Standard 110B as a sample. Samples and reference standards are measured under the same chemical and physical conditions. Variant samples are adjusted to a concentration of about 3 IMCU (International Milk-Coagulation Unit)/ml using 84 mM acetic acid buffer, pH 5.5. Then 20 µl of the enzyme preparation is added to 1 ml of preheated milk (32°C) in a glass tube placed in a water bath capable of maintaining a constant temperature of 32±1°C with constant stirring.

The total milk-clotting activity (efficiency) of rennet is calculated in International Milk-Clotting Units (IMCU) per ml relative to a standard having the same enzyme composition as the sample, according to the formula:

Sstandart x Tstundurt x Dsumple

Activity In IMCU/ml = ----------:----------:-----Dstundurt x Tsumple

Sstandard: The milk clotting activity of the international standard for rennet.

Tstandard: clotting time in seconds obtained for the standard dilution.

Dsample: dilution factor for the sample.

Dstandard: dilution factor for the standard.

Tsample: clotting time in seconds obtained for a diluted rennet enzyme sample, from the time enzyme was added to the flocculation time.

The μIMCU method can be used instead of the REMCAT method to determine milk clotting activity. Compared to REMCAT, the flocculation time for chymosin variants in the μIMCU assay is determined by measuring the OD (optical density) in 96-well microtiter plates at 800 nm in a UV/VIS (visible region) plate reader. A standard curve for various dilutions of a reference standard with known clotting efficiency is recorded for each plate. Samples are prepared by diluting the enzyme in 84 mM acetate buffer, 0.1% Triton X-100, pH 5.5. The reaction at 32° C. is started by adding 250 µl of a standard milk substrate containing 4% (w/w) low-temperature thickened and spray-dried skimmed milk powder and 7.5% (w/w) calcium chloride (pH®6). ,5) to 25 µl of the enzyme sample. The milk-clotting activity of the chymosin variants is then determined in International Milk-clotting Units (IMCU) per ml based on the flocculation time relative to the standard curve.

Determination of total protein content.

Preferably, total protein content can be determined using the Pierce BCA Protein Assay Kit from Thermo Scientific according to the supplier's instructions.

Calculation of specific coagulation activity.

The specific clotting activity (IMCU/mg total protein) was determined by dividing the clotting activity (IMCU/ml) by the total protein content (mg total protein per ml).

Determination of proteolytic activity.

Preferably, total proteolytic activity can be measured using fluorescently labeled Bodipy-FL casein as a substrate (EnzChek; Molecular Bioprobes, E6638). Casein derivatives heavily labeled with pH-insensitive green fluorescent Bodipy-FL quench the fluorescence of the conjugate. Protease-catalyzed hydrolysis releases fluorescent Bodipy-FL. This method is very sensitive, which is necessary for this experiment, since the standard has the lowest total proteolytic activity of all known coagulants. A 0.04 mg/ml substrate solution is prepared in 0.2 M phosphate buffer, pH 6.5, containing 100 mM NaCl, 5% glycerol and 0.1% Bridge. Chymosin variants are dissolved in 20 mM malonate buffer containing 100 mM NaCl, 5% glycerol and 0.1% Bridge. 20 µl each of the reference solution and chymosin variant were mixed in a black 384-well flat-bottomed Corning polystyrene plate and continuously measured for fluorescence in a fluorometer at 32°C for 10 hours. The slopes of the linear part of the fluorescence change curve were used to determine the total proteolytic activity.

Definition of C/R ratio.

The C/P ratio is calculated by dividing the clotting activity (C) by the proteolytic activity (P).

Statistical analysis of the effects of position and mutation on specific clotting activity and C/P ratio.

Preferably, a statistical approach with machine learning analysis and PCA analysis can be used to determine the effect of single mutations present in variants with multiple substitutions, for example, on specific milk clotting activity, as well as on the clotting ratio

- 6 040259 total and total proteolytic activity (C/P).

Preferred Embodiments of the Invention.

As indicated above and illustrated in the examples below, the present inventors have made a number of preferred chymosin polypeptide variants with improved clotting activity and/or C/P ratio compared to the corresponding parental polypeptide under comparable conditions.

In a preferred aspect, the present invention relates to an isolated variant chymosin polypeptide characterized in that:

(a) the isolated variant chymosin polypeptide has a specific milk-clotting activity (IMCU/mg total protein) that is at least 110% of the specific milk-clotting activity of its parent polypeptide and/or (b) the isolated variant chymosin polypeptide has a C/P ratio that is at least 200% C/P ratio of its parent polypeptide.

The parent polypeptide may have at least 80%, such as at least, for example, 80%, 85%, 95%, 97%, 98%, 99% or 100% sequence identity with the polypeptide of SEQ ID NO: 2 (camel chymosin).

A preferred isolated variant of the chymosin polypeptide may have a specific milk-clotting activity (IMCU/mg total protein) of at least 110% of the specific milk-clotting activity of the parent peptide and contain a substitution at one or more (several) of the following positions as specified in relation to the amino acid sequence of SEQ ID NO:2:

R242, L222, D59, S273,

K19, V309, S132, N249, 196, L166, H76, G251, Q280, Q56, M157, K231, M256, N291, more specifically, R242E, L222I, D59N, S273Y, K19T, V309I, S132A, N249D,

I96L, N249E, L166V, H76Q, N249D, G251D, Q280E, Q56H, M157L, K231N, M256L, N291Q.

Possibly, an isolated variant chymosin polypeptide may further comprise substitutions that alter the glycosylation pattern, such as, for example, substitutions at one or more positions of N100, N252 and/or N291, more specifically N100Q, N252Q and/or N291Q.

A preferred variant may contain one or more of the following combinations of substitutions, where each substitution is defined in relation to the amino acid sequence of SEQ ID NO: 2:

- 7 040259

Y11V, K19T, D59N, I96L, S164G, L166V, L222V, R242E, N249E, L253I:

Y11I, D59N, I96L, S164G, L166V, L222V, R242E, G251D, L253I;

Y11I, I96L, S164G, L222I, R242E;

Y11I, K19T, D59N, I96L, S164G, L222I, R242E, N249E, G251D;

H76Q, I96L, S164G, L222I, R242E, G251D, S273Y;

K19T, D59N, H76Q, S164G, L222I, N249D, S273Y;

K19T, D59N, H76Q, L166V, L222I, R242E, G251D, S273Y;

K19T, D59N, H76Q, S132A, L222I, G251D, S273Y, V309I;

Y21S, H76Q, S164G, L222I, R242E, G251D, S273Y;

D59N, S132A, S164G, L222I, R242E, N249D, G251D, S273Y;

D59N, H76Q, I96L, S132A, S164G, L166V, L222I, G251D, S273Y;

H76Q, S164G, LI66V, L222I, R242E, G251D, S273Y;

D59N, H76Q, S132A, S164G, LI66V, S273Y;

Y21S, D59N, H76Q, I96L, S164G, L222I, N249D, G251D, S273Y;

K19T, D59N, H76Q, S164G, R242E, N249D, G251D, S273Y;

K19T, D59N, I96L, S164G, L222I, G251D;

H76Q, L130I, L222I, S226T, G251D, S273Y;

D59N, H76Q, S164G, L222I, S226T, R242E;

Y21S, D59N, H76Q, I96L, L222I, S273Y;

H76Q, S164G, L222I, N249D, G251D, S273Y, V309I;

D59N, H76Q, S164G, L166V, L222I, N249D, G251D, S273Y, V309I;

D59N, I96L, L166V, L222I, R242E, G251D;

K19S, D59N, I96L, S164G, L222I, R242E, N249E, G251D;

Y11V, K19T, D59N, I96L, S164G, L166V, L222I, R242E, G251D, L253I;

K19T, D59N, I96L, S164G, L166I, L222I, R242E, N249D;

H76Q, I96L, S164G, L222I, R242E, G251D, S273;

K19T, I96L, L222I, R242E, L253I;

K19T, D59N, I96L, S164G, L222V, R242E, N249D, L253I;

I96L, S164G, L222I, R242E, G251D, S274Y;

N249D, N100Q, N291Q;

R242E, N100Q, N291Q;

R242E, G251D, Q280E, N100Q, N291Q;

R242E, N252D, N100Q, N291Q;

R242E, S273D, Q280E, N100Q, N291Q;

R242E, R254E, Q280E, N100Q, N291Q;

R242E, Q280E, N100Q, N291Q;

R242E, R254E, S273D, Q280E, N100Q, N291Q;

N252D, S273D, Q280E, N100Q, N291Q;

G251D, S273D, Q280E, N100Q, N291Q;

Y243E, Q280E, N100Q, N291Q;

Q56H, N252Q, N291Q;

R67Q, S132A, L222I, K231N, R242E, V248I;

R67Q, I96L, LI301, M157L, K231N, R242E;

R67Q, M157L, L222I, K231N, V248I;

R67Q, I96L, M157L, L222I, K231N;

R67Q, G70D, M157L, L222I, N291Q or

R67Q, L130I, M157L, R242E, M256L, N292H.

- 8 040259

In a suitable embodiment, a preferred isolated variant chymosin polypeptide of the present invention has a C/P ratio of at least 200% of the ratio

C/P of its parent polypeptide and contains a substitution at one or more of the following positions as indicated in relation to the amino acid sequence of SEQ ID NO: 2:

R242, 196, H76, SI64, S273, G251,

Yll, L222, L166, K19, Y21, S74, Y243, N249, Q280, F282, L295, N252, R254, G70, V136,

L222, K231, N291, more specifically R242E, I96L, H76Q, S164G, S273Y, G251D, Y11I,

R242D, L222V, Y11V, L166I, K19T, Y21S, S74D, Y243E, N249D, S273D, Q280E, F282E,

L295K, N252D, R254E, G70D, V136I, L222I, K231N, N291Q.

A preferred isolated variant chymosin polypeptide of the present invention may also contain one or more of the following combinations of substitutions, and with each substitution specified in relation to the amino acid sequence of SEQ ID NO: 2:

Y11V, K19T, D59N, I96L, S164G, L166V, L222V, R242E, N249E, L253I;

Y11I, D59N, I96L, S164G, L166V, L222V, R242E, G251D, L253I;

Y11I, I96L, S164G, L222I, R242E;

Y11I, K19T, D59N, I96L, S164G, L222I, R242E, N249E, G251D;

H76Q, I96L, S164G, L222I, R242E, G251D, S273Y;

Y21S, H76Q, S164G, L222I, R242E, G251D, S273Y;

H76Q, S164G, L166V, L222I, R242E, G251D, S273Y;

K19T, D59N, H76Q, S164G, R242E, N249D, G251D, S273Y;

Y21S, D59N, H76Q, I96L, S164G, L222I, N249D, G251D, S273Y;

D59N, H76Q, I96L, S132A, S164G, L166V, L222I, G251D, S273Y;

H76Q, S164G, L222I, N249D, G251D, S273Y, V309I;

D59N, H76Q, I96L, L130I, S164G, L222I, R242E, G251D;

H76Q, S164G, L166V, L222I, S226T, S273Y;

D59N, H76Q, I96L, S164G, L222I, S226T, N249D, G251D, S273Y;

K19T, D59N, H76Q, L166V, L222I, R242E, G251D, S273Y;

D59N, H76Q, S164G, L222I, R242E, S273Y, V309I;

H76Q, I96L, S164G, G251D, S273Y, V309I;

D59N, H76Q, L130I, S132A, S164G, L222I, R242E, G251D, S273Y;

D59N, H76Q, I96L, S132A, S164G, L222I, S226T, G251D, S273Y;

D59N, H76Q, S132A, S164G, LI66V, S273Y;

D59N, H76Q, S164G, L222I, S226T, R242E;

K19T, D59N, I96L, S164G, L222I, G251D;

D59N, H76Q, I96L, S164G, L222I, S226T, G251D, S273Y, V309I;

D59N, S132A, S164G, L222I, R242E, N249D, G251D, S273Y;

K19T, D59N, H76Q, S164G, L222I, N249D, S273Y;

K19T, D59N, S164G, L166V, L222I, S226T, G251D, S273Y;

Y21S, D59N, H76Q, S164G, L222I, S226T, G251D, S273Y, V309I;

K19T, Y21S, D59N, H76Q, S132A, S164G, L222I, G251D, S273Y;

D59N, H76Q, I96L, L130I, S164G, L222I, N249D, G251D, S273Y;

H76Q, L130I, L222I, S226T, G251D, S273Y;

D59N, H76Q, L130I, S164G, L166V, L222I, G251D, S273Y, V309I;

K19T, D59N, H76Q, L130I, S164G, L222I, S226T, G251D, S273Y;

D59N, H76Q, L130I, S164G, G251D, V309I;

K19T, Y21S, D59N, H76Q, L130I, S164G, L222I, S273Y;

K19T, D59N, H76Q, S132A, L222I, G251D, S273Y, V309I;

Y21S, D59N, S164G, L222I, R242E, G251D, S273Y, V309I;

D59N, H76Q, S226T, R242E, G251D, S273Y;

Y21S, D59N, H76Q, I96L, L222I, S273Y;

K19T, Y21S, H76Q, S164G, L222I, G251D, S273Y;

- 9 040259

K19T, D59N, H76Q, I96L, S164G, L166V, L222I, G251D, S273Y;

Y21S, D59N, H76Q, L130I, S132A, S164G, L222I, G251D, S273Y;

Y21S, D59N, H76Q, S164G, LI66V, N249D, G251D, S273Y;

Y11I, K19T, I96L, S164G, L222V, R242E, G251D;

H76Q, I96L, S164G, L222I, R242E, G251D, S273Y;

H76Q, I96L, S164G, L222I, R242E, G251D;

Y11V, I96L, S164G, L222I, R242E, N249D, L253I, I263L;

Y11V, K19T, D59N, I96L, S164G, L166V, L222I, R242E, G251D, L253I;

Y11V, K19T, E83S, I96L, S164G, L166V, L222I, R242E, G251D;

K19T, D59N, I96L, S164G, L166I, L222I, R242E, N249D;

I96L, S164G, L222I, R242E, N249D, G251D, I263L;

K19T, D59N, I96L, S164G, L222V, R242E, N249D, L253I;

I96L, S164G, L222I, R242E, G251D, S274Y;

K19T, I96L, S164G, L166V, L222I, R242E, N249D, G251D, I263V;

K19T, I96L, S164G, R242E, L253I;

Y11V, K19T, I96L, S164G, L166V, L222I, R242E;

D59N, I96L, S164G, L222I, R242E, L253I, I263L;

I96L, S164G, L222I, R242E, G251D;

K19S, D59N, I96L, S164G, L222I, R242E, N249E, G251D;

K19T, D59N, I96L, S164G, L166I, L222I, R242D, G251D, I263V;

I96L, S164G, LI66V, L222I, R242E, N249D, I263L;

K19T, D59N, I96L, S164G, L166V, L222I, R242D, G251D, L253I;

D59N, I96L, L166V, L222I, R242E, G251D;

K19T, D59N, I96V, S164G, L166V, L222I, R242E, I263L;

Y11I, K19T, D59N, S164G, L222I, G251D, 1263V;

K19T, D59N, I96L, S164G, L222I, N249E, G251D, L253V, I263L;

Y11V, E83S, I96L, S164G, L222I, R242E, G251D, L253I, I263L;

K19T, E83S, I96L, S164G, L222I, R242E, N249D, G251D, L253I;

K19T, E83S, I96L, S164G, L166V, L222I, R242E, N249D, G251D, L253I;

K19T, D59N, I96L, S164G, L222V, N249E, G251D, I263V;

Y11V, D59N, I96L, S164G, L222I, G251D, L253V;

Y11I, K19T, D59N, I96V, L222I, R242D, G251D;

K19T, E83T, I96L, S164G, L222I, R242E, L253V;

K19S, I96L, S164G, L166V, L222I, R242E;

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K19T, D59N, I96L, S164G, L222I, G251D;

K19T, I96L, S164N, L222I, R242E, I263L;

K19T, D59N, E83T, S164G, L166V, L222I, R242D, G251D;

K19T, E83S, I96L, S164G, L222I, R242E, G251D, L253I;

Y11V, E83S, I96L, S164G, L222I, R242E, L253I, I263L;

K19T, I96L, L222I, R242E, L253I;

K19T, I96L, S164G, L166V, L222I, N249D, I263L;

K19T, D59N, I96L, S164G, L166I, G251D, L253V;

Y11V, K19T, D59N, I96L, S164N, L166I, L222I, G251D;

R242E, Q280E, N100Q, N291Q;

R242E, N252D, N100Q, N291Q;

R242E, R254E, S273D, Q280E, N100Q, N291Q;

R242E, R254E, Q280E, N100Q, N291Q;

V32L, R67Q, L130I, M157L, K231N, M256L;

R67Q, L13OI, M157L, D158S, R242E, N291Q;

R67Q, V136I, M157L, L222I, V248I;

Y11V, R67Q, L130I, M157L, L222I, R242E;

R67Q, I96L, L130I, M157L, K231N, R242E;

R67Q, G70D, M157L, L222I, N291Q;

R67Q, S132A, L222I, K231N, R242E, V248I;

R67Q, L13OI, L222I, R242E, M256L;

R67Q, G70D, M157L, R242E, V248I;

R67Q, M157L, L222I, K231N, V248I;

R67Q, I96L, N100Q, L130I, M157L, N292H;

I45V, L130I, M157L, K231N, R242E or

R67Q, I96L, M157L, L222I, K231N.

Preferred Methods for Producing Isolated Chymosin Polypeptide Variants The present invention further provides methods for producing an isolated polypeptide according to the present invention.

These preferred methods may include the following steps:

(a) modification at one or more positions of a DNA sequence encoding a polypeptide having at least 80% sequence identity with SEQ ID NO: 2, where the modification includes a substitution, deletion or insertion at least one amino acid position;

(b) obtaining and isolating a variant polypeptide from step (a).

The parent polypeptide may have at least 85%, 95%, 97%, 98% or at least 99% sequence identity with the polypeptide of SEQ ID NO: 2 (camel chymosin).

In another preferred embodiment, the present invention relates to a method for producing an isolated chymosin polypeptide, wherein the variant contains one or more of the following substitutions, indicated in relation to the amino acid sequence of SEQ ID NO: 2:

D59, V309, S132, N249, L166, N249, Q56, M157, M256, R242,196, H76, S164, S273, G251, Yll, L166, K19, Y21, S74, Y243, N249, S273, Q280, F282, L295, N252, R254, Q294, G70, V136, L222, K231, N291 such as e.g. G251D, Y11I, R242D, L222V, Y11V, L166I, K19T, Y21S, S74D, Y243E, N249D, S273D, Q280E, F282E, L295K, N252D, R254E, Q294E, G70D, V136I, L222I, K231QN., N291QN., N291QN.

In another embodiment, the present invention relates to a method for obtaining an isolated variant chymosin polypeptide, where:

(a) the variant contains one or more combinations of the following substitutions, and wherein each substitution is indicated in relation to the amino acid sequence of SEQ ID NO: 2:

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Y11V, K19T, D59N, I96L, S164G, L166V, L222V, R242E, N249E, L253I;

Y11I, D59N, I96L, S164G, L166V, L222V, R242E, G251D, L253I;

Y11I, I96L, S164G, L222I, R242E;

Y11I, K19T, D59N, I96L, S164G, L222I, R242E, N249E, G251D;

H76Q, I96L, S164G, L222I, R242E, G251D, S273Y;

K19T, D59N, H76Q, S164G, L222I, N249D, S273Y;

K19T, D59N, H76Q, L166V, L222I, R242E, G251D, S273Y;

K19T, D59N, H76Q, S132A, L222I, G251D, S273Y, V309I;

Y21S, H76Q, S164G, L222I, R242E, G251D, S273Y;

D59N, S132A, S164G, L222I, R242E, N249D, G251D, S273Y;

D59N, H76Q, I96L, S132A, S164G, L166V, L222I, G251D, S273Y;

H76Q, S164G, LI66V, L222I, R242E, G251D, S273Y;

D59N, H76Q, S132A, S164G, LI 66V, S273Y;

K19T, D59N, H76Q, S164G, R242E, N249D, G251D, S273Y;

Y21S, D59N, H76Q, I96L, S164G, L222I, N249D, G251D, S273Y;

K19T, D59N, I96L, S164G, L222I, G251D;

D59N, H76Q, S164G, L222I, S226T, R242E;

H76Q, L130I, L222I, S226T, G251D, S273Y;

Y21S, D59N, H76Q, I96L, L222I, S273Y;

H76Q, S164G, L222I, N249D, G251D, S273Y, V309I;

D59N, I96L, LI66V, L222I, R242E, G251D;

Y11V, K19T, D59N, I96L, S164G, L166V, L222I, R242E, G251D, L253I;

K19S, D59N, I96L, S164G, L222I, R242E, N249E, G251D;

K19T, D59N, I96L, S164G, L166I, L222I, R242E, N249D;

H76Q, I96L, S164G, L222I, R242E, G251D, S273Y;

K19T, I96L, L222I, R242E, L253I;

K19T, D59N, I96L, S164G, L222V, R242E, N249D, L253I;

I96L, S164G, L222I, R242E, G251D, S274Y;

R242E, N252D, N100Q, N291Q;

R242E, R254E, Q280E, N100Q, N291Q;

R242E, Q280E, N100Q, N291Q;

R242E, R254E, S273D, Q280E, N100Q, N291Q;

R67Q, S132A, L222I, K231N, R242E, V248I;

R67Q, I96L, L130I, M157L, K231N, R242E;

R67Q, M157L, L222I, K231N, V248I;

R67Q, I96L, M157L, L222I, K23 IN or

R67Q, G70D, M157L, L222I, N291Q.

Another relevant aspect of the present invention relates to a method for producing a food or feed product, comprising adding an effective amount of an isolated chymosin polypeptide variant, as described herein, to the food(s) or feed(s) ingredient(s) and performing additional production steps to obtain a food or feed product, in particular when the food or feed product is a milk-based product or a food or feed product containing a chymosin polypeptide of the present invention.

Another relevant aspect of the present invention relates to a variant of the chymosin polypeptide of the present invention in a method for producing a milk-based product, such as, for example, cheese, such as, for example, draw cheese, cheddar, continental-type cheeses, soft cheese or white pickled cheese.

As discussed above, an isolated variant chymosin polypeptide as described herein can be used in accordance with the prior art, for example to produce a milk-based product of interest (such as, for example, a cheese product).

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As discussed above, one aspect of the invention relates to a method for producing a food or feed product, comprising adding an effective amount of an isolated variant chymosin polypeptide, as described in this description of the invention, to the food(s) or feed(s) ingredient(s) and carrying out the further steps obtaining a food or feed product.

Preferably, the food or feed product is a milk-based product and the method comprises adding an effective amount of an isolated chymosin polypeptide variant as described herein to milk and carrying out further manufacturing steps to produce a milk-based product.

The milk may be, for example, soy milk, sheep's milk, goat's milk, buffalo's milk, yak's milk, llama's milk, camel's milk or cow's milk.

The milk-based product may, for example, be a fermented dairy product such as quark or cheese.

As is known in the art, growth, purification, testing and processing can affect the activity of enzymes, and hence the enzyme of the present invention. Thus, the present invention relates to variants of the chymosin polypeptide, methods for their preparation and obtaining products containing them, where the variant chymosin polypeptide has improved coagulation activity and/or C/P ratio compared to the corresponding parental polypeptide under comparable conditions and preferably after production or processed in a different way under comparable conditions.

Examples

Example 1 Alignment and numbering in chymosin protein sequences and variant sequences.

Chymosin protein sequences were aligned using the ClustalW algorithm provided by EBI (EBI, Tools, Multiple Sequence Alignment, CLUSTALW, http://www.ebi.ac.uk/Tools/msa/clustalw2/) and as described in Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007). Bioinformatics 23(21), 2947-2948.

The ClustalW2 settings for multiple sequence alignments were: amino acid comparison matrix=BLOSUM, gap opening=10, gap length=0.5, gap spacing=8, infinite gaps, iteration=none, maximum iterations=1, bunching=NJ.

Bovine chymosin B preprochymosin (Genbank accession number P00794 - disclosed in this specification as SEQ ID NO: 1) was used as a reference sequence, where the N-terminal methionine is number 1 (MRCL......) and the C-terminal isoleucine (in the ...LAKAI protein sequence) is number 381. The variants were aligned with bovine B pre-prochymosin and the residues were numbered according to the corresponding bovine chymosin residue.

Example 2 Construction of chymosin variants.

Chymosin variants were constructed using various strategies.

When referring to camel chymosin, this document refers to camel chymosin containing the polypeptide of SEQ ID NO: 2.

Camel chymosin of SEQ ID NO: 2 can be considered as the relevant parental polypeptide having chymosin activity and used to generate camel chymosin variants.

When bovine chymosin is mentioned, this document refers to bovine chymosin containing the polypeptide of SEQ ID NO: 1.

The bovine chymosin of SEQ ID NO: 1 can be considered as the relevant parental polypeptide having chymosin activity and used to generate bovine chymosin variants.

Camel chymosin variants 180-269 and 367-461 were constructed based on the alignment of a large set of well known aspartic protease sequences having an identity of 25% or more compared to bovine chymosin B.

Variations were usually introduced in areas with high levels of amino acid variation between species, while conservative areas were not changed. Amino acid substitutions were selected based on phylogenetic, structural and experimental information to identify high probability changes to demonstrate positive effects on specific clotting activity and C/P ratio. Multiple variations were introduced into each variant construct to ensure that each single mutation was present in multiple variant constructs to minimize the effect of covariance between different substitutions.

Machine learning and statistical analysis were used to determine the relative contributions of amino acid substitutions to measured coagulation characteristics for chymosin variants (refs 14, 15).

Variants 271-366 were constructed based on detailed structural analysis of bovine chymosin (PDB code: 4AA8) and camel chymosin (PDB code: 4AA9). Variations were chosen based on

- 13 040259 the chemical nature of the respective amino acid side chains and their expected effect on either casein substrate binding or general enzymatic properties. Most of the amino acid substitutions in variants 271-346 were made at positions in the sequence, either within or in close structural proximity to the substrate-binding cleft, or in secondary building blocks that contact the bound casein substrate. In addition, changes have been made to positions on the protein surface that change the charge profile of these regions (ref. 5) and are therefore expected to affect enzyme performance. Variants 347-366 were made based on a different structural conformation of the N-terminal sequence in bovine and camel chymosin. Amino acid substitutions were made at positions in the substrate-binding cleft that interact with the N-terminus of camel chymosin.

Example 3. Obtaining the enzyme substance of the variant chymosin.

All chymosin variants were synthesized as synthetic genes and cloned into a fungal expression vector such as, for example, pGAMpR-C (described in WO 02/36752A2).

The vectors were transformed into E. coli and plasmid DNA was purified using standard molecular biology protocols known to those skilled in the art.

The plasmid variants were individually transformed into an Aspergillus niger or Aspergillus nidulans strain and the protein was produced essentially as described in WO 02/36752A2 and purified using standard chromatographic methods. For enzyme library screening, all chymosin variants were prepared in 20-60 ml fermentations. For a more detailed characterization of variants 433, 436, 453 and 457, the respective enzymes were fermented again on a 70 L scale.

As is known in the art, one skilled in the art can, based on their general knowledge, prepare and purify chymosin and chymosin variants such as the bovine and camel chymosin variants described herein.

Example 4. Determination of specific chymosin activity.

4.1. Determination of milk-clotting activity.

Milk-clotting activity was determined using the REMCAT method, which is a standard method developed by the International Dairy Federation (IDF method).

Milk-clotting activity was determined based on the time required for visible flocculation of a standard milk substrate obtained from low-temperature thickened and spray-dried skimmed milk powder with a calcium chloride solution of 0.5 g per liter (pH<6.5). The clotting time for the rennet sample was compared with the clotting time of a reference standard having a known milk-clotting activity and having the same enzyme composition according to the IDF HOB standard as the sample. Samples and reference standards were measured under the same chemical and physical conditions. Variant samples were adjusted to about 3 IMCU/ml using 84 mM acetic acid buffer, pH 5.5. Then, 20 μl of the enzyme preparation was added to 1 ml of preheated milk (32°C) in a glass analytical tube placed in a water bath capable of maintaining a constant temperature of 32±1°C with constant stirring.

The total clotting activity (efficiency) of rennet was calculated in International Milk Clotting Units (IMCU) per ml compared to a standard containing the same enzyme composition as the sample, according to the formula:

Sstandart x Tstandart x Dsample

Activity in IMCU/ml = ----------:----------:-----Dstandart x Tsample

Sstandard: The milk clotting activity of the international reference standard for rennet.

Tstandard: clotting time in seconds obtained for the standard dilution.

Dsample: dilution factor for the sample.

Dstandard: dilution factor for the standard.

Tsample: clotting time in seconds obtained for a dilute rennet sample from the time the enzyme was added to the time of flocculation.

To determine the clotting activity of the variants 1 and 3 libraries, as well as engineered variants, the pIMCU method was used instead of the REMCAT method. Compared to REMCAT, the flocculation time for chymosin variants in the pIMCU assay was determined by measuring the OD (optical density) in 96-well plates at 800 nm in a UV/VIS plate reader. A standard curve of various dilutions of a reference standard with known clotting activity was recorded for each plate. Samples were prepared by diluting the enzyme with 84 mM acetate buffer, 0.1% Triton X-100, pH 5.5 Reaction at 32° C. was started by adding 250 µl of standard milk substrate containing 4% (w/w) skimmed milk powder low temperature thickening and spray drying and 7.5% (w/w) calcium chloride (pH<6.5) to 25 µl of enzyme sample. The milk-clotting activity of the chymosin variants in International Milk-clotting Units (IMCU) per ml was determined based on the flocculation time for the sample compared to the standard curve.

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4.2. Determination of total protein content.

Total protein content was determined using the Pierce BCA Protein Assay Kit from

Thermo Scientific in accordance with the suppliers' instructions.

4.3. Calculation of specific coagulation activity.

Specific clotting activity (IMCU/mg total protein) was determined by dividing the clotting activity (IMCU/ml) by total protein content (mg total protein per ml).

Example 5 Determination of proteolytic activity.

Total proteolytic activity was measured using fluorescently labeled Bodipy-FL casein as a substrate (EnzChek; Molecular Bioprobes, E6638). Casein derivatives heavily labeled with pH-insensitive green fluorescent Bodipy-FL quench the fluorescence of the conjugate. Protease-catalyzed hydrolysis releases fluorescent Bodipy-FL. This method is very sensitive, which was necessary for this experiment because CHYMAX M has the lowest total proteolytic activity of all coagulants known to date.

A 0.04 mg/ml substrate solution was prepared in 0.2 M phosphate buffer, pH 6.5, containing 100 mM NaCl, 5% glycerol, and 0.1% Bridge. Chymosin variants were dissolved in 20 mM malonate buffer containing 100 mM NaCl, 5% glycerol, and 0.1% Bridge. 20 µl of substrate solution and chymosin variant were mixed in a black 384-well flat-bottomed Corning polystyrene plate and fluorescence was continuously determined in a fluorometer at 32°C for 10 h. The slopes of the linear part of the fluorescence change curve were used to determine the total proteolytic activity.

Example 6 Statistical Analysis of the Effects of Position and Mutation on Specific Clotting Activity and C/P Ratio.

To determine the effect of single mutations present in multiple substitution library variants 1-3 on the cleavage of k-casein between the Phe105 and Met106 positions, i.e. specific milk-clotting activity, as well as the ratio of coagulation and total proteolytic activity (C/P), used a statistical approach with machine learning analysis and X-ray analysis.

Results.

Library 1 of multiple replacements.

Camelid chymosin variants were generated, each with multiple substitutions compared to the wild type, and analyzed as described above. All variants have an amino acid sequence identical to camel chymosin (SEQ ID NO: 2), except for the variations shown in the table. Camel chymosin (CHY-MAX M) is included as a reference.

Clotting activities were determined using the μIMCU method.

Table 1. Enzymatic activity of camel chymosin variants

180-222. Values are given as % cleavage by wild-type camel chymosin (CHY-MAX M)

option CHY-MAX M mutations Cont.(C)1 100 Proteol.(R) 1 100 C/R 100 180 H76Q S132A S164G L222I N249D G251D 72 37 194 181 Y21S D59N H76Q S164G L166V N249D G251D S273Y 77 37 210 182 D59N H76Q S164G L222I R242E S273Y V309I 96 21 449 183 D59N H76Q L130I L166V L222I N249D G251D S273Y 84 55 152 184 Y21S D59N S164G L222I R242E G251D S273Y V309I 102 35 287 185 K19T Y21S D59N H76Q S132A S164G L222I G251D S273Y 97 29 334 186 D59N H76Q I96L L130I S164G L222I R242E G251D 85 16 524 187 H76Q S164G L166V L222I S226T S273Y 103 21 504 188 K19T D59N I96L S164G L222I G251D 126 31 403 189 Y21S H76Q S164G L222I R242E G251D S273Y 138 14 975 190 H76Q I96L S164G L222I R242E G251D S273Y 153 10 1479 191 H76Q S164G L222I N249D G251D S273Y V309I 112 19 606 192 K19T D59N H76Q S164G L222I N249D S273Y 152 42 363 193 Y21S D59N H76Q S164G L222I S226T G251D S273Y V309I 107 32 340 194 H76Q S164G L166V L222I R242E G251D S273Y 132 14 949 195 D59N H76Q I96L S164G L222I S226T N249D G251D S273Y 96 19 498 196 D59N H76Q L130I S164G L166V L222I G251D S273Y V309I 76 24 316 197 D59N S132A S164G L222I R242E N249D G251D S273Y 138 38 365 198 H76Q I96L S164G G251D S273Y V309I 71 16 443 199 D59N H76Q L130I S164G G251D V309I 54 18 309

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200 K19T D59N S164G L166V L222I S226T G251D S273Y 107 31 342 201 D59N H76Q I96L S132A S164G L222I S226T G251D S273Y 96 23 426 202 K19T D59N H76Q I96L S164G L166V L222I G251D S273Y 90 41 218 203 K19T D59N H76Q L130I S164G L222I S226T G251D S273Y 64 21 309 204 K19T D59N H76Q S132A L222I G251D S273Y V309I 141 48 294 205 H76Q L130I L222I S226T G251D S273Y 124 38 322 206 K19T Y21S D59N H76Q L130I S164G L222I S273Y 75 25 295 207 Y21S D59N H76Q I96L S164G L222I N249D G251D S273Y 129 17 762 208 K19T D59N H76Q S164G R242E N249D G251D S273Y 129 15 879 209 D59N H76Q S164G L222I S226T R242E 124 thirty 417 210 D59N H76Q I96L S132A S164G L166V L222I G251D S273Y 136 21 657 211 D59N H76Q S132A S164G L166V S273Y 131 31 423 212 Y21S D59N S164G L222I S226T N249D G251D S273Y 92 48 190 213 D59N H76Q L130I S132A S164G L222I R242E G251D S273Y 108 24 441 214 D59N H76Q S164G L166V L222I N249D G251D S273Y V309I 111 65 171 215 D59N H76Q I96L S164G L222I S226T G251D S273Y V309I 87 24 369 216 K19T D59N H76Q L166V L222I R242E G251D S273Y 146 thirty 494 217 Y21S D59N H76Q I96L L222I S273Y 118 52 228 218 D59N H76Q I96L L130I S164G L222I N249D G251D S273Y 75 23 323 219 L130I S164G L222I S273Y 46 38 121 220 K19T Y21S H76Q S164G L222I G251D S273Y 65 28 228 221 Y21S D59N H76Q L130I S132A S164G L222I G251D S273Y 65 31 213 222 D59N H76Q S226T R242E G251D S273Y 102 37 273

In table. 1 shows camel chymosin variants with data on specific clotting activity (C), non-specific proteolytic activity (P) and C/P ratio. Of the 43 variants, 17 showed a 10-50% increase in specific clotting activity compared to wild camel chymosin (CHY-MAX M). All variants have a significantly increased C/P ratio, with the best variant being 190, which shows an approximately 15-fold improvement over wild-type camel chymosin.

Mutation analysis of library 1 with multiple substitutions.

Statistical analysis of positional and mutational effects on specific clotting activity and C/P ratio was performed on the basis of proteolytic data for library 1. The most useful mutations for increasing specific clotting and C/P are shown in Table 1. 2 and 3, respectively.

Table 2. Mutational contribution (mean) to increase in specific clotting activity and standard deviations (sd) based on statistical analysis

mutation average sd R242E 1.98E-01 2.47E-02 L222I 1.09E-01 3.35E-02 D59N 6.06Е-02 3.12E-02 S273Y 6.06Е-02 3.47E-02 K19T 5.13E-02 2.65E-02 V309I 4.37E-02 2.92E-02 S132A 4.18E-02 2.46E-02 N249D 3.85E-02 2.54Е-02 I96L 3.38E-02 2.59E-02

Based on the results given in table. 2, it was concluded that mutations K19T, D59N, I96L, S132A, L222I, R242E, N249D, S273Y, and V309I increase the specific clotting activity of chymosin. Therefore, these mutations can be expected to reduce the dose of chymosin in cheese production.

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Table 3. Mutational contribution (mean) to increase in C/P ratio and standard deviations (sd) based on statistical analysis

mutation average sd R242E 2.12E-01 2.82E-02 I96L 1.20E-01 2.81E-02 H76Q 9.10Е-02 2.16E-02 S164G 8.59Е-02 2.19E-02 S273Y 7.77E-02 2.01Е-02 G251D 3.59Е-02 1.99E-02

Based on the results given in table. 3, it was concluded that the H76Q, I96L, S164G, R242E, G251D, and S273Y mutations increase the C/P ratio of chymosin. Therefore, these mutations would be expected to result in increased yield in cheese production using the respective chymosin variants.

Library 2 with multiple replacements.

Another set of camel chymosin variants, each with multiple substitutions compared to the wild type, was generated and analyzed as described above. All variants have an amino acid sequence identical to camel chymosin (SEQ ID NO: 2), except for the variations shown in the table. Camel chymosin (CHY-MAX M) is included as a reference.

Clotting activities were determined using the REMCAT method.

Table 4. Enzymatic activity of camel chymosin variants 223-269. Values are given as % cleavage of wild muna camel chymosin (CHY-MAX M)

option CHY-MAX M mutations Fold (C) 100 Proteol (P) 1 100 s/r 100 223 K19T D59N I96L S164G L222I G251D 89 37 242 224 Ylll K19T D59N 196V L222I R242D G251D 82 31 262 225 K19S D59N 196V S164G G251D 72 40 182

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226 K19S I96L S164G L166V L222I R242E 91 38 242 227 K19T D59N I96L S164G L166V L222I R242D G251D L253I 92 24 378 228 D59N I96L S164G L222I R242E L253I I263L 108 23 467 229 K19T D59N E83T I96L L222I G251D I263L 99 106 93 230 Y11I K19T D59N S164G L222I G251D I263V 54 16 343 231 K19T D59N I96L S164G L166I G251D L253V 63 thirty 206 232 K19T I96V S164G L222I N249D G251D L253I 56 29 193 233 K19T I96L L222I R242E L253I 125 57 220 234 K19T E83S I96L S164G L222I R242E G251D L253I 83 35 235 235 D59N E83T I96L S164N L222V G251D 42 53 80 236 K19S D59N I96L S164G L222I R242E N249E G251D 130 28 459 237 K19T I96L S164G L166V L222I N249D I263L 65 thirty 217 238 D59N I96L L166V L222I R242E G251D 178 51 347 239 K19T D59N E83T S164G L166V L222I R242D G251D 101 43 235 240 Y11I K19T D59N E83S I96L S164G L222I N249D 53 60 87 241 K19T E83T I96L S164G L222I R242E L253V 97 37 261 242 K19T D59N I96L S164G L166I L222I R242E N249D 129 21 623 243 Y11V K19T D59N I96L S164G L166V L222I R242E G251D L253I 130 17 759 244 K19T I96L S164N L222I R242E I263L 51 22 236 245 Y11V D59N I96L S164G L222I G251D L253V 63 24 265 246 K19T D59N I96V S164G L166V L222I R242E I263L 98 28 347 247 Y11V K19T D59N I96L S164N L166I L222I G251D 32 16 202 248 K19T I96L S164G L166V L222I R242E N249D G251D I263V 105 19 566 249 K19T I96L S164G R242E L253I 73 14 516 250 K19S D59N E83S I96L S164N L222I G251D 47 64 74 251 K19T D59N I96L S164G L222V N249E G251D I263V 79 27 293 252 K19T D59N I96L S164G L222I N249E G251D L253V I263L 69 21 332 253 Y11I K19T I96L S164G L222V R242E G251D 58 2 3265 254 I96L S164G L222I R242E N249D G251D I263L 82 14 601 255 K19T D59N I96L S164G L166I L222I R242D G251D I263V 108 25 427 256 K19T D59N I96L S164G L222V R242E N249D L253I 111 19 574 257 H76Q I96L S164G L222I R242E G251D S273Y 128 8 1597 258 K19T E83S I96L S164G L222I R242E N249D G251D L253I 95 thirty 315 259 I96L S164G L166V L222I R242E N249D I263L 104 26 405 260 Y11V K19T E83S I96L S164G L166V L222I R242E G251D 97 14 676 261 Y11V K19T I96L S164G L166V L222I R242E 94 19 491 262 Y11V E83S I96L S164G L222I R242E G251D L253I I263L 61 18 332 263 Y11V I96L S164G L222I R242E N249D L253I I263L 67 7 961 264 K19T I96L S164G L166V L222I R242E N249D I263L 75 50 149 265 Y11V E83S I96L S164G L222I R242E L253I I263L 62 28 222 266 K19T E83S I96L S164G L166V L222I R242E N249D G251D L253I 97 32 302 267 I96L S164G L222I R242E G251D S274Y 110 19 569 268 H76Q I96L S164G L222I R242E G251D 102 10 1054 269 I96L S164G L222I R242E G251D 101 22 465

In table. 4 shows camel chymosin variants with data on specific clotting activity (C), non-specific proteolytic activity (P), and C/P ratio. Of the 47 variants, 8 were identified with increased specific clotting activity from 10 to 78% compared to wild-type camel chymosin (CHY-MAX M). While 43 variants have significantly increased C/P ratios, the best variant is 253, which shows an approximately 33-fold improvement over wild-type camel chymosin.

Mutation Analysis of Library 2 with Multiple Substitutions Statistical analyzes of positional and mutational effects on specific clotting activity and C/P ratio were performed based on the proteolytic data for library 2. The most beneficial mutations for increasing specific clotting and C/P are shown in Table 1. 5 and 6, respectively.

Table 5. Mutational contribution (mean) to increase in specific clotting activity and standard deviations (sd) based on statistical analysis

mutation average sd R242E 4.00Е-01 3.19E-02 D59N 2.94Е-01 2.26E-02 N249E 1.47E-01 3.22E-02 L166V 1.27E-01 2.70E-02 S273Y 1.23E-01 2.94Е-02 L222I 1.07Е-01 3.53E-02 H76Q 5.93E-02 2.94Е-02 N249D 4.26E-02 2.38E-02

Based on the results given in table. 5, it was concluded that mutations D59N, H76Q,

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L166V, L222I, R242E, N249D, N249E and S273Y increase the specific coagulation activity of chymosin. Therefore, these mutations can be expected to reduce the dose of chymosin in cheese production.

Table 6. Mutational contribution (mean) to increase in C/P ratio and standard deviations (sd) based on statistical analysis

mutation average sd R242E 4.13E-01 2.20E-02 H76Q 2.50E-01 3.24Е-02 Y11I 2.49E-01 6.43E-02 S164G 2.27E-01 2.07Е-02 G251D 2.10E-01 2.65E-02 R242D 1.85E-01 2.69E-02 L222V 1.75E-01 4.53Е-02 Y11V 1.75E-01 2.83E-02 S273Y 8.29E-02 3.35E-02 L166I 7.64Е-02 2.91E-02 I96L 3.85E-02 2.59E-02 K19T 3.85E-02 2.43E-02

Based on the results given in table. 6, it was concluded that mutations Y11I, Y11V, K19T, H76Q, I96L, S164G, L166I, L222V, R242D, R242E, G251D and S273Y increase the C/P ratio of chymosin. Therefore, these mutations would be expected to result in increased yield in cheese production using the respective chymosin variants.

Structural changes in camel chymosin.

Camelid chymosin variants (SEQ ID NO: 2) were generated by substitution of amino acids at positions determined by protein structural analysis (Table 10). Mutations N100Q and N291Q were introduced at both the N-glycosylation sites of these variants and the reference camel chymosin (CamUGly) to obtain non-glycosylated, homogeneous protein samples.

Clotting activities were determined using the μIMCU method.

Table 7. Enzymatic activity of camel chymosin variants 271-308. Values are given as % cleavage of non-glycosylated camel chymosin (CamUGly) CamBov

option CamUGly mutations Fold (C) 100 Proteol (P) 100 C/R 100 N100Q. N291Q. 271 V221K N100Q. N291Q. 47 61 77 272 D290E N100Q. N291Q. 92 100 92 273 V136I N100Q. N291Q. 80 90 89 274 E240Q. N100Q. N291Q. 84 144 58 276 G289S N100Q. N291Q. 93 107 86 TP N292H N100Q. N291Q. 95 93 100 278 L295K N100Q. N291Q. 102 70 146 279 V136E N100Q. N291Q. 102 102 100 280 D290L N100Q. N291Q. 44 198 22 281 F119Y N100Q. N291Q. 8 45 18 282 Q.280E N100Q. N291Q. 79 72 110 283 F282E N100Q. N291Q. 93 80 116 284 N249D N100Q. N291Q. 118 84 140 285 R254S N100Q. N291Q. 47 94 50 286 R242E N1OOQ. N291Q. 114 67 170 288 V203R N100Q. N291Q. 99 113 88 289 N249R N100Q. N291Q. 76 108 70 290 H56K N100Q. N291Q. 99 133 74 291 S74D N100Q. N291Q. 94 87 108 292 A131D N100Q. N291Q. 17 39 44 293 Y190A N100Q. N291Q. 3 33 9 294 I297A N100Q. N291Q. 26 37 70 302 Y21S N100Q. N291Q. 97 87 111

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303 L130I N100Q. N291Q 77 82 95 306 G251D N100Q. N291Q. 100 81 123 307 Y243E N100Q. N291Q. 86 58 149 308 S273D N100Q N291Q 102 98 104

Based on the results given in table. 7, it was concluded that mutations Y21S, S74D, R242E, Y243E, N249D, G251D, S273D, Q280E, F282E and L295K increase the C/P ratio of chymosin. Mutations in R242E and N249D also result in increased specific clotting activity. Seven of the ten options with increased C/P ratios shown in Table. 7 carry mutations (R242E, N249D, G251D, Y243E, S273D, Q280E, F282E) in a distinct region on the protein surface that is located near the binding cleft as seen in FIG. 2. This region is thought to support binding of the κ-casein substrate through the interaction of the positively charged Arg96 sequence with His102 (refs 5, 16-18) at positions P10-P4 (ref 10). Negative charges introduced with mutations can enhance these interactions, resulting in increased specificity for casein (C/P). The results show that single amino acid substitutions in this region can significantly increase C/P.

Negative charge combinations in camel chymosin.

Additional variants of camel chymosin (SEQ ID NO: 2) have been made with combinations of mutations that introduce negative charges into the surface region described above (R242E, Y243E, G251D, N252D, R254E, S273D, Q280E). Mutations N100Q and N291Q were introduced into both the N-glycosylation sites of these variants and the camel chymosin reference (CamUGly) to obtain non-glycosylated homogeneous protein samples (Table 8).

Binding activities were determined using the μIMCU method.

Table 8. Enzymatic activity of camel chymosin variants 309-323. Values are given as % cleavage of non-glycosylated camel chymosin (CamUGly)

CamUGly variant mutations N100Q N291Q Fold (C) 100 | Proteol. (P) | | C/R 100 100 309 R242E Q.280E N100Q N291Q 133 59 225 310 R242E N252D N100Q N291Q 136 63 216 311 N252D Q.280E N100Q N291Q 108 96 112 312 Y243E Q.280E N100Q N291Q 112 71 158 313 Y243E N252D N100Q N291Q 91 77 117 314 R254E Q.280E N100Q N291Q 106 84 127 315 S273D Q.280E N100Q N291Q 77 51 150 316 R242E G251D N100Q N291Q 107 72 148 317 R242E G251D Q.280E N100Q N291Q 138 84 164 318 R242E S273D Q.280E N100Q N291Q 136 98 139 319 N252D S273D Q.280E N100Q N291Q 115 67 171 320 G251D S273D Q.280E N100Q N291Q 114 64 176 321 R242E R254E Q280E N100Q N291Q 134 66 202 322 R242E R254E S273D Q.280E N100Q N291Q 126 60 211 323 Y243E R254E S273D N100Q N291Q 103 71 144

All options presented in Table. 8 show an increase in the C/P ratio compared to non-glycosylated camel chymosin. Some of these variants (309, 310, 321, 322, 323) have even higher C/P than the best single negative charge mutation variant (286). It was concluded that the effect of increasing C/P caused by the introduction of negative charges into the P10-P4 interaction region in the chymosin structure can be further enhanced by combinations of the corresponding mutations.

Structural variations in bovine chymosin.

Bovine chymosin variants (SEQ ID NO: 1) were generated by amino acid substitutions at positions determined by protein structural analysis (Table 9). Mutations N252Q and N291Q were introduced at both N-glycosylation sites of these variants and reference bovine chymosin (BovUGly) to obtain non-glycosylated, homogeneous protein samples.

Clotting activities were determined using the μIMCU method.

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Table 9. Enzymatic activity of bovine chymosin variants 325-346. Values are given as % degradation of non-glycosylated bovine chymosin (BovUGly)

option BovUGly N252Q mutations N291Q Curl. (WITH) 100 Proteol (P) 100 C/R 100 325 V223F N252Q N291Q 54 130 41 327 A117S N252Q N291Q 75 76 96 329 Q242R N252Q N291Q 76 166 45 330 Q278K N252Q N291Q 94 112 83 332 H292N N252Q N291Q 96 71 133 333 Q294E N252Q N291Q 99 79 123 334 K295L N252Q N291Q 106 182 58 335 D249N N252Q N291Q 89 129 68 337 G244D N252Q N291Q 100 106 93 338 Q56H N252Q N291Q 110 140 77 339 L32I N252Q N291Q 86 124 69 340 K71E N252Q N291Q 44 50 86 341 R72T N252Q N291Q 103 172 59 342 Q83T N252Q N291Q 92 103 88 343 V113F N252Q N291Q 42 44 95 344 E133S N252Q N291Q 93 199 46 345 Y134G N252Q N291Q 106 115 91 346 K71A N252Q N291Q 79 131 60

The data in the table. 9 demonstrate that variations in Q56H, Y134G and K295L result in increased specific clotting activity and variations in H292N and Q294E result in increased C/P ratios. Both H292 and Q294 are located in a loop partially covering the substrate-binding cleft (FIG. 3), which explains the observed effect of the respective mutations at these positions on casein substrate specificity (C/P). In particular, while the H292N substitutions increased C/P and D249N, and K295L decreased the C/P of bovine chymosin, reverse effects on C/P were observed for the corresponding N292H, N249D, and L295K backmutations in camel chymosin (Table 7). This demonstrates that these amino acid changes have a similar effect on chymosin specificity across species.

N-terminal variations of camel chymosin.

Camelid chymosin variants (SEQ ID NO: 2) were generated by substitution of amino acids at positions determined by protein structural analysis of molecular interactions of the N-terminal sequence Y11-D13 in the substrate-binding cleft (Table 10). Mutations N100Q and N291Q were introduced at both N-glycosylation sites of these variants and reference camel chymosin (CamUGly) to obtain non-glycosylated, homogeneous protein samples.

Clotting activities were determined using the pIMCU method.

Table 10. Enzymatic activity of camel chymosin variants 347-366. Values are given as % cleavage of non-glycosylated camel chymosin (CamUGly)

option CamUGly mutations Curl. (WITH) 100 Proteol (P) 100 C/R 100 N100Q N291Q 347 Y11H N100Q N291Q 76 131 58 348 Code of Criminal Procedure N100Q N291Q 63 82 76 349 Y11R N100Q N291Q 55 277 20 350 Y11H D290E N100Q N291Q 74 105 71 351 Y11R D290E N100Q N291Q 62 101 62 352 Y11F N100Q N291Q 91 146 62 353 Y11I N100Q N291Q 96 83 116 354 Y11L N100Q N291Q 79 108 74 355 Y11V N100Q N291Q 101 64 157 356 L12F N100Q N291Q 96 147 66 357 L12I N100Q N291Q 83 91 91 359 D13N N100Q N291Q 88 131 67 360 D13Q N100Q N291Q 100 169 59 361 D13S N100Q N291Q 88 164 54 362 D13T N100Q N291Q 62 89 70 363 D13F N100Q N291Q 73 155 48 364 D13L N100Q N291Q 82 196 42 365 D13V N100Q N291Q 49 86 57 366 D13Y N100Q N291Q 74 99 75

Structural analysis of camel chymosin accompanied variations in the N-terminal sequence of the Y11-D13 21040259 strand, as well as at position D290, a potential Y11 interaction partner (Fig. 4). Because casein substrates compete with the N-terminal chymosin sequence for binding in the binding cleft, amino acid substitutions that alter the interactions between the binding cleft and the Y11-D13 motif are expected to affect enzymatic activity against various casein substrates and thus C/R ratio. The results of the respective variants 347-366 show a strong change in both specific clotting activity and C/P. In particular, options 353 and 355 exhibit increased C/P ratios. Therefore, it was concluded that the Y11I and Y11V amino acid substitutions lead to an increased C/P ratio. Because the chymosin binding cleft consists primarily of hydrophobic amino acids (ref. 9), both mutations can enhance N-terminal binding in the binding cleft through improved hydrophobic interactions and thus inhibit non-specific binding and hydrolysis of casein (P).

Library of 3 multiple substitutions.

Another set of camel chymosin variants, each with multiple substitutions compared to the wild type, was generated and analyzed as described above. All variants have an amino acid sequence identical to camel chymosin (SEQ ID NO: 2), except for the variations shown in the table. Camel chymosin (CHY-MAX M) is included as a reference.

Clotting activities were determined using the μIMCU method.

Table 11. Enzymatic activity of camel chymosin variants 367-416. Values are given as % cleavage of wild muna camel chymosin (CHY-MAX M)

option CHY-MAX M mutations Fold.(C) I 100 Proteol (P) I 100 C/R 100 367 R67Q N100Q L130I M157L V248I N291Q 46 64 72 368 N100Q L130I S132A M157L K231N 87 104 83 369 R67Q I96L L130I M157L L222I M256L 49 56 88 370 R67Q L130I S132A M157L R242E V248I 23 32 70 371 R67Q N100Q M157L R242E M256L 100 62 162 372 R67Q G70D M157L R242E V248I 89 32 276 373 V32L R67Q M157L L222I R242E 64 63 102 374 Y11V R67Q. M157L V248I M256L 71 45 158 375 R67Q V136I M157L L222I V248I 88 20 449 376 L130I M157L V248I M256L N291Q 90 80 112 377 R67Q I96L L130I M157L K231N R242E 124 37 339 378 V32L R67Q L130I M157L L222I K231N 52 103 51 379 L130I V136I M157L L222I N292H 55 47 118 380 R67Q G70D M157L L222I N291Q 117 34 339 381 V32L R67Q L130I K231N N292H 58 66 87 382 Y11V R67Q N100Q L130I V136I M157L 60 55 109 383 R67Q L130I L222I R242E M256L 78 27 290 384 R67Q M157L L222I V248I N292H 83 97 86 385 V32L R67Q M157L M256L N291Q 85 143 60 386 R67Q L130I S132A M157L L222I N292H 78 133 58 387 R67Q N100Q L130I M157L K231N N291Q 59 70 84 388 R67Q L130I K231N V248I N291Q 91 87 105 389 Y11V R67Q L130I M157L L222I K231N 63 47 134 390 I45V L130I M157L K231N R242E 108 43 253 391 V32L R67Q V136I M157L N291Q 104 84 124 392 R67Q N100Q L130I D158S V248I 70 67 105 393 I45V R67Q L130I M157L L222I K231N 79 54 147 394 V32L R67Q L130I S132A M157L V248I 74 130 57 395 Y11V R67Q L130I M157L N291Q N292H 74 83 90 396 R67Q N100Q L130I M157L L222I K231N 60 81 74 397 I45V R67Q G70D L130I S132A 68 75 90 398 I45V R67Q L130I V248I N292H 53 81 66 399 Y11V R67Q L130I M157L L222I R242E 106 28 373 400 R67Q N100Q D158S L130I M157L L222I 57 58 98 401 R67Q L130I V136I M157L K231N V248I 71 79 89 402 I45V R67Q L130I L222I N291Q 91 89 103 403 R67Q G70D L130I M157L K231N M256L 89 53 167 404 V32L R67Q L130I M157L D158S V248I 58 82 71 405 R67Q L130I M157L D158S R242E N291Q 92 16 556 406 R67Q L130I M157L D158S K231N N292H 53 74 72 407 R67Q L130I V248I M256L N292H 58 107 55 408 V32L R67Q I96L L130I M157L V248I 35 76 46 409 R67Q I96L N100Q L130I M157L N292H 96 36 263 410 V32L R67Q G70D N100Q M157L 68 66 104 411 V32L R67Q L130I M157L K231N M256L 102 18 574 412 R67Q I96L M157L L222I K231N 120 55 220

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413 R67Q M157L L222I K231N V248I 124 46 268 414 R67Q L130I M157L R242E M256L N292H 115 59 196 415 R67Q L222I K231N V248I 82 67 123 416 R67Q S132A L222I K231N R242E V248I 129 42 306

In table. 11 shows camel chymosin variants with data on specific clotting activity (C), non-specific proteolytic activity (P) and C/P ratio. Of the 50 variants, 6 showed a 10-29% increased specific clotting activity compared to wild-type camel chymosin (CHY-MAX M). While 23 variants have more than 10% increased C/P ratio, the best variant is 411, which shows an approximately 6-fold improvement over wild-type camel chymosin (CHY-MAX M). Mutational analysis of a library of 3 multiple substitutions.

Statistical analysis of positional and mutational effects on clotting activity (C) and C/P ratio was performed based on library 3 proteolytic data. The most beneficial mutations for increasing clotting and C/P are shown in Table 1. 12 and 13 respectively.

Table 12. Mutational contribution (mean) to increased clotting activity and standard deviations (sd) based on statistical analysis

mutation average sd R242E 4.63E-01 4.21E-02 I96L 2.31Е-01 4.82E-02 N291Q 1.67Е-01 3.97E-02 K231N 1.34Е-01 3.52E-02 M256L 1.28E-01 4D4E-02 S132A 1.04Е-01 3.35E-02 M157L 7.99E-02 3.49E-02

Based on the results given in table. 12, it was concluded that mutations I96L, S132A, M157L, K231N, R242E, M256L and N291Q increase the specific clotting activity of chymosin. Therefore, these mutations can be expected to reduce the dose of chymosin in cheese production.

Table 13. Mutational Contribution (Mean) to Increase in C/P Ratio and Standard Deviations (sd) Based on Statistical Analysis

mutation average sd R242E 6.66E-01 4.23 E-02 G70D 3.32E-01 5.72E-02 Y11V 2.06Е-01 3.61E-02 K231N 1D5E-01 2.92E-02 L222I 1.09E-01 3.71E-02 V136I 1.02E-01 4.53Е-02 I96L 9.84Е-02 6.02E-02 N291Q. 4.78E-02 4.20E-02

Based on the results given in table. 13, it was concluded that the Y11V, G70D, I96L, V136I, L222I, K231N, R242E, and N291Q mutations increase the C/P ratio of chymosin. Therefore, these mutations would be expected to result in increased yield in cheese production using the respective chymosin variants.

Library of 4 multiple substitutions.

Another set of camel chymosin variants, each with multiple substitutions compared to the wild type, was generated and analyzed as described above. All variants have an amino acid sequence identical to camel chymosin (SEQ ID NO: 2), except for the variations shown in the table. Camel chymosin (CHY-MAX M) is included as a reference.

Clotting activities were determined using the REMCAT method.

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Table 14. Enzymatic activity of camel chymosin variants 417-461. Values are given as % cleavage of wild muna camel chymosin (CHY-MAX M)

option CHY-MAX M mutations Fold (C) 100 Proteol (P) 1 100 C/R 100 417 Y11V K19T D59N S164G L166V L222I R242E N249E G251D 132 20 651 418 Y11V K19T D59N I96L S164G L166I L222I R242E N249E G251D 114 21 556 419 Y11I K19T D59N I96L S164G L166V L222I R242E N249E G251D 108 20 554 420 Y11I K19T D59N I96L S164G L166I L222I R242E G251D 98 eleven 898 421 Y11V K19T D59N I96L L166V L222V R242E N249E G251D L253I 132 84 156 422 Y11V K19T D59N I96L S164G L166V R242E 105 13 802 423 Y11V K19T D59N I96L S164G L222V R242E G251D 89 8 1131 424 Y11V K19T D59N I96L S164G L166I R242E N249E G251D L253I 93 8 1111 425 Y11V K19T D59N I96L S164G L166V L222V R242E N249E G251D 105 18 572 426 Y11V K19T D59N I96L S164G L166I L222V R242E N249E G251D L253I 93 18 512 427 Y11V K19T D59N L166V L222I R242E N249E G251D L253I 137 42 323 428 Y11V K19T D59N I96L S164G L166V L222I R242E N249E 120 15 803 429 Y11V K19T D59N S164G L166I L222I R242E G251D 107 17 630 430 Y11V K19T D59N I96L S164G R242E G251D 89 eleven 801 431 Y11V D59N I96L S164G L166I L222V R242E G251D L253I 79 28 283 432 Y11V D59N I96L S164G L166I L222I R242E G251D 102 24 432 433 Y11I D59N I96L S164G L166V L222V R242E G251D L253I 97 25 392 434 Y11V K19T D59N I96L S164G L222I R242E N249E G251D 99 33 301 435 Y11V K19T D59N I96L S164G L166I L222V R242E G251D 88 17 514 436 Y11V K19T D59N I96L S164G L166V L222V R242E N249E L253I 95 10 949 437 Y11V K19T D59N I96L S164G L166I L222V R242E N249E G251D 114 22 520 438 Y11I K19T I96L S164G L166V R242E N249E G251D 93 7 1262 439 Y11V K19T D59N I96L S164G L166V L222V R242E G251D 108 26 423 440 Y11V K19T D59N I96L S164G L222V R242E N249E G251D 105 9 1196 441 Y11I K19T L222V R242E N249E G251D 122 26 469 442 Y11V K19T I96L L222V R242E N249E G251D 105 21 503 443 Y11I K19T D59N I96L S164G L166V L222V R242E N249E G251D 105 18 595 444 Y11V K19T I96L S164G L166V L222V R242E N249E G251D 96 8 1242 445 Y11I K19T D59N I96L S164G L166I L222V R242E N249E G251D 82 12 707 446 Y11I I96L S164G L166V L222V R242E N249E G251D 95 16 579 447 Y11I K19T D59N I96L S164G L222V R242E N249E 90 eleven 790 448 Y11I K19T D59N I96L L222V R242E N249E G251D 153 40 381 449 Y11I K19T D59N I96L S164G L222I R242E 89 16 564 450 Y11I K19T D59N I96L S164G L166V R242E G251D 88 5 1686 451 Y11I K19T D59N S164G L166I L222V R242E G251D 93 21 440 452 Y11I I96L L222V R242E N249E G251D 122 22 566 453 Y11I I96L S164G L222I R242E 74 5 1375 454 Y11V K19T I96L L166V L222V R242E G251D 119 52 228 455 Y11I D59N I96L S164G L222I R242E G251D 105 9 1139 456 Y11I D59N I96L S164G L222V R242E N249E G251D 95 15 615 457 Y11I K19T D59N I96L S164G L222I R242E N249E G251D 101 7 1419 458 Y11I D59N I96L S164G L166V L222V R242E G251D 89 16 572 459 Y11V K19T D59N I96L L222V R242E G251D 143 62 230 460 Y11I K19T S164G L166I L222V R242E N249E G251D 80 13 625 461 Y11I D59N I96L S164G L166V L222V R242E N249E G251D 96 35 273

In table. 14 shows camel chymosin variants with data on specific clotting activity (C), non-specific proteolytic activity (P) and C/P ratio. Of the 45 variants, 11 showed a 14-53% increased specific clotting activity compared to wild-type camel chymosin (CHY-MAX M). While all 45 variants have an increased C/P ratio of over 10%, the best variant is 450, which shows an approximately 17-fold improvement over wild-type camel chymosin (CHY-MAX M).

Mutational library analysis of 4 multiple substitutions.

Statistical analysis of positional and mutational influences on clotting activity (C) and C/P ratio was performed based on library 4 proteolytic data. 15 and 16 respectively.

Table 15. Mutational contribution (mean) to increased clotting activity and standard deviations (sd) based on statistical

analysis mutation average sd D59N 3.99E-01 3.48E-02 L222I 2.05Е-01 2.64Е-02 L166V 1.92E-01 2.39E-02 N249E 1.45Е-01 1.88Е-02 G251D 9.79Е-02 2.29E-02

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Based on the results given in table. 15 it was concluded that mutations Y11V, D59N,

L166V, L222I, R242E, N249E and G251D increase the specific coagulation activity of chymosin.

Therefore, these mutations can be expected to reduce the dose of chymosin in cheese production.

Y11V 8.54Е-02 1.56E-02 R242E 5D4E-02 2D6E-02

Table 16. Mutational contribution (mean) to increase in C/P ratio and standard deviations (sd) based on statistical analysis

mutation average sd S164G 7.51Е-01 4.50E-02 K19T 2.85Е-01 4.93E-02 I96L 2.43Е-01 4D6E-02 R242E 2.25Е-01 7D2E-02 L253I 2.22E-01 4.61E-02 Y111 1,ZOE-01 4.93E-02 N249E 9.52Е-02 3.86E-02 Y11V 9.49Е-02 3.55Е-02

Based on the results shown in Table 16, it was concluded that the Y11I, Y11V, Y9T, I96L, S164G, R242E, N249E, and L253I mutations increase the C/P ratio of chymosin. Therefore, these mutations would be expected to result in increased yield in cheese production using the respective chymosin variants.

Selected variants of the library of 4 multiple substitutions were fermented again in 70 L, followed by purification and characterization of their proteolytic profile (Table 17).

Table 17. Enzymatic activity of selected variants of camel chymosin from 70 L of fermentation. Values are given as % cleavage of wild muna camel chymosin(CHY-MAX M)

option CHY-MAX M mutations Fold(S) 1 100 Proteol. (P) 1 100 C/R 100 433 Ylll D59N I96L S164G L166V L222V R242E G251D L253I 151 eleven 1356 436 Y11V K19T D59N I96L S164G L166V L222V R242E N249E L253I 188 9 2007 453 Ylll I96L S164G L222I R242E 153 8 1893 457 Ylll K19T D59N I96L S164G L222I R242E N249E G251D 217 7 3002

In table. 17 shows camel chymosin variants from a 70 L fermentation with data on specific clotting activity (C), non-specific proteolytic activity (P) and C/P ratio. All 4 variants show a 51%-117% increased specific clotting activity compared to wild-type camel chymosin (CHY-MAX M). While all 4 variants have over a 13-fold increase in C/P ratio, the best variant is 457, which shows an approximately 30-fold improvement over wild-type camel chymosin (CHY-MAX M).

- 25 040259

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Claims (15)

CLAIM 1. An isolated variant of the chymosin polypeptide, characterized in that: (a) this isolated variant chymosin polypeptide has a specific clotting activity (IMCU/mg total protein) that is at least 110% of the specific clotting activity of its parent polypeptide and/or (b) this isolated variant chymosin polypeptide has a C/P ratio (specific clotting activity/proteolytic activity) that is at least 200% of the C/P ratio of its parent polypeptide, wherein the isolated variant chymosin polypeptide contains a substitution at one or more (several) of the following positions as indicated in relation to the amino acid sequence SEQ ID NO: 2: N249, R242. 2. An isolated variant chymosin polypeptide according to claim 1, wherein the parent polypeptide has at least 80%, such as, for example, at least 82%, 85%, 95%, 97%, 98%, 99% or 100% sequence identity with the polypeptide of SEQ ID NO : 2 (camel chymosin). 3. An isolated chymosin polypeptide variant according to claim 1 or 2, wherein the substitutions are one or more of the substitutions indicated in relation to the amino acid sequence of SEQ ID NO: 2: R242E, R242D, N249D, N249E. 4. An isolated variant of the chymosin polypeptide according to claim 3, having a specific coagulation activity - 26 040259 a density (IMCU/mg of clotting protein) that is at least 110% of the specific clotting activity of the parent peptide and contains one or more of the following combinations of substitutions, and where each substitution is indicated in relation to the amino acid sequence of SEQ ID NO: 2: Y11V, K19T, D59N, I96L, S164G, L166V, L222V, R242E, N249E, L253I; Y11I, D59N, I96L, S164G, L166V, L222V, R242E, G251D, L253I; Y11I, I96L, S164G, L222I, R242E; Y11I, K19T, D59N, I96L, S164G, L222I, R242E, N249E, G251D; H76Q, I96L, S164G, L222I, R242E, G251D, S273Y; K19T, D59N, H76Q, S164G, L222I, N249D, S273Y; K19T, D59N, H76Q, L166V, L222I, R242E, G251D, S273Y; Y21S, H76Q, S164G, L222I, R242E, G251D, S273Y; D59N, S132A, S164G, L222I, R242E, N249D, G251D, S273Y; H76Q, S164G, L166V, L222I, R242E, G251D, S273Y; Y21S, D59N, H76Q, I96L, S164G, L222I, N249D, G251D, S273Y; K19T, D59N, H76Q, S164G, R242E, N249D, G251D, S273Y; D59N, H76Q, S164G, L222I, S226T, R242E; H76Q, S164G, L222I, N249D, G251D, S273Y, V309I; D59N, H76Q, S164G, L166V, L222I, N249D, G251D, S273Y, V309I; D59N, I96L, L166V, L222I, R242E, G251D; K19S, D59N, I96L, S164G, L222I, R242E, N249E, G251D; Y1IV, K19T, D59N, I96L, S164G, L166V, L222I, R242E, G251D, L253I; K19T, D59N, I96L, S164G, L166I, L222I, R242E, N249D; K19T, I96L, L222I, R242E, L253I; K19T, D59N, I96L, S164G, L222V, R242E, N249D, L253I; I96L, S164G, L222I, R242E, G251D, S274Y; N249D, N100Q, N291Q; R242E, N100Q, N291Q; R242E, G251D, Q280E, N100Q, N291Q; R242E, N252D, N100Q, N291Q; R242E, S273D, Q280E, N100Q, N291Q; R242E, R254E, Q280E, N100Q, N291Q; R242E, Q280E, N100Q, N291Q; R242E, R254E, S273D, Q280E, N100Q, N291Q; R67Q, S132A, L222I, K231N, R242E, V248I; R67Q, I96L, L130I, M157L, K231N, R242E ; or R67Q, L130I, M157L, R242E, M256L, N292H. 5. An isolated variant of a chymosin polypeptide according to any one of claims 1 to 4, having a C/P ratio of at least 200% of the C/P ratio of its parent polypeptide, and containing one or more combinations of the following substitutions, and where each substitution is indicated by in relation to the amino acid sequence of SEQ ID NO: 2: Y1II, D59N, I96L, S164G, L166V, L222V, R242E, G251D, L253I; - 27 040259 Y11V, K19T, D59N, I96L, S164G, L166V, L222V, R242E, N249E, L253I; Y11I, I96L, S164G, L222I, R242E; Y11I, K19T, D59N, I96L, S164G, L222I, R242E, N249E, G251D; H76Q, I96L, S164G, L222I, R242E, G251D, S273Y; Y21S, H76Q, S164G, L222I, R242E, G251D, S273Y; H76Q, S164G, L166V, L222I, R242E, G251D, S273Y; K19T, D59N, H76Q, S164G, R242E, N249D, G251D, S273Y; Y21S, D59N, H76Q, I96L, S164G, L222I, N249D, G251D, S273Y; D59N, H76Q, I96L, L130I, S164G, L222I, R242E, G251D; D59N, H76Q, I96L, S164G, L222I, S226T, N249D, G251D, S273Y; K19T, D59N, H76Q, L166V, L222I, R242E, G251D, S273Y; D59N, H76Q, S164G, L222I, R242E, S273Y, V309I; D59N, H76Q, L130I, S132A, S164G, L222I, R242E, G251D, S273Y; D59N, H76Q, S164G, L222I, S226T, R242E; D59N, S132A, S164G, L222I, R242E, N249D, G251D, S273Y; K19T, D59N, H76Q, S164G, L222I, N249D, S273Y; D59N, H76Q, I96L, L130I, S164G, L222I, N249D, G251D, S273Y; Y21S, D59N, S164G, L222I, R242E, G251D, S273Y, V309I; D59N, H76Q, S226T, R242E, G251D, S273Y; Y21S, D59N, H76Q, S164G, LI66V, N249D, G251D, S273Y; H76Q, I96L, S164G, L222I, R242E, G251D; Y11V, I96L, S164G, L222I, R242E, N249D, L253I, I263L; Y11V, K19T, D59N, I96L, S164G, L166V, L222I, R242E, G251D, L253I; Y11V, K19T, E83S, I96L, S164G, L166V, L222I, R242E, G251D; K19T, D59N, I96L, S164G, L166I, L222I, R242E, N249D; I96L, S164G, L222I, R242E, N249D, G251D, I263L; K19T, D59N, I96L, S164G, L222V, R242E, N249D, L253I; I96L, S164G, L222I, R242E, G251D, S274Y; K19T, I96L, S164G, L166V, L222I, R242E, N249D, G251D, I263V; K19T, I96L, S164G, R242E, L253I; D59N, I96L, S164G, L222I, R242E, L253I, I263L; I96L, S164G, L222I, R242E, G251D; - 28 040259 K19S, D59N, I96L, S164G, L222I, R242E, N249E, G251D; K19T, D59N, I96L, S164G, L166I, L222I, R242D, G251D, I263V; I96L, S164G, LI66V, L222I, R242E, N249D, I263L; K19T, D59N, I96L, S164G, L166V, L222I, R242D, G251D, L253I; D59N, I96L, L166V, L222I, R242E, G251D; K19T, D59N, I96V, S164G, L166V, L222I, R242E, I263L; K19T, D59N, I96L, S164G, L222I, N249E, G251D, L253V, I263L; Y11V, E83S, I96L, S164G, L222I, R242E, G251D, L253I, I263L; K19T, E83S, I96L, S164G, L222I, R242E, N249D, G251D, L253I; K19T, E83S, I96L, S164G, L166V, L222I, R242E, N249D, G251D, L253I; K19T, D59N, I96L, S164G, L222V, N249E, G251D, I263V; Y11I, K19T, D59N, I96V, L222I, R242D, G251D; K19T, E83T, I96L, S164G, L222I, R242E, L253V; K19S, I96L, S164G, L166V, L222I, R242E; K19T, I96L, S164N, L222I, R242E, I263L; K19T, D59N, E83T, S164G, L166V, L222I, R242D, G251D; K19T, E83S, I96L, S164G, L222I, R242E, G251D, L253I; Y11V, E83S, I96L, S164G, L222I, R242E, L253I, I263L; K19T, I96L, L222I, R242E, L253I; K19T, I96L, S164G, LI66V, L222I, N249D, I263L; R242E, Q280E, N100Q, N291Q; R242E, N252D, N100Q, N291Q; R242E, R254E, S273D, Q280E, N100Q, N291Q; R242E, R254E, Q280E, N100Q, N291Q; R67Q, L130I, M157L, D158S, R242E, N291Q; R67Q, V136I, M157L, L222I, V248I; Y11V, R67Q, L130I, M157L, L222I, R242E; R67Q, I96L, L130I, M157L, K231N, R242E; R67Q, S132A, L222I, K231N, R242E, V248I; R67Q, L130I, L222I, R242E, M256L; R67Q, G70D, M157L, R242E, V248I or I45V, L130I, M157L, K231N, R242E. 6. A method for producing an isolated chymosin polypeptide variant according to any one of claims 1 to 5, comprising the following steps: (a) making a modification at one or more positions of a DNA sequence encoding a polypeptide having at least 80% sequence identity with SEQ ID NO: 2, where the modification comprises a substitution at least one amino acid position; (b) isolating the modified polypeptide from step (a) to obtain a variant chymosin polypeptide from step (a). 7. The method of claim 6, wherein the parent polypeptide has at least 85%, 95%, 97%, 98%, or at least 99% sequence identity with the polypeptide of SEQ ID NO: 2 (camel chymosin). 8. A method for producing an isolated chymosin polypeptide variant according to claim 6 or 7, where: (a) the variant contains one or more of the following substitutions, indicated in relation to the amino acid sequence of SEQ ID NO: 2: N249, R242, such as, for example, N249E, N249D, R242E, R242D. 9. A method for producing an isolated chymosin polypeptide variant according to claim 7 or 8, where: (a) the variant contains one or more combinations of the following substitutions, and where each substitution is indicated in relation to the amino acid sequence of SEQ ID NO: 2: - 29 040259 Y1II, D59N, I96L, S164G, L166V, L222V, R242E, G251D, L253I; Y11V, K19T, D59N, I96L, S164G, L166V, L222V, R242E, N249E, L253I; Y11I, I96L, S164G, L222I, R242E; Y11I, K19T, D59N, I96L, S164G, L222I, R242E, N249E, G251D; H76Q, I96L, S164G, L222I, R242E, G251D, S273Y; K19T, D59N, H76Q, S164G, L222I, N249D, S273Y; K19T, D59N, H76Q, L166V, L222I, R242E, G251D, S273Y; Y21S, H76Q, S164G, L222I, R242E, G251D, S273Y; D59N, S132A, S164G, L222I, R242E, N249D, G251D, S273Y; H76Q, S164G, L166V, L222I, R242E, G251D, S273Y; K19T, D59N, H76Q, S164G, R242E, N249D, G251D, S273Y; Y21S, D59N, H76Q, I96L, S164G, L222I, N249D, G251D, S273Y; D59N, H76Q, S164G, L222I, S226T, R242E; H76Q, S164G, L222I, N249D, G251D, S273Y, V309I; D59N, I96L, L166V, L222I, R242E, G251D; Y11V, K19T, D59N, I96L, S164G, L166V, L222I, R242E, G251D, L253I; K19S, D59N, I96L, S164G, L222I, R242E, N249E, G251D; K19T, D59N, I96L, S164G, L166I, L222I, R242E, N249D; K19T, I96L, L222I, R242E, L253I; K19T, D59N, I96L, S164G, L222V, R242E, N249D, L253I; I96L, S164G, L222I, R242E, G251D, S274Y; R242E, N252D, N100Q, N291Q; R242E, R254E, Q280E, N100Q, N291Q; R242E, Q280E, N100Q, N291Q; R242E, R254E, S273D, Q280E, N100Q, N291Q; R67Q, S132A, L222I, K231N, R242E, V248I or R67Q, I96L, L130I, M157L, K231N, R242E. 10. A method of obtaining a food product, including adding an effective amount of the selected chymosin polypeptide variant according to any one of paragraphs. 1-5 to a food ingredient and carrying out further manufacturing steps to obtain a food product, where the food product is a milk-based product and the food ingredient is milk. 11. A method for producing a feed product, comprising adding an effective amount of an isolated chymosin polypeptide variant according to any one of claims 1 to 5 to a feed ingredient and carrying out further production steps to obtain a feed product, where the feed product is a milk-based product and the feed ingredient is milk. 12. A food product containing a variant chymosin polypeptide according to any one of claims 1-5. 13. A food product containing a chymosin polypeptide variant according to any one of claims 1 to 5. 14. Use of a chymosin polypeptide variant according to any one of claims 1 to 5 for cheese production. 15. The use of the chymosin polypeptide variant of claim 14 to produce draw cheese, cheddar, continental type cheeses, soft cheese or white pickled cheese.