(en)Variants of chymosin with improved milk-clotting properties.

1.ApplicationNumber: US-201515121286-A
1.PublishNumber: US-2017067041-A1
2.Date Publish: 20170309
3.Inventor: VAN DEN BRINK JOHANNES MAARTEN
JENSEN JESPER LANGHOLM
JACOBSEN JONAS
LUND MARTIN
JEPPESEN IBEN
JAECKEL CHRISTIAN

4.Inventor Harmonized: VAN DEN BRINK JOHANNES MAARTEN(DK)
JENSEN JESPER LANGHOLM(DK)
JACOBSEN JONAS(DK)
LUND MARTIN(DK)
JEPPESEN IBEN(DK)
JAECKEL CHRISTIAN(DK)

5.Country: US
6.Claims:
(en)Variants of chymosin with improved milk-clotting properties.

7.Description:
(en)FIELD OF THE INVENTION
The present invention relates to variants of chymosin with improved milk-clotting properties.
BACKGROUND ART
Enzymatic coagulation of milk by milk-clotting enzymes, such as chymosin and pepsin, is one of the most important processes in the manufacture of cheeses. Enzymatic milk coagulation is a two-phase process: a first phase where a proteolytic enzyme, chymosin or pepsin, attacks K-casein, resulting in a metastable state of the casein micelle structure and a second phase, where the milk subsequently coagulates and forms a coagulum.
Chymosin (EC 3.4.23.4) and pepsin (EC 3.4.23.1), the milk clotting enzymes of the mammalian stomach, are aspartic proteases belonging to a broad class of peptidases.
When produced in the gastric mucosal cells, chymosin and pepsin occur as enzymatically inactive pre-prochymosin and pre-pepsinogen, respectively. When chymosin is excreted, an N-terminal peptide fragment, the pre-fragment (signal peptide) is cleaved off to give prochymosin including a pro-fragment. Prochymosin is a substantially inactive form of the enzyme which, however, becomes activated under acidic conditions to the active chymosin by autocatalytic removal of the pro-fragment. This activation occurs in vivo in the gastric lumen under appropriate pH conditions or in vitro under acidic conditions.
The structural and functional characteristics of bovine, i.e. Bos taurus , pre-prochymosin, prochymosin and chymosin have been studied extensively. The pre-part of the bovine pre-prochymosin molecule comprises 16 aa residues and the pro-part of the corresponding prochymosin has a length of 42 aa residues. The active bovine chymosin comprises 323 aa is a mixture of two forms, A and B, both of which are active.
Chymosin is produced naturally in mammalian species such as bovines, camels, caprines, buffaloes, sheep, pigs, humans, monkeys and rats.
Bovine chymosin has for a number of years been commercially available to the dairy industry.
WO02/36752A2 (Chr. Hansen) describes recombinant production of camel chymosin.
WO2013/174840A1 (Chr. Hansen) describes mutants/variants of bovine and camel chymosin.
WO2013/164479A2 (DSM) describes mutants of bovine chymosin.
The references listed immediately below may in the present context be seen as references describing mutants of chymosin:
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 characterisation of chymosin pH optima mutants produced in Tricoderma 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; Supannee 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 mentioned above describe directly and unambiguously any of the chymosin mutants/variants as described/claimed below herein.
SUMMARY OF THE INVENTION
The problem to be solved by the present invention is to provide variants of chymosin with improved milk-clotting properties.
As discussed in working examples herein—the present inventors have identified a number of improved camel (see Example 6 herein) and bovine/camel (see Example 7 herein) chymosin variants.
Based on a comparative analysis of the camel and bovine variants—the present inventors identified a number of further amino acid positions that are herein important in the sense that by making a variant in one or more of these positions one may get an improved chymosin variant.
As known in the art—different natural wildtype chymosin polypeptide sequences obtained from different mammalian species (such as e.g. bovines, camels, sheep, pigs, or rats) are having a relatively high sequence similarity/identity.
In FIG. 1 herein this is exemplified by an alignment of herein relevant different chymosin sequences.
In view of this relatively close sequence relationship—it is believed that the 3D structures of different natural wildtype chymosins are also relatively similar.
In the present context—a natural obtained wildtype chymosin (such as bovine chymosin or camel chymosin) may herein be an example of a parent polypeptide—i.e. a parent polypeptide to which an alteration is made to produce a variant chymosin polypeptide of the present invention.
Without being limited to theory—it is believed that the herein discussed chymosin related amino acid positions are of general importance in any herein relevant chymosin enzyme of interest (e.g. chymosins of e.g. bovines, camels, sheep, pigs, rats etc)—in the sense that by making a variant in one or more of these positions one may get an improved chymosin variant in general (e.g. an improved bovine, camel, sheep, pig or rat chymosin variant).
As discussed herein—as a reference sequence for determining the amino acid position of a parent chymosin polypeptide of interest (e.g. camel, sheep, bovine etc) is herein used the public known bovine chymosin B preprochymosin sequence (Genbank accession number P00794—disclosed as SEQ ID NO: 1 herein).
The bovine chymosin B preprochymosin of SEQ ID NO: 1 may herein alternatively be termed Bovine ( Bos bovis ) chymosin B or simply bovine chymosin. The sequence is also shown in FIG. 1 herein.
Another herein relevant chymosin sequence is publically known Camelius dromedarius chymosin sequence of SEQ ID NO: 2 herein. It may herein alternatively be termed camel chymosin. The sequence is also shown in FIG. 1 herein.
In the present context it is believed that a parent chymosin polypeptide (e.g. from sheep or rat) that has at least 65% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin) may herein be seen as sufficient structural related to e.g. bovine or camel chymosin in order to be improved by making a variant in any of the amino acid positions as described herein.
Accordingly, a first aspect of the invention relates to a method for making an isolated chymosin polypeptide variant comprising the steps:
(a): making an alteration at one or more positions in a parent polypeptide having chymosin activity, wherein the alteration is comprising a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions 70; 75; 77; 79; 90; 102; 103; 108; 114; 117; 120; 124; 134; 154; 156, 163; 212; 222; 223; 224; 238; 246; 256; 261; K279V; L280; F281; R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309; R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336; 346; 361; 367 and 379; and

(b): producing and isolating the altered polypeptide of step (a) and thereby obtaining the isolated chymosin polypeptide variant, wherein the variant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined by an alignment of the parent polypeptide with the polypeptide of SEQ ID NO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used to determine the corresponding amino acid sequence in the parent polypeptide; and

(ii): the parent polypeptide has at least 65% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin), which is from amino acid position 59 to amino acid position 381 of SEQ ID NO: 1;

and with the proviso that the isolated chymosin polypeptide variant is NOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;
R125Q+G128N+H204R+Q246E+S284T;
Y185F+R213Q+Q246E;
V261A+V263I+G309W+L311I+Y326F;
G128D+L188I+Y326F;
G128N+R312S+S313Y+Y326F;
G128N+R312S+S313Y+Y326F;
D117N+V261A+R312S;
D216S+L224V+V263I+F281V+G309D;
Y79S+L224V+L311I; and
R119S+L224V+T297S.
The proviso above may be seen as relating to above discussed WO2013/174840A1 (Chr. Hansen)—since in this document is explicitly described these specific variants.
As understood by the skilled person in the present context—the proviso only relates to the specific mentioned variants.
For instance, a variant only comprising the Q246E substitution (i.e. not G309D, S329P and/or D337E) is not such a specific variant within the proviso—i.e. it is not disclaimed in the present context.
As known in the art—the skilled person may, based on his common general knowledge, routinely produce and purify chymosin and chymosin variants.
Said in other words, once the skilled person is in possession of a herein relevant parent polypeptide having chymosin activity of interest (e.g. from bovines, camels, sheep, pigs, or rats) it is routine work for the skilled person to make a variant of such a parent chymosin of interest.
A second aspect of the invention relates to an isolated chymosin polypeptide variant obtained by the method of first aspect or any herein relevant embodiments thereof.
The term “obtained” in relation to the second aspect above should be understood as that the isolated chymosin polypeptide variant has been obtained by the method of first aspect or any herein relevant embodiments thereof.
Accordingly, the term “obtained” in relation to the second aspect should not be understood as obtainable.
As discussed herein—in working examples herein were made variants using the polypeptide of SEQ ID NO: 1 (Bovine) as parent polypeptide—such variant may herein be termed bovine chymosin variants.
Accordingly, a third aspect of the invention relates to an isolated chymosin polypeptide variant comprising:
(a): an alteration at one or more positions in a parent polypeptide having chymosin activity, wherein the alteration is comprising a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions 70; 75; 77; 79; 90; 102; 103; 108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 222; 223; 224; 238; 246; 256; 261; K279V; L280; F281; R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309; R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336; 346; 361; 367 and 379; and

(b): wherein the variant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined by an alignment of the parent polypeptide with the polypeptide of SEQ ID NO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used to determine the corresponding amino acid sequence in the parent polypeptide; and

(ii): the parent polypeptide has at least 90% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin), which is from amino acid position 59 to amino acid position 381 of SEQ ID NO: 1; and

(iii): the isolated variant polypeptide has less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin);

and with the proviso that the isolated chymosin polypeptide variant is NOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;
R125Q+G128N+H204R+Q246E+S284T;
Y185F+R213Q+Q246E;
V261A+V263I+G309W+L311I+Y326F;
G128D+L188I+Y326F;
G128N+R312S+S313Y+Y326F;
G128N+R312S+S313Y+Y326F; and
D117N+V261A+R312S;
D216S+L224V+V263I+F281V+G309D
Y79S+L224V+L311I and
R119S+L224V+T297S.
As discussed herein—in working examples herein were made variants using the polypeptide of SEQ ID NO: 2 (camel chymosin) as parent polypeptide—such variant may herein be termed camel chymosin variant.
Accordingly, a fourth aspect of the invention relates to an isolated chymosin polypeptide variant comprising:
(a): an alteration at one or more positions in a parent polypeptide having chymosin activity, wherein the alteration is comprising a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions 70; 75; 77; 79; 90; 102; 103; 108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 222; 223; 224; 238; 246; 256; 261; K279V; L280; F281; R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309; R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336; 346; 361; 367 and 379; and

(b): wherein the variant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined by an alignment of the parent polypeptide with the polypeptide of SEQ ID NO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used to determine the corresponding amino acid sequence in the parent polypeptide; and

(ii): the parent polypeptide has at least 90% sequence identity with the mature polypeptide of SEQ ID NO: 2 (Camel chymosin), which is from amino acid position 59 to amino acid position 381 of SEQ ID NO: 2; and

(iii): the isolated variant polypeptide has less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 2 (camel chymosin);

and with the proviso that the isolated chymosin polypeptide variant is NOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;
R125Q+G128N+H204R+Q246E+S284T;
Y185F+R213Q+Q246E;
V261A+V263I+G309W+L311I+Y326F;
G128D+L188I+Y326F;
G128N+R312S+S313Y+Y326F;
G128N+R312S+S313Y+Y326F;
D117N+V261A+R312S;
D216S+L224V+V263I+F281V+G309D;
Y79S+L224V+L311I; and
R119S+L224V+T297S.
An isolated chymosin polypeptide variant as described herein may be used according to the art—e.g. to make a food or feed product of interest (such as e.g. a milk based product of interest that e.g. could be a cheese product).
Accordingly, a fifth aspect of the invention relates to a method for making a food or feed product comprising adding an effective amount of the isolated chymosin polypeptide variant as described herein to the food or feed ingredient(s) and carrying our further manufacturing steps to obtain the food or feed product.
Embodiment of the present invention is described below, by way of examples only.
DEFINITIONS
All definitions of herein relevant terms are in accordance of what would be understood by the skilled person in relation to the herein relevant technical context.
The term “chymosin” relates to an enzyme of the EC 3.4.23.4 class. Chymosin has a high specificity and it clots milk by cleavage of a single 105-Ser-Phe-I-Met-Ala-108 bond in kappa-chain of casein. An alternative name used in the art is rennin.
The term “chymosin activity” relates to chymosin activity of a chymosin enzyme as understood by the skilled person in the present context.
The skilled person knows how to determine herein relevant chymosin activity.
In working Example 4 herein is provided an example of a standard method to determine specific chymosin activity—alternatively termed clotting activity or milk clotting activity.
In working Example 5 herein is provided an example of a standard method to determine proteolytical activity.
As known in the art—the herein relevant so-called C/P ratio is determined by dividing the specific clotting activity (C) with the proteolytical activity (P).
As known in the art—a higher C/P ratio implies generally that the loss of protein during e.g. cheese manufacturing due to non-specific protein degradation is reduced, i.e. the yield of cheese is improved, and that the development of bitter taste in the cheese during maturation is reduced.
The term “isolated variant” means a variant that is modified by the hand of man. 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.
The term “mature polypeptide” means a peptide in its final form following translation and any post-translational modifications, such as N terminal processing, C terminal truncation, glycosylation, phosphorylation, etc. In the present context may a herein relevant mature chymosin polypeptide be seen as the active chymosin polypeptide sequence—i.e. without the pre-part and/or pro-part sequences. Herein relevant examples of a mature polypeptide are e.g. the mature polypeptide of SEQ ID NO: 1 (bovine chymosin), which is 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 from amino acid position 59 to amino acid position 381 of SEQ ID NO: 2.
The term “parent” or “parent polypeptide having chymosin activity” means a polypeptide to which an alteration is made to produce the enzyme variants of the present invention. The parent may be a naturally occurring (wild-type) polypeptide or a variant thereof.
The term “Sequence Identity” relates to the relatedness between two amino acid sequences or between two nucleotide sequences.
For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970 , J. Mol. Biol. 48: 443-453) as 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), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

For purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later.
The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “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 Gaps in Alignment).

The term “variant” means a peptide having chymosin activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1-3 amino acids adjacent to an amino acid occupying a position.
The amino acid may be natural or unnatural amino acids—for instance, substitution with e.g. a particularly D-isomers (or D-forms) of e.g. D-alanine could theoretically be possible.
The term “wild-type” chymosin peptide means a chymosin expressed by a naturally occurring organism, such as a mammalian (e.g. camel or bovine) found in nature.



DRAWINGS
FIG. 1 : An alignment of herein relevant different chymosin sequences. The shown “ Bos _ bovis _chymosin_B” is bovine chymosin of SEQ ID NO: 1 herein and the shown “ Camelus _ dromedarius ” is camel chymosin of SEQ ID NO: 2 herein. Using bovine chymosin of SEQ ID NO: 1 as reference sequence as described herein is can e.g. be seen that bovine chymosin has “V” in position 10 and camel chymosin has “A” in the same position 10. It may e.g. also be seen that bovine/Rat have “Q” in position 352 and Camel/ C. _ bactrianus have “E” in the same position 352.
In relation to the chymosin sequences shown in FIG. 1 —sheep has 94.5% sequence identity with bovine SEQ ID NO: 1 ; C. _ bactrianus has 83.2% sequence identity with bovine SEQ ID NO: 1; Camelus _ dromedarius (camel chymosin of SEQ ID NO: 2) has 84% sequence identity with bovine SEQ ID NO: 1; pig has 80.3% sequence identity with bovine SEQ ID NO: 1 and rat has 71.9% sequence with bovine identity SEQ ID NO: 1.
As understood by the skilled person in the present context—herein relevant sequence identity percentages of mature polypeptide sequences of e.g. sheep, C. _ bactrianus , camel, pig or rat chymosin with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin—i.e. amino acid positions 59 to 381 of SEQ ID NO: 1) are relatively similar to above mentioned sequence identity percentages.
FIG. 2 : The 3D structure of bovine chymosin—the 3D structure is public available. As an example are shown where the amino acid positions 296 and 294 are present in bovine Chymosin.
FIG. 3 : Show a graphical representation the REMCAT and Proteol values of a number of chymosin variants.
FIG. 4 : PCA plot of effect of individual substitutions. All position numbers are 15 lower than numbers used in text.



DETAILED DESCRIPTION OF THE INVENTION
Determining the Amino Acid Position of a Chymosin of Interest
As discussed above—as a reference sequence for determining the amino acid position of a herein relevant chymosin polypeptide of interest (e.g. camel, sheep, bovine etc.) is herein used the public known bovine chymosin sequence disclosed as SEQ ID NO: 1 herein.
For purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 1 (bovine chymosin) is used to determine the corresponding amino acid residue in another chymosin polypeptide. The amino acid sequence of another chymosin polypeptide is aligned with the polypeptide disclosed in SEQ ID NO: 1, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 1 is determined using the ClustalW algorithm as described in working Example 1 herein.
Identification of the corresponding amino acid residue in another chymosin polypeptide can be confirmed by using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970 , J. Mol. Biol. 48: 443-453) as 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), preferably version 3.0.0 or later.
Based on above well known computer programs—it is routine work for the skilled person to determine the amino acid position of a herein relevant chymosin polypeptide of interest (e.g. camel, sheep, bovine etc.).
In FIG. 1 herein is shown an example of an alignment.
Just as an example—in FIG. 1 can e.g. be seen that herein used bovine reference SEQ ID NO: 1 has a “G” in position 50 and “ Camelus _ dromedarius ” (SEQ ID NO: 2 herein) has an “A” in this position 50.
Nomenclature of Variants
In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. The accepted IUPAC single letter or three letter amino acid abbreviations are employed.
The specific variants discussed in this “nomenclature” section below may not be herein relevant variants of the present invention—i.e. this “nomenclature” section is just to describe the herein relevant used nomenclature as such.
Substitutions.
For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, a theoretical substitution of threonine with alanine at position 226 is designated as “Thr226Ala” or “T226A”. Multiple mutations are separated by addition marks (“+”), e.g., “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 another parent amino acid) with alanine at position 226.
Deletions.
For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is designated as “Gly195*” or “G195*”. Multiple deletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or “G195*+S411*”.
Insertions.
For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly the insertion of lysine after glycine at position 195 is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple amino acids is 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 glycine at position 195 is indicated as “Gly195GlyLysAla” or “G195GKA”.
In such cases the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would thus be:











Parent:
Variant:





195
195 195a 195b


G
G-K-A


















Multiple Alterations.
Variants comprising multiple alterations are separated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing a substitution of tyrosine and glutamic acid for arginine and glycine at positions 170 and 195, respectively.
Different Substitutions.
Where different substitutions can be introduced at a position, the different substitutions are separated by a comma, e.g., “Arg170Tyr,Glu” or “R170Y,E” represents a substitution of arginine with tyrosine or glutamic acid at position 170. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” or “Y167G,A+R170G,A” designates the following variants:
“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.
A Method for Making an Isolated Chymosin Polypeptide Variant
As discussed above—as known in the art, the skilled person may, based on his common general knowledge, routinely produce and purify chymosin and chymosin variants.
Said in other words, once the skilled person is in possession of a herein relevant parent polypeptide having chymosin activity of interest (e.g. from bovines, camels, sheep, pigs, or rats) it is routine work for the skilled person to make a variant of such a parent chymosin of interest.
An example of a suitable method to produce and isolate a chymosin (variant or parent) may be by well known e.g. fungal recombinant expression/production based technology as e.g. described in WO02/36752A2 (Chr. Hansen).
It is also routine work for the skilled person to make alteration at one or more positions in a parent polypeptide having chymosin activity, wherein the alteration is comprising a substitution, a deletion or an insertion in at least one amino acid position.
As known to the skilled person—this may e.g. be done by so-called site directed mutagenesis and recombinant expression/production based technology.
It is also routine work for the skilled person to determine if a herein relevant parent polypeptide (e.g. camel or bovine wildtype chymosin) and/or a herein relevant variant has chymosin activity or not.
As known in the art—chymosin activity may be determined by the so-called C/P ratio, which is determined by dividing the specific clotting activity (C) with the proteolytical activity (P).
As known in the art—a higher C/P ratio implies generally that the loss of protein during e.g. cheese manufacturing due to non-specific protein degradation is reduced, i.e. the yield of cheese is improved, and that the development of bitter taste in the cheese during maturation is reduced.
In working example 4 herein is described a suitable method to determine the specific clotting activity (C) and in working example 5 herein is described a suitable method to determine proteolytical activity (P).
Preferably, an isolated chymosin polypeptide variant as described herein is a variant, wherein the variant has a chymosin activity giving a higher C/P ratio as compared to the C/P ratio of bovine chymosin comprising the mature polypeptide of SEQ ID NO: 1 herein.
Preferably, an isolated chymosin polypeptide variant as described herein is a variant, wherein the variant has a chymosin activity giving a higher C/P ratio as compared to the C/P ratio of camel chymosin comprising the mature polypeptide of SEQ ID NO: 2 herein.
More preferably, an isolated chymosin polypeptide variant as described herein is a variant, wherein the variant has
a chymosin activity giving a higher C/P ratio as compared to the C/P ratio of bovine chymosin comprising the mature polypeptide of SEQ ID NO: 1 herein; and a chymosin activity giving a higher C/P ratio as compared to the C/P ratio of camel chymosin comprising the mature polypeptide of SEQ ID NO: 2 herein.

As discussed above—as a reference sequence for determining the amino acid position of a herein relevant chymosin polypeptide of interest (e.g. camel, sheep, bovine etc) is herein used the public known bovine chymosin sequence disclosed as SEQ ID NO: 1 herein.
As discussed above—based on e.g. the computer sequence alignment programs discussed herein—it is routine work for the skilled person to determine the herein relevant amino acid position of a herein relevant chymosin polypeptide of interest (e.g. camel, sheep, bovine etc).
The term “the parent polypeptide has at least 65% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin)” of e.g. the method of the first aspect herein may be seen as relating to a sequence based limitation of the parent chymosin polypeptide used to make a herein relevant variant thereof.
Said in other words—a mature parent chymosin polypeptide (e.g. sheep or pig) that has at least 65% sequence identity with the mature Bovine chymosin is believed to be sufficient structural identical to e.g. Bovine or Camel chymosin in order to be herein relevant—i.e. in the present context it is believed that a mature parent chymosin polypeptide (e.g. from e.g. sheep or rat) that has at least 65% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin) may herein be seen as sufficient structural related to e.g. bovine or camel chymosin in order to be improved by making a variant in any of the amino acid positions as described herein.
The camel chymosin polypeptide of SEQ ID NO: 2 has 84% sequence identity with the bovine polypeptide of SEQ ID NO: 1 (i.e. the complete SEQ ID NO: 1 from position 1 to 381, which includes pre and pro sequence).
As understood by the skilled person in the present context—a herein relevant parent polypeptide having chymosin activity may already e.g. be a variant of e.g. a corresponding wildtype chymosin.
For instance, a camel chymosin variant with e.g. 5-10 alterations (e.g. substitutions) as compared to wildtype camel chymosin polypeptide of SEQ ID NO: 2 will still be a parent polypeptide that has at least 65% sequence identity with the mature polypeptide of SEQ ID NO: 1 (Bovine) as required in e.g. first aspect herein.
Said in other words, a herein relevant isolated chymosin polypeptide variant may comprise alterations (e.g. substitutions) in other position than the positions of e.g. the first aspect herein.
In relation to the chymosin sequences shown in FIG. 1 herein—sheep has 94.5% sequence identity with bovine SEQ ID NO: 1 ; C. _ bactrianus has 83.2% sequence identity with bovine SEQ ID NO: 1; pig has 80.3% sequence identity with bovine SEQ ID NO: 1 and rat has 71.9% sequence with bovine identity SEQ ID NO: 1.
As understood by the skilled person in the present context—herein relevant sequence identity percentages of e.g. mature sheep, C. _ bactrianus , camel, pig or rat chymosin with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin—i.e. amino acid positions 59 to 381 of SEQ ID NO: 1) are relatively similar to above mentioned sequence identity percentages.
Preferred Variants:
As discussed above—e.g. the first aspect relates to an isolated chymosin polypeptide variant, wherein the alteration is comprising a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions 70; 75; 77; 79; 90; 102; 103; 108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 223; 224; 238; 246; 256; 261; K279V; L280; F281; R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309; R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336; 346; 361; 367 and 379.
A preferred embodiment relates to an isolated chymosin polypeptide variant, wherein the alteration comprises a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions of e.g. the first aspect herein.
It may be preferred that at least one alteration is a substitution—i.e. a herein relevant preferred embodiment relates to an isolated chymosin polypeptide variant, wherein the alteration is comprising a substitution in at least one amino acid position corresponding to any of positions e.g. the first aspect herein.
Preferably, an isolated chymosin polypeptide variant, wherein the alteration is comprising a substitution in at least one amino acid position corresponding to any of positions L70M; F75Y; K77T; Y79S; V90L; D102N; I103V; K120Q; F124Y; H134Q; I154L; D156V; L163E; S212A; S222G; M223E; L224V; L238I; Q246E; V256I; V261A; K279V; L280I; F281A; R300D,E,S,T,N,Q; R312D,E,S,T,N,Q; E320T; R324V; D325Q; Y326F; K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; S331Y; Q346E; I361L; V367I; or K379P.
Preferably, the substitution is wherein the substitution is Q246E; K279V; R300Q; R312S; Y326F or K336D,E,S,T,N,Q, wherein a preferred K336 substitution is K336Q.
As understood by the skilled person in the present context—if the parent chymosin polypeptide already has e.g. “V” in position 156 then is does not make sense to talk about making the substitution 156V for this specific parent chymosin polypeptide. As can be seen in FIG. 1 herein—rat wildtype chymosin has “V” in position 156—the substitution 156V may be seen as herein irrelevant for the specific rat chymosin polypeptide sequence of FIG. 1 .
As understood by the skilled person in the present context—if the parent chymosin polypeptide does not have e.g. “D” in position 156 then is does not make sense to talk about making the substitution D156V for this specific parent chymosin polypeptide. As can be seen in FIG. 1 herein—rat wildtype chymosin has “V” in position 156—the substitution D156V may therefore be seen as herein irrelevant for the specific rat chymosin polypeptide sequence of FIG. 1 .
In a preferred embodiment, the substitution is wherein the substitution is:
H134Q+Q246E+Y326F;
D117N+L280I+G309D;
H134Q+D156V+G309D;
D156V+Q246E+L280I;
D117N+H134Q+L280I;
D156V+G309D+Y326F;
D117N+D156V+D325M;
L280I+D325M+Y326F;
D117N+Q246E+Y326F;
D117N+H134Q+D325M;
N310Q+N349Q+K279V;
R300Q+N307D;
N307D+G309D;
N307D+R312S;
R300Q+K336Q;
N307D+K336Q;
G309D+R312S;
R300Q+N307D+G309D+R312S+K336Q;
N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q;
L280I+G309D+S331Y+T342S+D325Q;
L280I+G309D+L224V+E320T+T235S;
L280I+G309W+K77T+R324I;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+V213F+E320T+V90L;
L280I+G309D+Q220S+L224V+H134Q;
L280I+G309W+L238I+T342S;
L280I+G309W+F75Y+Y79S;
L280I+G309D+F75Y+S331Y+Q346E;
L280I+G309D+L224V+I103V+L238I;
L280I+G309D+F124Y+Q346E+I154L;
L280I+G309D+I154L+V261A+V367I;
L280I+G309D+Y79S+L224V+S212A;
L280I+G309D+L70M+T342S;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309D+Y79S+T342S+I154L;
L280I+G309D+Y79S+I103V+F281A;
L280I+G309D+V256I+V261A+K379P;
L280I+G309D+Q346E+K77T+T235S;
L280I+G309D+H239N+R324I+D325Q;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+Y326F+L70M+D325Q;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309W+S212A+V261A;
L280I+G309D+S331Y+L224V+Y326F;
L280I+G309D+K120Q+M223E+H239N;
L280I+G309D+H239N+R324I+D325Q;
L280I+G309W+L238I+T342S;
L280I+G309D+V213F+E320T+V90L;
L280I+G309D+L70M+T342S;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+Y79S+L224V+S212A;
L280I+G309D+S331Y+L224V+Y326F;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309W+L238I+T342S;
L280I+G309D+V213F+E320T+V90L; or
L280I+G309W+S212A+V261A.
In a more preferred embodiment, the substitution is wherein the substitution is:
D117N+L280I+G309D;
L280I+D325M+Y326F;
D117N+Q246E+Y326F;
R300Q+N307D+G309D+R312S+K336Q; or
N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q.
Preferred Parent Polypeptide Having Chymosin Activity:
Preferably, the parent polypeptide has at least 70% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin), more preferably the parent polypeptide has at least 75% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).
Just as an example—a herein suitable relevant parent polypeptide could e.g. be bovine chymosin A—as known in the art bovine chymosin A may only have one amino acid difference as compared to bovine chymosin B of SEQ ID NO: 1 herein.
As discussed above—in working examples herein were made variants using the polypeptide of SEQ ID NO: 1 (Bovine) as parent polypeptide—such variant may herein be termed bovine chymosin variants.
Accordingly, in a preferred embodiment—the parent polypeptide has at least 90% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin), more preferably the parent polypeptide has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin) and even more preferably the parent polypeptide has at least 97% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin). It may be preferred that the parent polypeptide is the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).
As understood by the skilled person in the present context—a herein relevant parent polypeptide having chymosin activity may already e.g. be a variant of e.g. a corresponding wildtype chymosin.
For instance, a bovine chymosin variant with e.g. 5-10 alterations (e.g. substitutions) as compared to mature wildtype bovine chymosin polypeptide of SEQ ID NO: 1 will still be a parent polypeptide that has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 1 (Bovine chymosin).
The mature polypeptide of SEQ ID NO: 1 (Bovine) is 323 amino acids long—accordingly, a bovine chymosin variant with e.g. 25 amino acid substitutions as compared to mature wildtype bovine chymosin polypeptide of SEQ ID NO: 1 will not be a parent polypeptide that has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 1 (Bovine chymosin).
Said in other words and in general—a herein relevant isolated chymosin polypeptide variant may comprise alterations (e.g. substitutions) in other positions than the positions of e.g. the first aspect herein.
As discussed above—in working examples herein were made variants using the polypeptide of SEQ ID NO: 2 (Camel) as parent polypeptide—such variant may herein be termed camel chymosin variant.
Accordingly, in a preferred embodiment—the parent polypeptide has at least 90% sequence identity with the mature polypeptide of SEQ ID NO: 2 (Camel chymosin), more preferably the parent polypeptide has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 2 (Camel chymosin) and even more preferably the parent polypeptide has at least 97% sequence identity with the mature polypeptide of SEQ ID NO: 2 (Camel chymosin). It may be preferred that the parent polypeptide is the mature polypeptide of SEQ ID NO: 2 (Camel chymosin).
As understood by the skilled person in the present context—a parent polypeptide that has at least 90% sequence identity with the mature polypeptide of SEQ ID NO: 2 (Camel) is still within the SEQ ID NO: 1 (Bovine) based sequence identity requirement of point (ii) of first aspect herein—i.e. it will be a parent polypeptide that has at least 65% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).
An Isolated Variant of Bovine Chymosin:
As discussed above—in working examples herein were made variants using the polypeptide of SEQ ID NO: 1 (Bovine) as parent polypeptide—such variant may herein be termed bovine chymosin variants.
As discussed above—the third aspect accordingly relates to an isolated chymosin polypeptide variant comprising:
(a): an alteration at one or more positions in a parent polypeptide having chymosin activity, wherein the alteration is comprising a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions 70; 75; 77; 79; 90; 102; 103; 108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 222; 223; 224; 238; 246; 256; 261; K279V; L280; F281; R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309; R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336; 346; 361; 367 and 379; and

(b): wherein the variant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined by an alignment of the parent polypeptide with the polypeptide of SEQ ID NO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used to determine the corresponding amino acid sequence in the parent polypeptide; and

(ii): the parent polypeptide has at least 90% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin), which is from amino acid position 59 to amino acid position 381 of SEQ ID NO: 1; and

(iii): the isolated variant polypeptide has less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin);

and with the proviso that the isolated chymosin polypeptide variant is NOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;
R125Q+G128N+H204R+Q246E+S284T;
Y185F+R213Q+Q246E;
V261A+V263I+G309W+L311I+Y326F;
G128D+L188I+Y326F;
G128N+R312S+S313Y+Y326F;
G128N+R312S+S313Y+Y326F; and
D117N+V261A+R312S;
D216S+L224V+V263I+F281V+G309D
Y79S+L224V+L311I and
R119S+L224V+T297S.
The above described definitions and preferred embodiments are also relevant for this aspect.
Preferably, an isolated bovine chymosin polypeptide variant as described herein is a variant, wherein the variant has a chymosin activity giving a higher C/P ratio as compared to the C/P ratio of bovine chymosin comprising the mature polypeptide of SEQ ID NO: 1.
In a preferred embodiment—the parent polypeptide has at least 92% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin), more preferably the parent polypeptide has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin) and even more preferably the parent polypeptide has at least 97% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin). It may be preferred that the parent polypeptide is the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).
As understood by the skilled person in the present context—an isolated chymosin variant may comprise alterations (e.g. substitutions) in other amino acid positions than given above.
For instance, a bovine chymosin variant with e.g. 5-10 alterations (e.g. substitutions) as compared to wildtype bovine chymosin polypeptide of SEQ ID NO: 1 will still be a parent polypeptide that has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 1 (Bovine chymosin).
It may be preferred that the isolated bovine chymosin variant comprises less than 30 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO: 1 (bovine chymosin) or it may be preferred that the isolated bovine chymosin variant comprises less than 20 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO: 1 (bovine chymosin) or it may be preferred that the isolated bovine chymosin variant comprises less than 10 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO: 1 (bovine chymosin) or it may be preferred that the isolated bovine chymosin variant comprises less than 5 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).
As understood by the skilled person in the present context—the term “the isolated variant polypeptide has less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin)” of point (iii) above relates to that the herein described isolated bovine chymosin variant shall of course not have a polypeptide sequence that is 100% identical to the public known wild-type bovine chymosin sequence of SEQ ID NO: 1.
A preferred embodiment relates to an isolated bovine chymosin polypeptide variant, wherein the alteration comprises a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions of the third aspect.
It may be preferred that at least one alteration is a substitution—i.e. a herein relevant preferred embodiment relates to an isolated chymosin polypeptide variant, wherein the alteration is comprising a substitution in at least one amino acid position corresponding to any of positions of the third aspect.
Preferably, the substitution is L70M; F75Y; K77T; Y79S; V90L; D102N; I103V; N108D; D117N; F114Y; K120Q; F124Y; H134Q; I154L; D156V; L163E; S212A; M223E; L224V; L238I; Q246E; V256I; V261A; K279V; L280I; F281A; R300D,E,S,T,N,Q; R312D,E,S,T,N,Q; E320T; R324V; D325Q; Y326F; K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; S331Y; Q346E; I361L; V367I; or K379P.
Preferably, the substitution is Q246E; K279V; R300Q; R312S; Y326F or K336D,E,S,T,N,Q, wherein a preferred K336 substitution is K336Q.
In a preferred embodiment, the substitution is:
H134Q+Q246E+Y326F;
D117N+L280I+G309D;
H134Q+D156V+G309D;
D156V+Q246E+L280I;
D117N+H134Q+L280I;
D156V+G309D+Y326F;
D117N+D156V+D325M;
L280I+D325M+Y326F;
D117N+Q246E+Y326F;
D117N+H134Q+D325M;
N310Q+N349Q+K279V;
R300Q+N307D;
N307D+G309D;
N307D+R312S;
R300Q+K336Q;
N307D+K336Q;
G309D+R312S;
R300Q+N307D+G309D+R312S+K336Q;
N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q;
L280I+G309D+S331Y+T342S+D325Q;
L280I+G309D+L224V+E320T+T235S;
L280I+G309W+K77T+R324I;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+V213F+E320T+V90L;
L280I+G309D+Q220S+L224V+H134Q;
L280I+G309W+L238I+T342S;
L280I+G309W+F75Y+Y79S;
L280I+G309D+F75Y+S331Y+Q346E;
L280I+G309D+L224V+I103V+L238I;
L280I+G309D+F124Y+Q346E+I154L;
L280I+G309D+I154L+V261A+V367I;
L280I+G309D+Y79S+L224V+S212A;
L280I+G309D+L70M+T342S;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309D+Y79S+T342S+I154L;
L280I+G309D+Y79S+I103V+F281A;
L280I+G309D+V256I+V261A+K379P;
L280I+G309D+Q346E+K77T+T235S;
L280I+G309D+H239N+R324I+D325Q;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+Y326F+L70M+D325Q;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309W+S212A+V261A;
L280I+G309D+S331Y+L224V+Y326F;
L280I+G309D+K120Q+M223E+H239N;
L280I+G309D+H239N+R324I+D325Q;
L280I+G309W+L238I+T342S;
L280I+G309D+V213F+E320T+V90L;
L280I+G309D+L70M+T342S;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+Y79S+L224V+S212A;
L280I+G309D+S331Y+L224V+Y326F;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309W+L238I+T342S;
L280I+G309D+V213F+E320T+V90L; or
L280I+G309W+S212A+V261A.
In a more preferred embodiment, the substitution is wherein the substitution is:
D117N+L280I+G309D;
L280I+D325M+Y326F;
D117N+Q246E+Y326F;
R300Q+N307D+G309D+R312S+K336Q; or
N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q.
An Isolated Variant of Camel Chymosin:
As discussed above—in working examples herein were made variants using the polypeptide of SEQ ID NO: 2 (camel chymosin) as parent polypeptide—such variant may herein be termed camel chymosin variant.
As discussed above—the fourth aspect accordingly relates to an isolated chymosin polypeptide variant comprising:
(a): an alteration at one or more positions in a parent polypeptide having chymosin activity, wherein the alteration is comprising a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions 70; 75; 77; 79; 90; 102; 103; 108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 223; 224; 238; 246; 256; 261; K279V; L280; F281; R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309; R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336; 346; 361; 367 and 379; and

(b): wherein the variant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined by an alignment of the parent polypeptide with the polypeptide of SEQ ID NO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used to determine the corresponding amino acid sequence in the parent polypeptide; and

(ii): the parent polypeptide has at least 90% sequence identity with the mature polypeptide of SEQ ID NO: 2 (Camel chymosin), which is from amino acid position 59 to amino acid position 381 of SEQ ID NO: 2; and

(iii): the isolated variant polypeptide has less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 2 (camel chymosin);

and with the proviso that the isolated chymosin polypeptide variant is NOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;
R125Q+G128N+H204R+Q246E+S284T;
Y185F+R213Q+Q246E;
V261A+V263I+G309W+L311I+Y326F;
G128D+L188I+Y326F;
G128N+R312S+S313Y+Y326F;
G128N+R312S+S313Y+Y326F;
D117N+V261A+R312S;
D216S+L224V+V263I+F281V+G309D;
Y79S+L224V+L311I; and
R119S+L224V+T297S.
The above described definitions and preferred embodiments are also relevant for this aspect.
Preferably, an isolated camel chymosin polypeptide variant as described herein is a variant, wherein the variant has a chymosin activity giving a higher C/P ratio as compared to the C/P ratio of camel chymosin comprising the mature polypeptide of SEQ ID NO: 2.
In a preferred embodiment—the parent polypeptide has at least 92% sequence identity with the mature polypeptide of SEQ ID NO: 2 (camel chymosin), more preferably the parent polypeptide has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 2 (camel chymosin) and even more preferably the parent polypeptide has at least 97% sequence identity with the mature polypeptide of SEQ ID NO: 2 (camel chymosin). It may be preferred that the parent polypeptide is the mature polypeptide of SEQ ID NO: 2 (Camel chymosin).
As understood by the skilled person in the present context—an isolated chymosin variant may comprise alterations (e.g. substitutions) in other amino acid positions than given above.
For instance, a camel chymosin variant with e.g. 5-10 alterations (e.g. substitutions) as compared to wildtype camel chymosin polypeptide of SEQ ID NO: 2 will still be a parent polypeptide that has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 2 (camel chymosin).
It may be preferred that the isolated camel chymosin variant comprises less than 30 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO: 2 (camel chymosin) or it may be preferred that the isolated camel chymosin variant comprises less than 20 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO: 2 (camel chymosin) or it may be preferred that the isolated camel chymosin variant comprises less than 10 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO: 2 (camel chymosin) or it may be preferred that the isolated camel chymosin variant comprises less than 5 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO: 2 (camel chymosin).
As understood by the skilled person in the present context—the term “the isolated variant polypeptide has less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 2 (camel chymosin)” of point (iii) above relates to that the herein described isolated camel chymosin variant shall of course not have a polypeptide sequence that is 100% identical to the public known wild-type camel chymosin sequence of SEQ ID NO: 2.
A preferred embodiment relates to an isolated camel chymosin polypeptide variant, wherein the alteration comprises a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions of the fourth aspect.
It may be preferred that at least one alteration is a substitution—i.e. a herein relevant preferred embodiment relates to an isolated chymosin polypeptide variant, wherein the alteration is comprising a substitution in at least one amino acid position corresponding to any of positions of the fourth aspect.
Preferably, the substitution is L70M; F75Y; K77T; Y79S; V90L; D102N; I103V; K120Q; F124Y; I154L; L163E; S212A; M223E; L224V; L238I; Q246E; V256I; V261A; K279V; R300D,E,S,T,N,Q; R312D,E,S,T,N,Q; E320T; R324V; Y326F; K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; S331Y; Q346E; I361L; V367I; or K379P.
Preferably, the substitution is Q246E; K279V; R300Q; R312S; Y326F or K336D,E,S,T,N,Q, wherein a preferred K336 substitution is K336Q.
In a preferred embodiment, the substitution is:
H134Q+Q246E+Y326F;
D117N+L280I+G309D;
H134Q+D156V+G309D;
D156V+Q246E+L280I;
D117N+H134Q+L280I;
D156V+G309D+Y326F;
D117N+D156V+D325M;
L280I+D325M+Y326F;
D117N+Q246E+Y326F;
D117N+H134Q+D325M;
N310Q+N349Q+K279V;
R300Q+N307D;
N307D+G309D;
N307D+R312S;
R300Q+K336Q;
N307D+K336Q;
G309D+R312S;
R300Q+N307D+G309D+R312S+K336Q;
N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q;
L280I+G309D+S331Y+T342S+D325Q;
L280I+G309D+L224V+E320T+T235S;
L280I+G309W+K77T+R324I;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+V213F+E320T+V90L;
L280I+G309D+Q220S+L224V+H134Q;
L280I+G309W+L238I+T342S;
L280I+G309W+F75Y+Y79S;
L280I+G309D+F75Y+S331Y+Q346E;
L280I+G309D+L224V+I103V+L238I;
L280I+G309D+F124Y+Q346E+I154L;
L280I+G309D+I154L+V261A+V367I;
L280I+G309D+Y79S+L224V+S212A;
L280I+G309D+L70M+T342S;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309D+Y79S+T342S+I154L;
L280I+G309D+Y79S+I103V+F281A;
L280I+G309D+V256I+V261A+K379P;
L280I+G309D+Q346E+K77T+T235S;
L280I+G309D+H239N+R324I+D325Q;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+Y326F+L70M+D325Q;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309W+S212A+V261A;
L280I+G309D+S331Y+L224V+Y326F;
L280I+G309D+K120Q+M223E+H239N;
L280I+G309D+H239N+R324I+D325Q;
L280I+G309W+L238I+T342S;
L280I+G309D+V213F+E320T+V90L;
L280I+G309D+L70M+T342S;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+Y79S+L224V+S212A;
L280I+G309D+S331Y+L224V+Y326F;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309W+L238I+T342S;
L280I+G309D+V213F+E320T+V90L; or
L280I+G309W+S212A+V261A.
In a more preferred embodiment, the substitution is wherein the substitution is:
D117N+L280I+G309D;
L280I+D325M+Y326F;
D117N+Q246E+Y326F;
R300Q+N307D+G309D+R312S+K336Q; or
N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q.
A Method for Making a Milk Based Product
As discussed above—an isolated chymosin polypeptide variant as described herein may be used according to the art—e.g. to make a milk based product of interest (such as e.g. a cheese product).
As discussed above—an aspect of the invention relates to a method for making a food or feed product comprising adding an effective amount of the isolated chymosin polypeptide variant as described herein to the food or feed ingredient(s) and carrying our further manufacturing steps to obtain the food or feed product.
Preferably, the food or feed product is a milk based product and wherein the method comprises adding an effective amount of the isolated chymosin polypeptide variant as described herein to milk and carrying our further manufacturing steps to obtain the milk based product.
The milk may e.g. be soy milk, sheep milk, goat milk, buffalo milk, yak milk, lama milk, camel milk or cow milk.
The milk based product may e.g. be a fermented milk product, a quark or a cheese.
Aspects/Embodiments Herein—Presented in Claim Format
Herein described aspects and preferred embodiments of the invention may be presented/described in a so-called claim format—this is done below.
1. A method for making an isolated chymosin polypeptide variant comprising the steps:

(a): making an alteration at one or more positions in a parent polypeptide having chymosin activity, wherein the alteration is comprising a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions 70; 75; 77; 79; 90; 102; 103; 108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 222; 223; 224; 238; 246; 256; 261; K279V; L280; F281; R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309; R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336; 346; 361; 367 and 379; and

(b): producing and isolating the altered polypeptide of step (a) and thereby obtaining the isolated chymosin polypeptide variant, wherein the variant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined by an alignment of the parent polypeptide with the polypeptide of SEQ ID NO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used to determine the corresponding amino acid sequence in the parent polypeptide; and

(ii): the parent polypeptide has at least 65% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin), which is from amino acid position 59 to amino acid position 381 of SEQ ID NO: 1;

and with the proviso that the isolated chymosin polypeptide variant is NOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;
R125Q+G128N+H204R+Q246E+S284T;
Y185F+R213Q+Q246E;
V261A+V263I+G309W+L311I+Y326F;
G128D+L188I+Y326F;
G128N+R312S+S313Y+Y326F;
G128N+R312S+S313Y+Y326F;
D117N+V261A+R312S;
D216S+L224V+V263I+F281V+G309D;
Y79S+L224V+L311I; and
R119S+L224V+T297S.
2. The method for making an isolated chymosin polypeptide variant of claim 1 , wherein the isolated chymosin polypeptide variant has:
a chymosin activity giving a higher C/P ratio as compared to the C/P ratio of bovine chymosin comprising the mature polypeptide of SEQ ID NO: 1; and a chymosin activity giving a higher C/P ratio as compared to the C/P ratio of camel chymosin comprising the mature polypeptide of SEQ ID NO: 2.

3. The method for making an isolated chymosin polypeptide variant of any of the preceding claims, wherein the alteration comprises a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions of claim 1 .

4. The method for making an isolated chymosin polypeptide variant of any of the preceding claims, wherein the alteration comprises a substitution in at least one amino acid position corresponding to any of positions of claim 1 .

5. The method for making an isolated chymosin polypeptide variant of claim 4 , wherein the substitution is L70M; F75Y; K77T; Y79S; V90L; D102N; I103V; N108D; D117N; F114Y; K120Q; F124Y; H134Q; I154L; D156V; L163E; S212A; S222G; M223E; L224V; L238I; Q246E; V256I; V261A; K279V; L280I; F281A; R300D,E,S,T,N,Q; R312D,E,S,T,N,Q; E320T; R324V; D325Q; Y326F; K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; S331Y; Q346E; I361L; V367I; or K379P.

6. The method for making an isolated chymosin polypeptide variant of claim 5 , wherein the substitution is Q246E; K279V; R300Q; R312S; Y326F or K336D,E,S,T,N,Q.

7. The method for making an isolated chymosin polypeptide variant of claim 6 , wherein the substitution is K336Q.

8. The method for making an isolated chymosin polypeptide variant of claim 4 , wherein the substitution is:


H134Q+Q246E+Y326F;
D117N+L280I+G309D;
H134Q+D156V+G309D;
D156V+Q246E+L280I;
D117N+H134Q+L280I;
D156V+G309D+Y326F;
D117N+D156V+D325M;
L280I+D325M+Y326F;
D117N+Q246E+Y326F;
D117N+H134Q+D325M;
N310Q+N349Q+K279V;
R300Q+N307D;
N307D+G309D;
N307D+R312S;
R300Q+K336Q;
N307D+K336Q;
G309D+R312S;
R300Q+N307D+G309D+R312S+K336Q;
N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q;
L280I+G309D+S331Y+T342S+D325Q;
L280I+G309D+L224V+E320T+T235S;
L280I+G309W+K77T+R324I;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+V213F+E320T+V90L;
L280I+G309D+Q220S+L224V+H134Q;
L280I+G309W+L238I+T342S;
L280I+G309W+F75Y+Y79S;
L280I+G309D+F75Y+S331Y+Q346E;
L280I+G309D+L224V+I103V+L238I;
L280I+G309D+F124Y+Q346E+I154L;
L280I+G309D+I154L+V261A+V367I;
L280I+G309D+Y79S+L224V+S212A;
L280I+G309D+L70M+T342S;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309D+Y79S+T342S+I154L;
L280I+G309D+Y79S+I103V+F281A;
L280I+G309D+V256I+V261A+K379P;
L280I+G309D+Q346E+K77T+T235S;
L280I+G309D+H239N+R324I+D325Q;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+Y326F+L70M+D325Q;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309W+S212A+V261A;
L280I+G309D+S331Y+L224V+Y326F;
L280I+G309D+K120Q+M223E+H239N;
L280I+G309D+H239N+R324I+D325Q;
L280I+G309W+L238I+T342S;
L280I+G309D+V213F+E320T+V90L;
L280I+G309D+L70M+T342S;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+Y79S+L224V+S212A;
L280I+G309D+S331Y+L224V+Y326F;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309W+L238I+T342S;
L280I+G309D+V213F+E320T+V90L; or
L280I+G309W+S212A+V261A.
9. The method for making an isolated chymosin polypeptide variant of claim 4 , wherein the substitution is:
D117N+L280I+G309D;
L280I+D325M+Y326F;
D117N+Q246E+Y326F;
R300Q+N307D+G309D+R312S+K336Q; or
N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q.
10. The method for making an isolated chymosin polypeptide variant of any of the preceding claims, wherein the parent polypeptide has at least 75% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).

11. The method for making an isolated chymosin polypeptide variant of claim 10 , wherein the parent polypeptide has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).

12. The method for making an isolated chymosin polypeptide variant of any of claims 1 to 9 , wherein the parent polypeptide has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 2 (Camel chymosin), which is from amino acid position 59 to amino acid position 381 of SEQ ID NO: 2.

13. An isolated chymosin polypeptide variant obtained by the method of any of claims 1 to 12 .

14. An isolated chymosin polypeptide variant comprising:

(a): an alteration at one or more positions in a parent polypeptide having chymosin activity, wherein the alteration is comprising a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions 70; 75; 77; 79; 90; 102; 103; 108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 222; 223; 224; 238; 246; 256; 261; K279V; L280; F281; R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309; R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336; 346; 361; 367 and 379; and

(b): wherein the variant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined by an alignment of the parent polypeptide with the polypeptide of SEQ ID NO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used to determine the corresponding amino acid sequence in the parent polypeptide; and

(ii): the parent polypeptide has at least 90% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin), which is from amino acid position 59 to amino acid position 381 of SEQ ID NO: 1; and

(iii): the isolated variant polypeptide has less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin);

and with the proviso that the isolated chymosin polypeptide variant is NOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;
R125Q+G128N+H204R+Q246E+S284T;
Y185F+R213Q+Q246E;
V261A+V263I+G309W+L311I+Y326F;
G128D+L188I+Y326F;
G128N+R312S+S313Y+Y326F;
G128N+R312S+S313Y+Y326F;
D117N+V261A+R312S;
D216S+L224V+V263I+F281V+G309D
Y79S+L224V+L311I and
R119S+L224V+T297S.
15. The isolated chymosin polypeptide variant of claim 14 , wherein the isolated variant has a chymosin activity giving a higher C/P ratio as compared to the C/P ratio of bovine chymosin comprising the mature polypeptide of SEQ ID NO: 1.

16. The isolated chymosin polypeptide variant of any of claims 14 to 15 , wherein the parent polypeptide has at least 97% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).

17. The isolated chymosin polypeptide variant of any of claims 14 to 16 , wherein the isolated bovine chymosin variant comprises less than 10 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).

18. The isolated chymosin polypeptide variant of any of claims 14 to 17 , wherein the alteration comprises a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions of claim 14 .

19. The isolated chymosin polypeptide variant of any of claims 16 to 18 , wherein the alteration comprises a substitution in at least one amino acid position corresponding to any of positions of claim 14 .

20. The isolated chymosin polypeptide variant of claim 19 , wherein the substitution is L70M; F75Y; K77T; Y79S; V90L; D102N; I103V; N108D; D117N; F114Y; K120Q; F124Y; H134Q; I154L; D156V; L163E; S212A; S222G; M223E; L224V; L238I; Q246E; V256I; V261A; K279V; L280I; F281A; R300D,E,S,T,N,Q; G309D,W; R312D,E,S,T,N,Q; E320T; R324V; D325Q; Y326F; K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; S331Y; Q346E; I361L; V367I; or K379P.

21. The isolated chymosin polypeptide variant of claim 20 , wherein the substitution is Q246E; K279V; R300Q; R312S; Y326F or K336D,E,S,T,N,Q.

22. The isolated chymosin polypeptide variant of claim 21 , wherein the substitution is K336Q.

23. The isolated chymosin polypeptide variant of claim 19 , wherein the substitution is:

H134Q+Q246E+Y326F;
D117N+L280I+G309D;
H134Q+D156V+G309D;
D156V+Q246E+L280I;
D117N+H134Q+L280I;
D156V+G309D+Y326F;
D117N+D156V+D325M;
L280I+D325M+Y326F;
D117N+Q246E+Y326F;
D117N+H134Q+D325M;
N310Q+N349Q+K279V;
R300Q+N307D;
N307D+G309D;
N307D+R312S;
R300Q+K336Q;
N307D+K336Q;
G309D+R312S;
R300Q+N307D+G309D+R312S+K336Q;
N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q;
L280I+G309D+S331Y+T342S+D325Q;
L280I+G309D+L224V+E320T+T235S;
L280I+G309W+K77T+R324I;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+V213F+E320T+V90L;
L280I+G309D+Q220S+L224V+H134Q;
L280I+G309W+L238I+T342S; L280I+G309W+F75Y+Y79S;
L280I+G309D+F75Y+S331Y+Q346E;
L280I+G309D+L224V+I103V+L238I;
L280I+G309D+F124Y+Q346E+I154L;
L280I+G309D+I154L+V261A+V367I;
L280I+G309D+Y79S+L224V+S212A;
L280I+G309D+L70M+T342S;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309D+Y79S+T342S+I154L;
L280I+G309D+Y79S+I103V+F281A;
L280I+G309D+V256I+V261A+K379P;
L280I+G309D+Q346E+K77T+T235S;
L280I+G309D+H239N+R324I+D325Q;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+Y326F+L70M+D325Q;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309W+S212A+V261A;
L280I+G309D+S331Y+L224V+Y326F;
L280I+G309D+K120Q+M223E+H239N;
L280I+G309D+H239N+R324I+D325Q;
L280I+G309W+L238I+T342S;
L280I+G309D+V213F+E320T+V90L;
L280I+G309D+L70M+T342S;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+Y79S+L224V+S212A;
L280I+G309D+S331Y+L224V+Y326F;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309W+L238I+T342S;
L280I+G309D+V213F+E320T+V90L; or
L280I+G309W+S212A+V261A.
24. The isolated chymosin polypeptide variant of claim 19 , wherein the substitution is:
D117N+L280I+G309D;
L280I+D325M+Y326F;
D117N+Q246E+Y326F;
R300Q+N307D+G309D+R312S+K336Q; or
N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q.
25. An isolated chymosin polypeptide variant comprising:

(a): an alteration at one or more positions in a parent polypeptide having chymosin activity, wherein the alteration is comprising a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions 70; 75; 77; 79; 90; 102; 103; 108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 222; 223; 224; 238; 246; 256; 261; K279V; L280; F281; R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; G309; R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; 320; 324; D325Q; 326; 331; 336; 346; 361; 367 and 379; and

(b): wherein the variant has chymosin activity;

and wherein:

(i): the amino acid position of the parent polypeptide is determined by an alignment of the parent polypeptide with the polypeptide of SEQ ID NO: 1 (bovine chymosin)—i.e. the polypeptide of SEQ ID NO: 1 is used to determine the corresponding amino acid sequence in the parent polypeptide; and

(ii): the parent polypeptide has at least 90% sequence identity with the mature polypeptide of SEQ ID NO: 2 (Camel chymosin), which is from amino acid position 59 to amino acid position 381 of SEQ ID NO: 2; and

(iii): the isolated variant polypeptide has less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 2 (camel chymosin);

and with the proviso that the isolated chymosin polypeptide variant is NOT a specific variant selected from the group consisting of:

Q246E+G309D+S329P+D337E;
R125Q+G128N+H204R+Q246E+S284T;
Y185F+R213Q+Q246E;
V261A+V263I+G309W+L311I+Y326F;
G128D+L188I+Y326F;
G128N+R312S+S313Y+Y326F;
G128N+R312S+S313Y+Y326F;
D117N+V261A+R312S;
D216S+L224V+V263I+F281V+G309D;
Y79S+L224V+L311I; and
R119S+L224V+T297S.
26. The isolated chymosin polypeptide variant of claim 25 , wherein the isolated variant has a chymosin activity giving a higher C/P ratio as compared to the C/P ratio of camel chymosin comprising the mature polypeptide of SEQ ID NO: 2.

27. The isolated chymosin polypeptide variant of any of claims 25 to 26 , wherein the parent polypeptide has at least 97% sequence identity with the mature polypeptide of SEQ ID NO: 2 (camel chymosin).

28. The isolated chymosin polypeptide variant of any of claims 25 to 26 , wherein the isolated camel chymosin variant comprises less than 10 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO: 2 (camel chymosin).

29. The isolated chymosin polypeptide variant of any of claims 25 to 28 , wherein the alteration comprises a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions of claim 23 .

30. The isolated chymosin polypeptide variant of any of claims 25 to 29 , wherein the alteration comprises a substitution in at least one amino acid position corresponding to any of positions of positions of claim 23 .

31. The isolated chymosin polypeptide variant of claim 30 , wherein the substitution is L70M; F75Y; K77T; Y79S; V90L; D102N; I103V; N108D; D117N; F114Y; K120Q; F124Y; H134Q; I154L; D156V, L163E; S212A; S222G; M223E; L224V; D325Q; L238I; Q246E; V256I; V261A; K279V; L280I; F281A; R300D,E,S,T,N,Q; G309D,W; R312D,E,S,T,N,Q; E320T; R324V; Y326F; K336D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; S331Y; Q346E; I361L; V367I; or K379P.

32. The isolated chymosin polypeptide variant of claim 31 , wherein the substitution is Q246E; K279V; R300Q; R312S; Y326F or K336D,E,S,T,N,Q.

33. The isolated chymosin polypeptide variant of claim 32 , wherein the substitution is K336Q.

34. The isolated chymosin polypeptide variant of claim 30 , wherein the substitution is:

H134Q+Q246E+Y326F;
D117N+L280I+G309D;
H134Q+D156V+G309D;
D156V+Q246E+L280I;
D117N+H134Q+L280I;
D156V+G309D+Y326F;
D117N+D156V+D325M;
L280I+D325M+Y326F;
D117N+Q246E+Y326F;
D117N+H134Q+D325M;
N310Q+N349Q+K279V;
R300Q+N307D;
N307D+G309D;
N307D+R312S;
R300Q+K336Q;
N307D+K336Q;
G309D+R312S;
R300Q+N307D+G309D+R312S+K336Q;
N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q;
L280I+G309D+S331Y+T342S+D325Q;
L280I+G309D+L224V+E320T+T235S;
L280I+G309W+K77T+R324I;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+V213F+E320T+V90L;
L280I+G309D+Q220S+L224V+H134Q;
L280I+G309W+L238I+T342S;
L280I+G309W+F75Y+Y79S;
L280I+G309D+F75Y+S331Y+Q346E;
L280I+G309D+L224V+I103V+L238I;
L280I+G309D+F124Y+Q346E+I154L;
L280I+G309D+I154L+V261A+V367I;
L280I+G309D+Y79S+L224V+S212A;
L280I+G309D+L70M+T342S;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309D+Y79S+T342S+I154L;
L280I+G309D+Y79S+I103V+F281A;
L280I+G309D+V256I+V261A+K379P;
L280I+G309D+Q346E+K77T+T235S;
L280I+G309D+H239N+R324I+D325Q;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+Y326F+L70M+D325Q;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309W+S212A+V261A;
L280I+G309D+S331Y+L224V+Y326F;
L280I+G309D+K120Q+M223E+H239N;
L280I+G309D+H239N+R324I+D325Q;
L280I+G309W+L238I+T342S;
L280I+G309D+V213F+E320T+V90L;
L280I+G309D+L70M+T342S;
L280I+G309D+H134Q+V213F+F281A;
L280I+G309D+Y79S+L224V+S212A;
L280I+G309D+S331Y+L224V+Y326F;
L280I+G309D+H134Q+M223E+L70M;
L280I+G309W+L238I+T342S;
L280I+G309D+V213F+E320T+V90L; or
L280I+G309W+S212A+V261A.
35. The isolated chymosin polypeptide variant of claim 30 , wherein the substitution is:
D117N+L280I+G309D;
L280I+D325M+Y326F;
D117N+Q246E+Y326F;
R300Q+N307D+G309D+R312S+K336Q; or
N158Q+N349Q+R300Q+N307D+G309D+R312S+K336Q.
36: A method for making a food or feed product comprising adding an effective amount of the isolated chymosin polypeptide variant according to any of claims 14 to 35 to the food or feed ingredient(s) and carrying our further manufacturing steps to obtain the food or feed product.

37: The method for making a food or feed product of claim 35 , wherein the product is a milk based product and wherein the method comprises adding an effective amount of the isolated chymosin polypeptide variant according to any of claims 14 to 36 to milk and carrying our further manufacturing steps to obtain the milk based product.

38: The method for making a milk based product of claim 37 , wherein the milk is soy milk, sheep milk, goat milk, buffalo milk, yak milk, lama milk, camel milk or cow milk.

39: The method for making a milk based product of any of claims 36 to 38 , wherein the milk based product is a fermented milk product, a quark or a cheese.

EXAMPLES
Example 1
Alignment and Numbering of Chymosin Protein Sequences and Variant Sequences
Chymosin protein sequences were aligned using the ClustalW algorithm as provided by the EBI (EBI, tools, multiple sequence alignment, CLUSTALW″, http://www.ebi.ac.uk/Tools/msa/clustalw2/) and as described in Larkin M A, Blackshields G, Brown N P, Chenna R, McGettigan P A, McWilliam H, Valentin F, Wallace I M, Wilm A, Lopez R, Thompson J D, Gibson T J, Higgins D G (2007). Bioinformatics 23(21), 2947-2948.
ClustalW2 settings for multiple sequence alignments were Protein weight Matrix=BLOSUM, GAP open=10, GAP EXTENSION=0.05, GAP DISTANCES=8, No End Gaps, ITERATION=none, NUMITER=1, CLUSTERING=NJ
As a reference sequence the bovine chymosin B preprochymosin was used (Genbank accession number P00794—disclosed herein as SEQ ID NO: 1), where the N-terminal Methionin has number 1 (MRCL . . . ) and the C-terminal Isoleucin (in the protein sequence . . . LAKAI) has number 381. Variants were aligned against the bovine B pre-pro-chymosin and residues were numbered according to the corresponding bovine chymosin residue.
Example 2
Design of Chymosin Variants
Chymosin variants were designed using different strategies.
When there is referred to camel chymosin there is referred to camel chymosin comprising the polypeptide of SEQ ID NO: 2 herein.
Camel chymosin of SEQ ID NO: 2 may be seen as a herein relevant parent polypeptide having chymosin activity used to make camel chymosin variants thereof.
When there is referred to bovine chymosin there is referred to bovine chymosin comprising the polypeptide of SEQ ID NO: 1 herein.
Bovine chymosin of SEQ ID NO: 1 may be seen as a herein relevant parent polypeptide having chymosin activity used to make bovine chymosin variants thereof.
Variants of camel chymosin were designed based on an alignment of a large set of public known aspartic protease sequences having an identity of 25% or more compared to bovine chymosin B.
Variations were generally introduced in hypervariable regions, while conserved regions were not changed. Multiple variations were introduced in each variant construct, ensuring that each single mutation was present in multiple variant constructs (for discussion of results—see example 6 below).
Variants of bovine chymosin were designed based on a comparison of bovine and camel chymosin. Bovine residues were e.g. changed to the camel counterpart (for discussion of results—see example 7 below).
Example 3
Preparation of Chymosin Variant Enzyme Material
All chymosin variants were synthesized as synthetic genes and cloned into a fungal expression vector corresponding essentially to pGAMpR-C (described in WO02/36752A2)
The vectors were transformed into E. coli and plasmid DNA was purified using standard molecular biology protocols, known to the person skilled in the art.
The variant plasmids were individually transformed into an Aspergillus niger or Aspergillus nidulans strain and protein was produced essentially as described in WO02/36752A2 and purified using standard chromatography techniques.
As known in the art—the skilled person may, based on his common general knowledge, produce and purify chymosin and chymosin variants—such as herein described bovine and camel chymosin variants.
Example 4
Determination of Specific Chymosin Activity
4.1 Determination of Clotting Activity
Milk clotting activity was determined using the REMCAT method, which is the standard method developed by the International Dairy Federation (IDF method) Milk clotting activity is determined from the time needed for a visible flocculation of a standard milk substrate prepared from a low-heat, low fat milk powder with a calcium chloride solution of 0.5 g per liter (pH 6.5). The clotting time of a rennet sample is compared to that of a reference standard having known milk-clotting activity and having the same enzyme composition by IDF Standard 110B as the sample. Samples and reference standards were measured under identical chemical and physical conditions. Variant samples were adjusted to approximately 3 IMCU/ml using an 84 mM acetic acid pH 5.5 buffer. Hereafter, 200 μl enzyme was added to 10 ml preheated milk (32° C.) in a glass test tube placed in a water bath, capable of maintaining a constant temperature of 32° C.±1° C. under constant stirring.
The total milk-clotting activity (strength) of a rennet was 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:





Strength





in






IMCU
/
ml


=


Sstandard
×
Tstandard
×
Dsample


Dstandard
×
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 the diluted rennet sample from addition of enzyme to time of flocculation


For clotting activity determination of camel variant evaluation in Example 9, the pIMCU method was used instead of the REMCAT method. As compared to REMCAT, flocculation time of chymosin variants in the pIMCU assay was determined by OD measurements in 96-well microtiter plates at 800 nm in a UV/VIS plate reader. A standard curve of various dilutions of a reference standard with known clotting strength was recorded on each plate. Samples were prepared by diluting enzyme in 84 mM acetate buffer, 0.1% triton X-100, pH 5.5. Reaction at 32° C. was started by adding 250 uL of a standard milk substrate containing 4% (w/w) low-heat, low fat milk powder and 7.5% (w/w) calcium chloride (pH 6.5) to 25 uL enzyme sample. Milk clotting activity of chymosin variants in International Milk-Clotting Units (IMCU) per ml was determined based on sample flocculation time relative to the standard curve.
4.2 Determination of Total Protein Content
Total protein content was determined using the Pierce BCA Protein Assay Kit from Thermo Scientific following the instructions of the providers.
4.3 Calculation of Specific Clotting Activity
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).
Example 5
Determination of Proteolytic Activity
General proteolytic activity was measured using fluorescently labelled Bodipy-FL casein as a substrate (EnzChek; Molecular Bioprobes, E6638). Casein derivatives heavily labeled with pH-insensitive green-fluorescent Bodipy-FL result in almost complete quenching of the conjugate's fluorescence. Protease catalyzed hydrolysis releases fluorescent Bodipy-FL. This method is very sensitive which was essential for this experiment as CHYMAX M has the lowest general proteolytical activity of all coagulants known to date.
The assay was conducted in a 0.2 M phosphate buffer adjusted to the desired pH at a final substrate concentration of 0.04 mg/ml. Prior to mixing 1 part of substrate with 1 part of enzyme, both prepared in the phosphate buffer, all enzyme variants where normalized to 50 IMCU/ml (according to Example 4). The substrate and enzyme were mixed in a 96-well Nunc Fluoro microtitter plates, sealed and incubated at 32° C. for 60 min. After incubation the sealing was removed and the fluorescence recorded in a fluorimeter. For variants evaluated in Examples 9 and 10, 1 part of substrate was mixed with 1 part of non-normalized enzyme samples in 386-well Nunc Fluoro microtitter plates and the fluorescence was continuously recorded in a fluorimeter at 32 C for 10 hours. Slopes of the linear part of fluorescence increase were used to determine general proteolytic activity.
Example 6
Evaluation of Camel Chymosin Variants
For all variants the specific clotting activity (IMCU/mg of total protein) was determined at pH 6.5 according to Example 4 and the proteolytical activity was determined according to example 5 at pH 6.5 The C/P ratio was determined for all variants at pH 6.5 by dividing the specific clotting activity (IMCU/mg) with the proteolytical activity.
As a reference the camel wildtype gene was included.
Variants with Multiple Substitutions
It can be concluded that there are clear combinatorial effects, where different substitutions have an effect on the respective effects.












IMCU/




mg
Proteol
C/P












1
H134Q, Q246E, Y326F
104%
211%
49%

2
D117N, L280I, G309D
122%
66%
185%

3
H134Q, D156V, G309D
117%
179%
66%

4
D156V, Q246E, L280I
105%
199%
53%

5
D117N, H134Q, L280I
67%
683%
10%

6
D156V, G309D, Y326F
100%
115%
87%

8
D117N, D156V, D325M
127%
457%
28%

9
L280I, D325M, Y326F
113%
94%
121%

10
D117N, Q246E, Y326F
127%
121%
105%

11
D117N, H134Q, D325M
134%
192%
69%

Ref
camel
100%
100%
100%































It can be concluded that variants 1, 2, 3, 4, 8, 9, 10 and 11 have a higher specific milk clotting activity, with variants 2, 8, 10 and 11 having the strongest improvement
It can be concluded that variants 2 and 9 have a reduced proteolytical activity.
It can be concluded that variants 2, 9 and 10 have an increased C/P ratio.
Based on this variant 2 is the most preferred variant, while variants 9 and 10 also show preferred characteristics.
Individual Mutations
As all variants included multiple mutations, the data of the ranked variants were investigated in more details using statistical methods and 3D structure analysis, to determine the individual amino acid changes that have a positive or negative effect.
The effects of the individual amino acid changes can be summarized as follows but depend much upon the other amino acid changes in the different variants.
Based on these the preferred mutations are D117N, Q246E, G 309D, Y326F and L280I.















C
P
C/P





H134Q
+
−−
−−
Exposed lobe


Q246E
+
−−
−
Backbone


Y326F
+
−
+/−
Backbone


D117N
++
−−
−
Backbone lobe


L280I
+
+/−
+/−
In cleft


G309D
+
−−
+/−
Outside small lobe


D156V
+
−−
−−
Backbone


D325M
++
−−
−−
Backbone
























The term “+” refers to a positive amino acid exchange—i.e. “++” is more positive than “+”.
The term “−” refers to a negative amino acid exchange—i.e. “−−” is more negative than “−”.
The term “positive” refers to a positive effect on the cheese making properties of the variants, i.e. improved clotting activity (“C”) and increased C/P ratio are considered to be positive (“+” or “++”) while increased general proteolytical activity (“P”) is considered to be a negative property (“−” or “−−”). The qualification “+/−” indicates a relatively neutral effect
The descriptions of the right column of the table relates to where the individual mutations are situated in the 3D structure of camel chymosin. The 3D structure of camel chymosin is publicly available.
Conclusions:
The results above demonstrate that following individual mutations in camel chymosin were preferred (i.e. with improved C/P ratio as compared to camel wildtype chymosin): D117N, Q246E, G 309D, Y326F, L280I.
The results above demonstrate that following multiple substitutions/mutations in camel chymosin were preferred (i.e. with improved C/P ratio as compared to camel wildtype chymosin):
D117N+L280I+G309D;
L280I+D325M+Y326F;
D117N+Q246E+Y326F.
Example 7
Evaluation of Camel and Bovine Chymosin Variants
For all variants the specific clotting activity (IMCU/mg of total protein) was determined at pH 6.5 according to Example 4, while the general or aspecific proteolytical activity was determined as described in example 5.
The C/P ratio was determined for all variants at pH 6.5 by dividing the specific clotting activity (IMCU/mg) with the proteolytical activity.
As a reference a camel wildtype gene was included.
For better comparison all variants were made in a background that did not have active N-glycosylation sites, the so called Ugly variants. These were made by changing the N in the two potential N-glycosylation sites into a Q.
For further results, see FIG. 3 .
Description of the Variants
In variant J2, K279 was replaced by V in bovine non-glycosylated chymosin
In variant J32, the flap region from bovine non-glycosylated chymosin was replaced by the flap region from Pepsin.
In variant J72, the negative patch from bovine chymosin was used to replace the corresponding regions in camel chymosin. In variant J44, R300 was replaced in camel chymosin by Q, the corresponding amino acid in bovine chymosin. This mutation is also found in variant J72.










Relative to camel







RemCat
Prot
C/P











J2
BovUgly
N310Q, N349Q, K279V
54%
227%
24%

J22
BovUgly
Pepsin positive patch
15%
115%
13%

J32
BovUgly
K279V, L80I, K129E,
63%
63%
10%



P130T, H134T, Q141T,



V171F, E191S, Y192G,



N310Q, N349Q

J44
CamUGly
R300Q
123%
114%
108%

J72
CamUgly
N158Q, N349Q,
125%
58%
215%



R300Q, N307D,



G309D, R312S, K336Q

CamUgly
Camel
N158Q, N349Q
100%
98%
102%

BovUgly
Bovine
N310Q, N349Q
40%
208%
19%

Camel

N/A
100%
100%
100%

































Conclusions:
Mutation of the Lysin at position 279 of bovine chymosin resulted in a variant that showed comparable proteolytical activity and an increased specific clotting activity as compared to bovine chymosin (variant J2). Accordingly, it can be concluded that Valine at position 279 is the preferred amino acid.
The effect of glycosylation of Camel chymosin on the cheese making properties is neglectible. Comparison of the unglycosylated camel variant with the wild-type camel chymosin indicates no significant changes. However, introduction of the negative patch reason from bovine chymosin in camel chymosin (variant J72) shows a positive effect on the specific clotting activity, while the general proteolytical activity is approximately 2 fold reduced, resulting in a doubling of the C/P ratio. Introduction of the single mutation R300Q from this patch (variant J44) shows a similar positive effect on clotting activity as seen for variant J72. Q is concluded to be the preferred amino acid in position 300.
The negative patch region in bovine chymosin is expected to have an important effect for positioning of the enzyme outward the correct cleavage site, thus improving the enzymes specificity. The effect is expected to be mostly charge related, i.e. any change that increases the negative charge in this reason will result in increased specificity.
Below is shown an alignment of the negative charged region of bovine and camel chymosin. Only charged residues are indicated.
RxxxxxxNxGxxRxxxxxxxxxxxxxxxxxxxxxxxK Camel
QxxxxxxDxDxxSxxxxxxxxxxxxxxxxxxxxxxxQ Bovine
With respect to position numbers and using the Camel as reference the numbering is starting from the right:
R300
N307
G309
R312
K336
Example 8
Evaluation of Camel Variants
A number of different variants, each having multiple substitutions as compared to the wild type camel chymosin, was analyzed.
For all variants the specific clotting activity (IMCU/mg of total protein) was determined at pH 6.5 according to Example 4, while the aspecific proteolytical activity was determined as described in Example 5 by measuring proteolytical activity per 100 IMCU.
As a reference a camel wildtype gene was included.
Analysis of Variants
The variants indicated in the table have an amino acid sequence identical to the camel chymosin gene (indicated by camel wt), except for the variations mentioned for each variant.
Clotting activity is mentioned as IMCU per mg of total protein. Improved clotting activities are indicated with one or more “+” symbols. Proteolytical activity is expressed in artificial units per 100 IMCU. Improved variants, i.e. variants with reduced proteolytical activities, are indicated with one or more “+” symbols. More “+” symbols indicate a stronger improvement. In the “Overall” column “+” symbols indicate variants that have generally improved properties, i.e. a low proteolytical activity with a high clotting activity.





TABLE 1



analysis of camel chymosin variants







Clotting
Proteolytical



IMCU/mg
Au/100 IMCU
Overall
















1
L280I
G309D
E141S
Q220S
R324I
196

161852



2
L280I
G309W
F75Y
Y79S

419
++
43355

3
L280I
G309D
H134Q
S222G
S331Y
299

36409

4
L280I
G309D
K120Q
M223E
H239N
250

13642
+++

5
L280I
G309D
Q220S
V213F
T342S
231

139775

6
L280I
G309D
H134Q
V213F
F281A
376
+
23575
+

7
L280I
G309D
S331Y
L224V
Y326F
318

12257
++++
+

8
L280I
G309D
Y326F
V241I
E305T
353
+
33477

9
L280I
G309D
S331Y
F124Y
I346L
338

37156

10
L280I
G309D
M223E
L224V
L273V
324

36425

11
L280I
G309D
H134Q
M223E
L70M
386
+
10664
++++
++

12
L280I
G309D
F75Y
S331Y
Q346E
418
++
40393

13
L280I
G309D
L224V
I103V
L238I
412
++
50010

14
L280I
G309W
L238I
T342S

420
+++
21087
+
++

15
L280I
G309D

L70M
T342S
395
++
22743
+

16
L280I
G309D
Y79S
L224V
S212A
403
++
23684
+

17
L280I
G309D
V213F
E320T
V90L
426
+++
21956
+
++

18
L280I
G309D
L163E
S222G
V261A
246

97468

19
L280I
G309W
S212A
V261A

344

10865
++++
+

20
L280I
G309D
Q220S
L224V
H134Q
425
+++
35156

21
L280I
G309W
K77T
R324I

434
+++
45616

22
L280I
G309W
I361L
I103V

324

32966

23
L280I
G309D
E141S
R324V
V367I
360
+
77215

24
L280I
G309D
Y79S
L273V
L163E
317

62132

25
L280I
G309D
I154L
T235S
K379P
333

93587

26
L280I
G309D
F75Y
T342S
V261A
361
+
108877

27
L280I
G309D
V90L
K379P
V318T
317

52280

28
L280I
G309D
V256I
V90L
E141S
289

81720

29
L280I
G309D
I154L
V261A
V367I
405
++
59055

30
L280I
G309D
Y326F
L273V
V90L
312

54833

31
L280I
G309D
H134Q
L163E
V318T
344

43594

32
L280I
G309D
Y79S
H134Q
Y326F
337

30815

33
L280I
G309D
Y79S
I103V
F281A
379
+
104307

34
L280I
G309D
V256I
V261A
K379P
378
+
39517

35
L280I
G309D
S331Y
L238I
I154L
293

60312

36
L280I
G309D
S222G
R324V
I154L
223

62784

37
L280I
G309D
H239N
F124Y
V90L
312

55432

38
L280I
G309D
H239N
R324I
D325Q
377
+
17261
++

39
L280I
G309W
K120Q
V367I

354

75440

40
L280I
G309D
Y326F
L70M
9325Q
373
+
72792

41
L280I
G309D
L224V
E320T
T235S
446
+++
32453

42
L280I
G309D
S331Y
T342S
D325Q
475
++++
70103

43
L280I
G309D
F124Y
Q346E
I154L
410
++
33586

44
L280I
G309D
V261A
R324V
F281A
198

34974

45
L280I
G309D
I361L
S212A
V318T
343

64876

46
L280I
G309D
Y79S
T342S
I154L
382
+
122413

47
L280I
G309D
Q346E
K77T
T235S
377
+
34716

48
L280I
G309D
K120Q
Y326F
K77T
264

46463

Camel wt





366
+
15.664
+++

Bovine wt





208

62.662










































































High specific clotting activity is essential for a good milk clotting enzymes. In total 21 variants with an increased specific clotting activity, relative to the camel chymosin, were identified and included in Table 2 below.





TABLE 2



Camel chymosin variants with increased Clotting activity







Clotting
Proteolytical
Overall
















42
L280I
G309D
S331Y
T342S
D325Q
475
++++
70.103



41
L280I
G309D
L224V
E320T
T235S
446
+++
30.953

21
L280I
G309W
K77T
R324I

434
+++
45.616

17
L280I
G309D
V213F
E320T
V90L
426
+++
21.956
+
++

20
L280I
G309D
Q220S
L224V
H134Q
425
+++
35.156

14
L280I
G309W
L238I
T342S

420
+++
21.087
+
++

2
L280I
G309W
F75Y
Y79S

419
++
43.355

12
L280I
G309D
F75Y
S331Y
Q346E
418
++
40.393

13
L280I
G309D
L224V
I103V
L238I
412
++
50.010

43
L280I
G309D
F124Y
Q346E
I154L
410
++
33.586

29
L280I
G309D
I154L
V261A
V367I
405
++
59.055

16
L280I
G309D
Y79S
L224V
S212A
403
++
23.684
+

15
L280I
G309D

L70M
T342S
395
++
22.743
+

11
L280I
G309D
H134Q
M223E
L70M
386
+
10.664
++++
++

46
L280I
G309D
Y79S
T342S
I154L
382
+
122.413

33
L280I
G309D
Y79S
I103V
F281A
379
+
104.307

34
L280I
G309D
V256I
V261A
K379P
378
+
39.517

47
L280I
G309D
Q346E
K77T
T235S
377
+
34.716

38
L280I
G309D
H239N
R324I
D325Q
377
+
17.261
++

6
L280I
G309D
H134Q
V213F
F281A
376
+
23.575
+

40
L280I
G309D
Y326F
L70M
D325Q
373
+
72.792

Camel





366
+
15.664
+++

wt














































Reduced proteolytical activity is a perquisite for a good milk clotting enzymes. In total 10 variants with a reduced proteolytical activity, relative to the camel chymosin, were identified (see Table 3 below).





TABLE 3



Camel chymosin variants with reduced proteolytical activity







Clotting
Proteolytical
Overall
















11
L280I
G309D
H134Q
M223E
L70M
386
+
10.664
++++
++

19
L280I
G309W
S212A
V261A

344

10.865
++++
+

7
L280I
G309D
S331Y
L224V
Y326F
318

12.257
++++
+

4
L280I
G309D
K120Q
M223E
H239N
250

13.642
+++

38
L280I
G309D
H239N
R324I
D325Q
377
+
17.261
++

14
L280I
G309W
L238I
T342S

420
+++
21.087
+
++

17
L280I
G309D
V213F
E320T
V90L
426
+++
21.956
+
++

15
L280I
G309D

L70M
T342S
395
++
22.743
+

6
L280I
G309D
H134Q
V213F
F281A
376
+
23.575
+

16
L280I
G309D
Y79S
L224V
S212A
403
++
23.684
+

Camel





366
+
15.664
+++

wt



































Based on an overall analysis five variants were identified that had improved properties for both milk clotting and proteolytical activities. These five variants are indicated in table 4 below.





TABLE 4



Camel chymosin variants with increased clotting activity and

decreased proteolytical activity







Clotting
Proteolytical
Overall
















7
L280I
G309D
S331Y
L224V
Y326F
318

12.257
++++
+

11
L280I
G309D
H134Q
M223E
L70M
386
+
10.664
++++
++

14
L280I
G309W
L238I
T342S

420
+++
21.087
+
++

17
L280I
G309D
V213F
E320T
V90L
426
+++
21.956
+
++

19
L280I
G309W
S212A
V261A

344

10.865
++++
+

Camel





366
+
15.664
+++

wt































Statistical Analysis of the Effects of Individual Mutations
A statistical, PCA based, analysis was used to identify single mutations with positive effects on either proteolytical activity, milk clotting activity, or both. In the table below, mutations resulting in increased clotting activity, decreased proteolytical activity or both increased clotting and decreased proteolytical activity are summarized. The PCA plot is indicated in the FIG. 4 .





TABLE 5



single substitutions having positive effects on clotting,

proteolytical activity or on both







Clotting +




proteolytical
Clotting
Proteolytical





H134Q
I103V
R324V


L224V
F75Y
K120Q


Q346E
D325Q
M223E


L70M
I154L
S331Y


G309W
I361L
K379P


E320T
Y79S
L163E


L238I
D117N


V90L
L280I


V367I
V261A


V256I


K77T


S212A


F124Y




































Positional Effects
It was expected that most mutations that would have an effect on clotting activity or on general proteolytical activity (i.e. specificity) would be located in or close to the catalytical cleft. The substrate is entering the catalytical cleft and it is also here that cleavage takes place.
Suprisingly, only few of the substitutions that were shown to have a positive effect on clotting activity and/or specificity were located in this region (for example L280I L70M and F75Y). Many mutations that had a positive effect were found on other parts of the molecule
Substitutions Resulting in Improved Clotting Activity
Most of the substitutions resulting in improved clotting activity were located in the body of the enzyme and are likely to have caused conformational changes in the molecule. Substitution F75Y is located at the entrance of the cleft and is rather subtle, resulting in increased polarity.





TABLE6



substitutions giving improved clotting










I103V
Lobe, back


F75Y
Cleft entrance


D325Q
Backbone


I154L
Backbone


I361L
Body


Y79S
Backbone


D117N
Side


L280I
Close to cleft


V261A
Side






























Substitutions Resulting in Reduced Proteolytical Activity
Most of the substitutions are located in the body of the molecule. The resulting conformational changes might result in increased accessibility for the substrate. Two mutations were found at the lobes that mark the entrance of the catalytical cleft. The L163E substitution increases the negative charge. This strengthens the results from example 7, showing the importance of charge in these positions.





TABLE 6



Mutations resulting in reduced proteolytical activity










R324V
Backbone


K120Q
Side


M223E
Body


S331Y
Lobe


K379P
Backbone


L163E
Lobe



























Substitutions Resulting in Improved Clotting and Reduced Proteolytical Activity
Some of the substitutions that result in an overall improvement of the milk clotting capabilities result in charge changes that are likely to be involved in substrate recognition. These include H134Q resulting in higher positive, as well as the Q346E substitution resulting in more negative charge. Other substitutions with positive effects on both clotting and specificity are most likely resulting in more general conformational changes of the chymosin molecule.





TABLE 7



Mutations giving improved clotting and reduced proteolytical activity










H134Q
Outside flap


L224V
Backbone


Q346E
Entrance cleft


L70M
Cleft


G309W
Side lobe


E320T
Backbone


L238I
Backbone


V90L
Close to cleft


V367I
Backbone


V256I
Backbone


K77T
Side protruding


S212A
Backbone


F124Y
Backbone


































Example 9
Evaluation of Camel Variants
Variant Characterization
Camel chymosin variants evaluated in Example 7 regarding their milk clotting (C) and general proteolytic (P) activities were produced again and evaluated regarding their casein cleavage specificity C/P (Table 1 below). The C/P ratio is a measure for a coagulant's efficiency in cheese making, i.e., the yield of cheese curd obtained from a certain volume of milk. Milk clotting and general proteolytic activities were determined as described in Examples 4 and 5, respectively. In this example, however, proteolytic activity was measured without normalization for clotting activity.
Camel chymosin was analyzed as reference. C/P values of all variants are shown as relative values to wild type camel chymosin. An impact of total protein concentration in the enzyme samples on C/P was detected, and C/P values were corrected for this correlation accordingly.





TABLE 1



Analysis of camel chymosin variants







variant
mutations
Clotting (C)
Proteolytical (P)
C/P













1
L280I
G309D
E141S
Q220S
R324I
92%
125%
25%

2
L280I
G309W
F75Y
Y79S

108%
129%
78%

3
L280I
G309D
H134Q
S222G
S331Y
103%
34%
271%

4
L280I
G309D
K120Q
M223E
H239N
96%
81%
85%

5
L280I
G309D
Q220S
V213F
T342S
75%
113%
42%

6
L280I
G309D
H134Q
V213F
F281A
62%
31%
339%

7
L280I
G309D
S331Y
L224V
Y326F
91%
110%
143%

8
L280I
G309D
Y326F
V241I
E305T
135%
114%
94%

9
L280I
G309D
S331Y
F124Y
I346L
98%
123%
81%

10
L280I
G309D
M223E
L224V
L273V
93%
78%
105%

11
L280I
G309D
H134Q
M223E
L70M
116%
68%
246%

12
L280I
G309D
F75Y
S331Y
Q346E
155%
83%
172%

13
L280I
G309D
L224V
I103V
L238I
136%
89%
128%

14
L280I
G309W
L238I
T342S

124%
159%
89%

15
L280I
G309D

L70M
T342S
93%
152%
35%

16
L280I
G309D
Y79S
L224V
S212A
137%
91%
100%

17
L280I
G309D
V213F
E320T
V90L
133%
163%
46%

18
L280I
G309D
L163E
S222G
V261A
72%
49%
182%

19
L280I
G309W
S212A
V261A

104%
122%
138%

20
L280I
G309D
Q220S
L224V
H134Q
201%
52%
315%

21
L280I
G309W
K77T
R324I

160%
102%
139%

22
L280I
G309W
I361L
I103V

108%
132%
79%

24
L280I
G309D
Y79S
L273V
L163E
91%
76%
112%

25
L280I
G309D
I154L
T235S
K379P
112%
118%
112%

26
L280I
G309D
F75Y
T342S
V261A
108%
90%
141%

27
L280I
G309D
V90L
K379P
V318T
95%
135%
55%

28
L280I
G309D
V256I
V90L
E141S
109%
146%
139%

29
L280I
G309D
I154L
V261A
V367I
157%
95%
156%

30
L280I
G309D
Y326F
L273V
V90L
99%
119%
58%

31
L280I
G309D
H134Q
L163E
V318T
95%
59%
247%

32
L280I
G309D
Y79S
H134Q
Y326F
105%
66%
219%

33
L280I
G309D
Y79S
I103V
F281A
124%
66%
342%

34
L280I
G309D
V256I
V261A
K379P
146%
102%
134%

36
L280I
G309D
S222G
R324V
I154L
76%
68%
161%

37
L280I
G309D
H239N
F124Y
V90L
102%
125%
67%

38
L280I
G309D
H239N
R324I
D325Q
90%
143%
127%

39
L280I
G309W
K120Q
V367I

103%
94%
139%

40
L280I
G309D
Y326F
L70M
D325Q
96%
207%
10%

41
L280I
G309D
L224V
E320T
T235S
116%
102%
134%

42
L280I
G309D
S331Y
T342S
D325Q
145%
102%
158%

43
L280I
G309D
F124Y
Q346E
I154L
135%
94%
176%

44
L280I
G309D
V261A
R324V
F281A
71%
63%
137%

45
L280I
G309D
I361L
S212A
V318T
116%
122%
100%

46
L280I
G309D
Y79S
T342S
I154L
137%
102%
115%

47
L280I
G309D
Q346E
K77T
T235S
124%
107%
123%

48
L280I
G309D
K120Q
Y326F
K77T
90%
86%
113%

Camel wt





100%
100%
100%






































































A total of 30 out of 46 characterized variants show improved C/P compared to wild type camel chymosin (Table 2 below). A more than 3-fold improvement was observed for the three top variants 33, 6 and 20.





TABLE 2



Camel chymosin variants with improved C/P







variant
mutations
Clotting (C)
Proteolytical (P)
C/P













33
L280I
G309D
Y79S
I103V
F281A
124%
66%
342%

6
L280I
G309D
H134Q
V213F
F281A
62%
31%
339%

20
L280I
G309D
Q220S
L224V
H134Q
201%
52%
315%

3
L280I
G309D
H134Q
S222G
S331Y
103%
34%
271%

31
L280I
G309D
H134Q
L163E
V318T
95%
59%
247%

11
L280I
G309D
H134Q
M223E
L70M
116%
68%
246%

32
L280I
G309D
Y79S
H134Q
Y326F
105%
66%
219%

18
L280I
G309D
L163E
S222G
V261A
72%
49%
182%

43
L280I
G309D
F124Y
Q346E
I154L
135%
94%
176%

12
L280I
G309D
F75Y
S331Y
Q346E
155%
83%
172%

36
L280I
G309D
S222G
R324V
I154L
76%
68%
161%

42
L280I
G309D
S331Y
T342S
D325Q
145%
102%
158%

29
L280I
G309D
I154L
V261A
V367I
157%
95%
156%

7
L280I
G309D
S331Y
L224V
Y326F
91%
110%
143%

26
L280I
G309D
F75Y
T342S
V261A
108%
90%
141%

21
L280I
G309W
K77T
R324I

160%
102%
139%

28
L280I
G309D
V256I
V90L
E141S
109%
146%
139%

39
L280I
G309W
K120Q
V367I

103%
94%
139%

19
L280I
G309W
S212A
V261A

104%
122%
138%

44
L280I
G309D
V261A
R324V
F281A
71%
63%
137%

34
L280I
G309D
V256I
V261A
K379P
146%
102%
134%

41
L280I
G309D
L224V
E320T
T235S
116%
102%
134%

13
L280I
G309D
L224V
I103V
L238I
136%
89%
128%

38
L280I
G309D
H239N
R324I
D325Q
90%
143%
127%

47
L280I
G309D
Q346E
K77T
T235S
124%
107%
123%

46
L280I
G309D
Y79S
T342S
I154L
137%
102%
115%

48
L280I
G309D
K120Q
Y326F
K77T
90%
86%
113%

24
L280I
G309D
Y79S
L273V
L163E
91%
76%
112%

25
L280I
G309D
I154L
T235S
K379P
112%
118%
112%

10
L280I
G309D
M223E
L224V
L273V
93%
78%
105%

Camel wt





100%
100%
100%






















































Statistical Analysis of the Positional and Mutational Effects on C/P
A statistical, PCA based, analysis was used to identify single mutations with positive effects on the specificity of milk clotting over general casein proteolysis (C/P) of camel chymosin. The following mutations were found to be beneficial for high C/P ratios:
H134Q, F281A, I103V, V256I, I154L, S222G, L224V, Q346E, S331Y, K77T, V367I, G309D, V261A, D325Q, L280I, D117N, L163E, S212A
Example 10
Evaluation of Camel Variants
Variant Characterization
Based on the positional and mutational effects determined in Example 7, another set of camel chymosin variants was generated with multiple substitutions as compared to wild type camel chymosin and evaluated regarding their casein substrate specificity (C/P) as described in Example 9 (Table 1 below).





TABLE 1



Analysis of camel chymosin variants







variant
mutations
Clotting (C)
Proteolytical (P)
C/P


















1
L70M
Y79S
D117N
H134Q
M223E
V256I
L280I
G309D
Q346E

132%
116%
117%

2
L70M
Y79S
D117N
H134Q
M223E
L280I
G309W
S331Y


131%
56%
194%

3
L70M
D117N
H134Q
M223E
V256I
L280I
G309D
S331Y
K379P

109%
75%
135%

4
L70M
D117N
H134Q
S212A
M223E
V261A
L280I
G309D
V367I

83%
115%
108%

5
L70M
D117N
H134Q
D156V
L280I





135%
108%
137%

6
L70M
K77T
V90L
D117N
H134Q
D202Q
M223E
L280I
G309D

135%
113%
124%

7
L70M
Y79S
D117N
H134Q
M223E
V261A
L280I
G309D
E320T

141%
124%
143%

8
L70M
V109L
H134Q
M223E
G309D





82%
86%
87%

9
L70M
D117N
F124Y
H134Q
M223E
L238I
L280I
G309D
V367I

105%
97%
115%

10
L70M
D117N
H134Q
S212A
M223E
L280I
G309W
Q346E


101%
79%
133%

11
L70M
D117N
H134Q
D156V
M223E
L280I
G309D
E320T
Q346E

153%
101%
119%

12
L70M
V109L
D117N
H134Q
L224V
L280I
G309D



98%
71%
128%

13
L70M
D117N
H134Q
D202Q
M223E
V261A
L280I



116%
144%
126%

14
L70M
D117N
D202Q
M223E
L224V
L280I
G309D



85%
126%
111%

15
L70M
K77T
D117N
H134Q
S212A
M223E
V256I
L280I
G309D

154%
130%
129%

16
L70M
H134Q
D156V
M223E
L280I
G309W




136%
131%
137%

17
L70M
V90L
D117N
H134Q
M223E
L238I
V256I
L280I
G309D

121%
101%
97%

18
L70M
D117N
H134Q
S212A
M223E
S331Y




124%
76%
151%

19
L70M
V109L
D117N
F124Y
H134Q
M223E
V261A
L280I
G309W

96%
98%
128%

20
L70M
V90L
H134Q
M223E
L280I
E320T




138%
110%
98%

21
L70M
N108D
D117N
H134Q
M223E
G309W
E320T



187%
151%
138%

22
V109L
D117N
H134Q
M223E
L238I
L280I
G309D
E320T


110%
93%
106%

23
L70M
D117N
H134Q
M223E
G309D
Q346E
V367I
K379P


67%
102%
118%

24
L70M
N108D
D117N
V261A
L280I
G309D




95%
117%
102%

25
L70M
D117N
H134Q
L238I
L280I
G309W
K379P



97%
92%
113%

26
L70M
Y79S
D117N
M223E
L280I
K379P




137%
123%
129%

27
D117N
H134Q
M223E
L224V
V256I
L280I




132%
102%
127%

28
L70M
K77T
N108D
D117N
H134Q
M223E
L280I
Q346E


167%
106%
166%

29
L70M
Y79S
N108D
D117N
F124Y
H134Q
D202Q
M223E
L280I
G309D
183%
57%
151%

Bovine wt










100%
100%
100%





















































A total of 26 out of 29 variants show improved C/P ratios, as compared to wild type camel chymosin. A 2-fold improvement was observed for the best variant (Table 2, below).





TABLE 2



Camel chymosin variants with improved C/P







variant
mutations
Clotting (C)
Proteolytical (P)
C/P


















2
L70M
Y79S
D117N
H134Q
M223E
L280I
G309W
S331Y


131%
56%
194%

28
L70M
K77T
N108D
D117N
H134Q
M223E
L280I
Q346E


167%
106%
166%

18
L70M
D117N
H134Q
S212A
M223E
S331Y




124%
76%
151%

29
L70M
Y79S
N108D
D117N
F124Y
H134Q
D202Q
M223E
L280I
G309D
183%
57%
151%

7
L70M
Y79S
D117N
H134Q
M223E
V261A
L280I
G309D
E320T

141%
124%
143%

21
L70M
N108D
D117N
H134Q
M223E
G309W
E320T



187%
151%
138%

5
L70M
D117N
H134Q
D156V
L280I





135%
108%
137%

16
L70M
H134Q
D156V
M223E
L280I
G309W




136%
131%
137%

3
L70M
D117N
H134Q
M223E
V256I
L280I
G309D
S331Y
K379P

109%
75%
135%

10
L70M
D117N
H134Q
S212A
M223E
L280I
G309W
Q346E


101%
79%
133%

15
L70M
K77T
D117N
H134Q
S212A
M223E
V256I
L280I
G309D

154%
130%
129%

26
L70M
Y79S
D117N
M223E
L280I
K379P




137%
123%
129%

12
L70M
V109L
D117N
H134Q
L224V
L280I
G309D



98%
71%
128%

19
L70M
V109L
D117N
F124Y
H134Q
M223E
V261A
L280I
G309W

96%
98%
128%

27
D117N
H134Q
M223E
L224V
V256I
L280I




132%
102%
127%

13
L70M
D117N
H134Q
D202Q
M223E
V261A
L280I



116%
144%
126%

6
L70M
K77T
V90L
D117N
H134Q
D202Q
M223E
L280I
G309D

135%
113%
124%

11
L70M
D117N
H134Q
D156V
M223E
L280I
G309D
E320T
Q346E

153%
101%
119%

23
L70M
D117N
H134Q
M223E
G309D
Q346E
V367I
K379P


67%
102%
118%

1
L70M
Y79S
D117N
H134Q
M223E
V256I
L280I
G309D
Q346E

132%
116%
117%

9
L70M
D117N
F124Y
H134Q
M223E
L238I
L280I
G309D
V367I

105%
97%
115%

25
L70M
D117N
H134Q
L238I
L280I
G309W
K379P



97%
92%
113%

14
L70M
D117N
D202Q
M223E
L224V
L280I
G309D



85%
126%
111%

4
L70M
D117N
H134Q
S212A
M223E
V261A
L280I
G309D
V367I

83%
115%
108%

22
V109L
D117N
H134Q
M223E
L238I
L280I
G309D
E320T


110%
93%
106%

24
L70M
N108D
D117N
V261A
L280I
G309D




95%
117%
102%

Camel wt










100%
100%
100%


















































Statistical Analysis of the Positional and Mutational Effects on C/P
A statistical, PCA based, analysis was used to identify single mutations with positive effects on the specificity of milk clotting over general casein proteolysis (C/P) of camel chymosin. The following mutations were found to be beneficial for high C/P ratios:
S331Y, Y79S, K77T, D117N, H134Q, N108D, G309W, L224V, D156V, L280I, M223E, V367I, F114Y
Example 11
Evaluation of Camel Variants
A statistical, PCA based, analysis was performed on the combined set of variants from Examples 9 and 10, and single mutations were identified with positive effects on the specificity of milk clotting over general casein proteolysis (C/P) of camel chymosin. The following mutations were found to be beneficial for high C/P ratios:
F281A, H134Q, I103V, S331Y, S222G, I154L, L280I, G309D, D117N, L224V, N108D, L163E, G309W, K77T, Y79S
These mutations agree well with the beneficial mutations determined in Examples 9 and 10.
Structural Evaluation of Positional and Mutational Effects on C/P
As seen in Example 8, the majority of beneficial mutations are again located distant from the substrate binding cleft. Only L280I and F281A are located directly in the cleft (Gilliland et al. 1990). 1280 points into the hydrophobic core of the C-terminal lobe. This mutation might therefore lead to subtle conformational changes of the binding cleft and, thus, influence substrate specificity. Position 281 is part of the S2 binding site and interacts with the P2 position in the casein substrate. A mutation in this position is very likely to have an impact on casein binding and, thus, proteolysis. Mutations G309W and S331Y are positioned on the surface of the C-terminal lobe in a region that has been described to interact with K-casein to aid substrate binding in the catalytic cleft (Gilliland et al. 1990). These mutations might therefore have a positive impact on substrate binding. I154L and D156V, and L163E represent changes to the core of the N-terminal lobe, possibly leading to subtle structural rearrangements of the enzyme with impact on catalytic activity. Mutations S222G and L224V introduce changes into the beta sheet that might interact with the protein N-terminus in its activated form (Langholm Jensen et al.). Potential effects on the activation state of the enzyme could result in shifted casein substrate specificity. The remaining hit mutations K77T, Y79S, I103V, N108D, D117N, and H134Q are located on the surface of the N-terminal lobe and, with exception of I103V, represent exchanges of polar amino acids. These changes on the surface of the enzyme most probably influence interactions with casein molecules leading to improved specificity in favor of κ-casein.
REFERENCES


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