Variants of chymosin with improved milk-clotting properties

Patent metadata

Field	Value
Publication number	US-11555182-B2
Application number	US-202117208104-A
Country	US
Kind code	B2
Filing date	Mar 22, 2021
Priority date	May 19, 2016
Publication date	Jan 17, 2023
Grant date	Jan 17, 2023

Abstract

Variants of chymosin with improved milk clotting properties.

First claim

The invention claimed is:

1. An isolated chymosin polypeptide variant comprising an alteration in one or more amino acid positions relative to a parent polypeptide having chymosin activity, wherein the alteration comprises a substitution, a deletion, or an insertion at amino acid position M157 of the parent polypeptide, and a further alteration selected from (a) a substitution, deletion, or insertion at one or more amino acid positions of the parent polypeptide selected from G70, 5132, V136, K231, and N291, and (b) an L222I substitution relative to the parent polypeptide, wherein the variant does not comprise an L222V substitution relative to the parent polypeptide, and wherein:
(i) the amino acid position of the parent polypeptide is determined by alignment of the parent polypeptide with the polypeptide of SEQ ID NO:2 (camel chymosin),
(ii) the parent polypeptide has at least 80% amino acid sequence identity with SEQ ID NO: 2,
(iii) the variant has fewer than 30 amino acid alterations as compared to SEQ ID NO: 2, as determined by an alignment of the amino acid sequence of the variant with the amino acid sequence of SEQ ID NO: 2, and
(iv) the variant has one or both of (i) a higher ratio of specific clotting activity to proteolytic activity (“C/P ratio”) and (ii) at least 85% of the specific clotting activity of its parent polypeptide.

2. The isolated chymosin polypeptide variant of claim 1 , having an additional alteration in its amino acid sequence relative to the parent polypeptide selected from a substitution, deletion, or insertion at one or more of amino acid positions Y11, K19, Y21, Q56, D59, S74, H76, 196, 5164, L166, 8242, Y243, N249, G251, N252, 8254, M256, 5273, Q280, F282, L295, and V309, of the parent polypeptide, wherein the amino acid position of the parent polypeptide is determined by alignment of the parent polypeptide with the polypeptide of SEQ ID NO:2.

3. The isolated chymosin polypeptide variant of claim 1 , having an additional alteration in its amino acid sequence relative to the parent polypeptide selected from a substitution, deletion, or insertion at one or more of amino acid positions V32, R67, N100, and L130 of the parent polypeptide, wherein the amino acid position of the parent polypeptide is determined by alignment of the parent polypeptide with the polypeptide of SEQ ID NO:2.

4. The isolated chymosin polypeptide variant of claim 1 , wherein the alteration at position M157 is the substitution M157L.

5. The isolated chymosin polypeptide variant according to claim 1 , wherein the further alteration is one or more selected from S132A, G70D, V136I, L222I, K231N, and N291Q.

6. The isolated chymosin polypeptide variant according to claim 2 , wherein the additional alteration is one or more selected Y11I, Y11V, K19T, Y21S, Q56H, D59N, S74D, H76Q, I96L, S164G, L166V, R242E, R242D, Y243E, N249E, N249D, G251D, N252D, R254E, M256L, S273D, S273Y, Q280E, F282E, L295K, and V309I.

7. The isolated chymosin polypeptide variant according to claim 3 , wherein the additional alteration is one or more selected from of V32L, R67Q, N100Q, and L130I.

8. An isolated chymosin polypeptide variant according to claim 1 , wherein the alteration comprises substitutions selected from:
N100Q, L130I, S132A, M157L, and K231N;
R67Q, G70D, M157L, L222I, and N291Q; and
V32L, R67Q, V136I, M157L, and N291Q.

9. An isolated chymosin polypeptide variant according to claim 1 , wherein the alteration comprises substitutions selected from:
R67Q, I96L, L130I, M157L, K231N, and R242E;
R67Q, M157L, L222I, K231N, and V248I;
R67Q, I96L, M157L, L222I, and K231N;
R67Q, N100Q, M157L, R242E, M256L;
R67Q, G70D, M157L, R242E, and V248I;
V32L, R67Q, M157L, L222I, and R242E;
Y11V, R67Q, M157L, V248I, and M256L;
R67Q, V136I, M157L, L222I, and V248I;
L130I, M157L, V248I, M256L, and N291Q;
L130I, V136I, M157L, L222I, and N292H;
Y11V, R67Q, N100Q, L130I, V136I, and M157L;
V32L, R67Q, M157L, M256L, and N291Q;
Y11V, R67Q, L130I, M157L, L222I, and K231N;
I45V, L130I, M157L, K231N, and R242E;
I45V, R67Q, L130I, M157L, L222I, and K231N;
Y11V, R67Q, L130I, M157L, L222I, and R242E;
R67Q, G70D, L130I, M157L, K231N, and M256L;
R67Q, L130I, M157L, D158S, R242E, and N291Q;
R67Q, I96L, N100Q, L130I, M157L, and N292H;
V32L, R67Q, G70D, N100Q, and M157L;
V32L, R67Q, L130I, M157L, K231N, and M256L; and
R67Q, L130I, M157L, R242E, M256L, and N292H.

10. A method for making an isolated chymosin polypeptide variant according to claim 1 , comprising:
making an alteration at one or more positions in a parent polypeptide having chymosin activity, wherein the alteration comprise a substitution, a deletion, or an insertion at amino acid position M157 of the parent polypeptide, and a further alteration selected from (a) a substitution, deletion, or insertion at one or more amino acid positions of the parent polypeptide selected from G70, 5132, V136, K231, and N291, and (b) an L222I substitution relative to the parent polypeptide, to obtain the variant, wherein the variant does not comprise an L222V substitution relative to the parent polypeptide, and wherein:
(i) the amino acid position of the parent polypeptide is determined by alignment of the parent polypeptide with SEQ ID NO:2 (camel chymosin),
(ii) the parent polypeptide has at least 80% amino acid sequence identity with SEQ ID NO: 2, and
(iii) the variant has fewer than 30 amino acid alterations as compared to SEQ ID NO: 2, as determined by an alignment of the amino acid sequence of the variant with the amino acid sequence of SEQ ID NO: 2.

11. The method according to claim 10 , further comprising making an additional alteration in the amino acid sequence of the variant relative to the parent polypeptide selected from a substitution, deletion, or insertion at one or more of amino acid positions Y11, K19, Y21, Q56, D59, S74, H76, 196, 5164, L166, R242, Y243, N249, G251, N252, R254, M256, 5273, Q280, F282, L295, and V309, of the parent polypeptide, wherein the amino acid position of the parent polypeptide is determined by alignment of the parent polypeptide with the polypeptide of SEQ ID NO:2.

12. The method according to claim 10 , further comprising making an additional alteration in the amino acid sequence of the variant relative to the parent polypeptide selected from a substitution, deletion, or insertion at one or more of amino acid positions V32, R67, N100, and L130 of the parent polypeptide, wherein the amino acid position of the parent polypeptide is determined by alignment of the parent polypeptide with the polypeptide of SEQ ID NO:2.

13. The method according to claim 10 , wherein the alteration at position M157 is the substitution M157L.

14. The method according to claim 10 , wherein the further alteration is one or more selected from S132A, G70D, V136I, L222I, K231N, and N291Q.

15. The method according to claim 11 , wherein the additional alteration is one or more selected from Y11I, Y11V, K19T, Y21S, Q56H, D59N, S74D, H76Q, I96L, S164G, L166V, R242E, R242D, Y243E, N249E, N249D, G251D, N252D, R254E, M256L, S273D, S273Y, Q280E, F282E, L295K, and V309I.

16. The method according to claim 2 , wherein the additional alteration is one or more selected from V32L, R67Q, N100Q, and L130I.

17. The method according to claim 10 , wherein the alteration comprises substitutions selected from:
N100Q, L130I, S132A, M157L, and K231N;
R67Q, G70D, M157L, L222I, and N291Q; and
V32L, R67Q, V136I, M157L, and N291Q.

18. The method according to claim 10 , wherein the alteration comprises substitutions selected from:
R67Q, I9

Assignees

• Chr Hansen As

Inventors

• Jaeckel Christian
• Lund Martin
• Van Den Brink Johannes Maarten

Classifications

• A23C19/072

  Cheddar type {or similar hard cheeses without eyes} · CPC title

• C12N9/6483 Primary

  Chymosin (3.4.23.4), i.e. rennin · CPC title

• C12Y304/23004

  Chymosin (3.4.23.4), i.e. rennin · CPC title

• A23C19/076

  Soft unripened cheese, e.g. cottage or cream cheese · CPC title

• A23C19/04

  characterised by the use of specific enzymes of vegetable or animal origin (A23C19/032 takes precedence) · CPC title