Variants of chymosin with improved milk-clotting properties

Patent metadata

Field	Value
Publication number	US-9930899-B2
Application number	US-201314402567-A
Country	US
Kind code	B2
Filing date	May 22, 2013
Priority date	May 25, 2012
Publication date	Apr 3, 2018
Grant date	Apr 3, 2018

Abstract

Variants of chymosin with improved milk-clotting properties.

First claim

The invention claimed is:

1. A method for making an isolated chymosin polypeptide variant comprising:
(a) producing a chymosin polypeptide variant having an alteration at one or more positions in its amino acid sequence relative to a parent polypeptide having chymosin activity, wherein the parent polypeptide has at least 65% sequence identity with the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO: 1 (bovine chymosin), wherein the alteration comprises a substitution at an amino acid position corresponding to position 117 as determined by an alignment of the amino acid sequence of the parent polypeptide with the amino acid sequence of the polypeptide of SEQ ID NO: 1 (bovine chymosin), and
(b) isolating the polypeptide variant of step (a), thereby obtaining the isolated chymosin polypeptide variant,
wherein the variant has fewer than 30 amino acid alterations in the region from amino acid position 59 to amino acid position 381 as compared to the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO: 1 (bovine chymosin) or as compared to the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO: 2 (Camel chymosin), as determined by an alignment of the amino acid sequence of the variant with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2, respectively, and has chymosin activity.

2. The method of claim 1 , wherein the isolated chymosin polypeptide variant has:
a chymosin activity giving a higher clotting activity to proteolytical activity (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 of claim 1 , wherein the alteration comprises the amino acid substitution D117N.

4. The method of claim 1 wherein the alteration comprises the amino acid substitutions Y79S, D117N and I321L.

5. The method of claim 1 , wherein the parent polypeptide has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).

6. The method of claim 1 , wherein the parent polypeptide has at least 95% sequence identity with the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO: 2 (Camel chymosin).

7. An isolated chymosin polypeptide variant obtained by a method that comprises:
(a) producing a chymosin polypeptide variant having an alteration at one or more positions in its amino acid sequence relative to a parent polypeptide having chymosin activity, wherein the parent polypeptide has at least 65% sequence identity with the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO: 1 (bovine chymosin), wherein the alteration comprises a substitution at an amino acid position corresponding to position 117 as determined by an alignment of the amino acid sequence of the parent polypeptide with the amino acid sequence of the polypeptide of SEQ ID NO: 1 (bovine chymosin); and
(b) isolating the polypeptide variant of step (a), thereby obtaining the isolated chymosin polypeptide variant,
wherein the variant has fewer than 30 amino acid alterations in the region from amino acid position 59 to amino acid position 381 as compared to the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO: 1 (bovine chymosin) or as compared to the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO: 2 (Camel chymosin), as determined by an alignment of the amino acid sequence of the variant with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2, respectively, and has chymosin activity.

8. An isolated chymosin polypeptide variant having an alteration at one or more positions in its amino acid sequence relative to a parent polypeptide having chymosin activity, wherein the parent polypeptide has at least 90% sequence identity with the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO: 1 (bovine chymosin), wherein the alteration comprises a substitution at an amino acid position corresponding to position 117 as determined by an alignment of the amino acid sequence of the parent polypeptide with the amino acid sequence of the polypeptide of SEQ ID NO: 1 (bovine chymosin), wherein:
the variant has chymosin activity;
the variant has less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin); and
the variant has fewer than 30 amino acid alterations in the region from amino acid position 59 to amino acid position 381 as compared to the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO: 1 (bovine chymosin), as determined by an alignment of the amino acid sequence of the variant with the amino acid sequence of SEQ ID NO: 1.

9. The isolated chymosin polypeptide variant of claim 8 , wherein
the parent polypeptide has at least 97% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin); and
the isolated chymosin polypeptide variant comprises fewer than 10 amino acid alterations in the region from amino acid position 59 to amino acid position 381 as compared to the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO: 1 (bovine chymosin), as determined by an alignment of the amino acid sequence of the variant with the amino acid sequence of SEQ ID NO: 1.

10. The isolated chymosin polypeptide variant of claim 8 , wherein the alteration comprises the amino acid substitution D117N.

11. The isolated chymosin polypeptide variant of claim 8 , wherein the alteration comprises the amino acid substitutions Y79S, D117N, and I321L.

12. An isolated chymosin polypeptide variant having an alteration at one or more positions in its amino acid sequence relative to a parent polypeptide having chymosin activity, wherein the parent polypeptide has at least 90% sequence identity with the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO: 2 (Camel chymosin), wherein the alteration comprises a substitution at an amino acid position corresponding to position 117 as determined by an alignment of the amino acid sequence of the parent polypeptide with the amino acid sequence of the polypeptide of SEQ ID NO: 1 (bovine chymosin), wherein:
the variant has chymosin activity; and
the variant has less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 2 (Camel chymosin); and
the variant has fewer than 30 amino acid alterations in the region from amino acid position 59 to amino acid position 381 as compared to the mature polypeptide from amino acid position 59 to amino acid position 381 of SEQ ID NO: 2 (Camel chymosin), as determined by an alignment of the amino acid sequence of the variant with the amino acid sequence of SEQ ID NO: 2.

13. The isolated chymosin polypeptide variant of claim 12 , wherein the alteration comprises the amino acid substitutions Y79S, D117N and I321L.

14. A method for making a milk-based food or feed product comprising adding an effective amount of the isolated chymosin polypeptide variant according to claim 8 to food or feed ingredient(s) comprising milk.

15. The method for making a milk-based food or feed product according to claim 14 , wherein the milk is selected from the group consisting of soya milk, sheep milk, goat milk, buffalo milk, yak milk, lama milk, camel milk and cow milk.

16. The method for making a milk-based food or feed product according

Assignees

• Chr Hansen As

Inventors

• Van Den Brink Johannes Maarten
• Lund Martin
• Jacobsen Jonas
• Hansen Sari Charlotte
• Jeppesen Iben

Classifications

• A23C19/0326

  Rennet produced by fermentation, e.g. microbial rennet; Rennet produced by genetic engineering · CPC title

• A23K20/189

  Enzymes · CPC title

• C12N15/52 Primary

  Genes encoding for enzymes or proenzymes · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title

• A23C9/1209 Primary

  Proteolytic or milk coagulating enzymes, e.g. trypsine · CPC title