Chymosine enzyme variants

Patent metadata

Field	Value
Publication number	US-10253305-B2
Application number	US-201715782528-A
Country	US
Kind code	B2
Filing date	Oct 12, 2017
Priority date	May 3, 2012
Publication date	Apr 9, 2019
Grant date	Apr 9, 2019

Abstract

A variant polypeptide having chymosin activity, wherein the variant has an amino acid sequence which, when aligned with the chymosin comprising the sequence set out in SEQ ID NO: 2, comprises at least one substitution of an amino acid residue corresponding to any of amino acids 2, 22, 40, 48, 50, 51, 53, 61, 62, 76, 88, 98, 99, 109, 112, 117, 125, 126, 135, 144, 160, 161, 163, 187, 189, 194, 200, 201, 202, 203, 221, 223, 240, 242, 244, 254, 267, 271, 273, 278, 280, 284, 289, 292, 294 or 295
said positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having chymosin activity. Such a variant polypeptide may be used in the preparation of a cheese.

First claim

The invention claimed is:

1. A polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 2, wherein the polypeptide has chymosin activity; increased specificity, as compared to the polypeptide of SEQ ID NO: 2; and comprises at least one substitution corresponding to a substitution in the polypeptide of SEQ ID NO: 2 selected from D98V, D98I, D98S, D98L, D98T, D98A, D98G, S273F, S273T, S273A, S273G, S273P, S273Q, and S273N.

2. The polypeptide of claim 1 , wherein the polypeptide has decreased proteolysis in a cheese matrix and/or increased productivity as compared with the polypeptide of SEQ ID NO:2.

3. The polypeptide of claim 2 , wherein the polypeptide has reduced curd targeting or reduced thermostability as compared with the polypeptide of SEQ ID NO:2.

4. The polypeptide of claim 2 , further comprising at least one substitution of an amino acid residue corresponding to a position in the polypeptide of SEQ ID NO:2 selected from
48, 50, 51, 61, 62, 109, 117, 126, 135, 144, 160, 161, 201, 202, 203, 221, 223, 240, 242, 244, 254, 267, 280, 292, and 295.

5. The polypeptide of claim 2 , further comprising at least one substitution of an amino acid residue at a position corresponding to a position in the polypeptide of SEQ ID NO:2 selected from
48, 50, 51, 53, 109, 126, 135, 144, 160, 201, 221, 242, 267, and 280,

and wherein the polypeptide has reduced temperature stability as compared with the polypeptide of SEQ ID NO:2.

6. The polypeptide of claim 1 , wherein the polypeptide has increased specific activity or increased expression level as compared with the polypeptide of SEQ ID NO:2.

7. The polypeptide of claim 6 , further comprising at least one substitution of an amino acid residue at a position corresponding to a position in the polypeptide of SEQ ID NO:2 selected from
98, 109, 112, 117, 126, 135, 144, 161, 202, 203, 221, 223, 240, 244, 254, 273, and 295,

and wherein the polypeptide has increased specific activity as compared with the polypeptide of SEQ ID NO:2.

8. The polypeptide of claim 6 , further comprising at least one substitution of an amino acid residue at a position corresponding to a position in the polypeptide of SEQ ID NO:2 selected from
50, 53, 109, 135, 144, 160, 201, 242, 267, 280, and 295,

and wherein the polypeptide has an increased expression level as compared with the polypeptide of SEQ ID NO:2.

9. The polypeptide of claim 1 , further comprising at least one substitution of an amino acid residue at a position corresponding to a position in the polypeptide of SEQ ID NO:2 selected from
2, 22, 40, 76, 88, 98, 99, 112, 125, 144, 163, 187, 189, 194, 200, 223, 271, 278, 284, 289, and 294,

and wherein the polypeptide has decreased specificity as compared with the polypeptide of SEQ ID NO:2.

10. The polypeptide of claim 1 , further comprising at least one substitution of an amino acid residue at a position corresponding to a position in the polypeptide of SEQ ID NO:2 selected from
48, 50, 53, 144, 160, 201, 242, 267, and 280,

and wherein the polypeptide has increased clotting activity at low pH as compared with the polypeptide of SEQ ID NO:2.

11. The polypeptide of claim 1 , further comprising at least one substitution of an amino acid residue at a position corresponding to a position in the polypeptide of SEQ ID NO:2 selected from
51, 126, 135, and 221,

and wherein the polypeptide has decreased proteolytic activity at low pH as compared with the polypeptide of SEQ ID NO:2.

12. The polypeptide of claim 1 , further comprising at least one substitution of an amino acid residue at a position corresponding to a position in the polypeptide of SEQ ID NO:2 selected from
51, 109, 126, 135, and 221,

and wherein the polypeptide has decreased proteolytic activity at low temperature as compared with the polypeptide of SEQ ID NO:2.

13. The polypeptide of claim 1 which comprises one or more additional substitutions.

14. The polypeptide of claim 1 having at least 95% sequence identity with the polypeptide of SEQ ID NO:2.

15. A composition comprising the polypeptide according to claim 1 .

16. The polypeptide of claim 1 capable of being used in preparation of a cheese.

17. A process for preparing a cheese, wherein said method comprises clotting milk by adding an effective amount of the polypeptide of claim 1 to milk and further processing the clotted milk to manufacture cheese.

Assignees

• Dsm Ip Assets Bv

Inventors

• Dekker Petrus Jacobus Theodorus
• De Jong Rene Marcel
• Muijlwijk Cornelis Marinus

Classifications

• A23C19/0684

  Soft uncured Italian cheeses, e.g. Mozarella, Ricotta, Pasta filata cheese; Other similar stretched cheeses · CPC title

• A23C19/032 Primary

  characterised by the use of specific microorganisms, or enzymes of microbial origin · CPC title

• C12N9/6483 Primary

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

• A23C2220/202

  Genetic engineering of microorganisms used in dairy technology · CPC title

• A23C19/0326

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