Variants of chymosin with improved milk-clotting properties

An isolated chymosin polypeptide variant of a parent polypeptide having chymosin activity, wherein the amino acid sequence of the variant differs from the amino acid sequence of the parent by having an alteration comprising a substitution at at least one amino acid position of the parent sequence corresponding to one or both of positions 70 and 75, wherein the corresponding position in the parent sequence is determined by alignment of the parent sequence with SEQ ID NO:1 (bovine chymosin),

• wherein the variant has chymosin activity;
• and wherein:
• (i) the amino acid sequence of the parent polypeptide has at least 90% sequence identity with one or both of (a) the amino acid sequence from position 59 to position 381 of SEQ ID NO:1 (the mature bovine chymosin polypeptide) and (b) the amino acid sequence from position 59 to amino acid position 381 of 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:1 or SEQ ID NO:2, respectively;
• and
• (ii) the amino acid sequence of the isolated variant polypeptide has less than 100% sequence identity with the amino acid sequence from position 59 to position 381 of SEQ ID NO:1 and has less than 100% sequence identity with the amino acid sequence from position 59 to position 381 of SEQ ID NO:2.

A method for making an isolated chymosin polypeptide variant comprising:

• (a) producing and isolating a chymosin polypeptide variant of a parent polypeptide having chymosin activity, wherein the amino acid sequence of the variant differs from the amino acid sequence of the parent by having an alteration comprising a substitution at at least one amino acid position of the parent sequence corresponding to one or both of positions 70 and 75, wherein the corresponding position in the parent sequence is determined by alignment of the parent sequence with SEQ ID NO:1 (bovine chymosin), thereby obtaining the isolated chymosin polypeptide variant, wherein the variant has chymosin activity;
• wherein:
• the amino acid sequence of the parent polypeptide has at least 65% sequence identity with the amino acid sequence from position 59 to position 381 of SEQ ID NO:1 (the mature bovine chymosin polypeptide);
• 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 one or both of (a) the amino acid sequence from position 59 to amino acid position 381 of SEQ ID NO:1 and (b) the amino acid sequence from position 59 to amino acid position 381 of 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:1 or SEQ ID NO:2, respectively.