Variants of chymosin with improved milk-clotting properties

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.