Protein engineering of the milk-clotting aspartic proteinases

Abstract

Calf Chymosin and a fungal protease from Mucor pusillus (Mucor rennin) are members of the aspartic proteinases used as milk-coagulants in cheese industry. A system for production of recombinant chymosin as inclusion bodies in Escherichia coli cells and its refolding into the active form was established. Another expression system for production of Mucor rennin in Saccharomyces cerevisiae was also established. Mucor rennin was efficiently excreted from the yeast host as a heavily glycosylated form. Glycosylation affected both the secretion and the enzyme properties. Site-directed mutagenesis of the Tyr residue at position 75 in chymosin and Mucor rennin revealed its crucial role in catalytic function of the aspartic proteinases. The results also suggested possibility to improve practical properties of the milk-clotting enzymes by site-directed mutagenesis.

Key Words:

• Chymosin
• EC 3.4.23.4
• Mucor pusillus rennin
• inclusion body
• secretion
• glycosylation
• site-directed mutagenesis
• subsite
• heat stability