PURIFICATION AND CHARACTERIZATION OF A MILK CLOTTING PROTEASE FROM MUCOR-BACILLIFORMIS

An acid protease having milk clotting activity has been isolated from Mucor bacilliformis cultures. The enzyme was basically purified by ion ic exchange chromatography. An average yield of 29 mg purified product was obtained from 100 mL crude extract. As purity criteria, SDS-PAGE, reverse-phase HPLC, and N-terminal analysis were performed. The prote ase is a protein composed of a single polypeptide chain with glycine a t the N-terminus. The mol wt is approx 32,000, and its amino acid comp osition is very similar to those of other fungal proteases. As expecte d, its clotting activity was drastically inhibited by pepstatin A acti on. On the other hand, its instability against heat treatment and its clotting/proteolytic activity ratio indicate that it may be considered as a potential substitute for bovine chymosin.