PURIFICATION AND CHARACTERISTICS OF MILK CLOTTING PROTEASE FROM ASPERGILLUS-USTUS

A strain of mold having milk clotting protease was isolated from soil, and identified as Aspergillus ustus. The crude enzyme from wheat bran culture was purified by salting-out and several column chromatographic methods. In the final stage, the enzyme was refined to a unique protein of which milk clotting activity (MCA) was 18.7 fold of the crude enzyme. The molecular weight was estimated to be 86 kDa. MCA increased with a decrease of pH within the pH range 5.0 to 6.75 but proteolytic activity (PA) was optimum at pH 6.0, and both activities were stable in pH 6.3-6.0. Optimum temperature was 63-degrees-C for MCA and 50-degrees-C for PA. Inactivation by heating was observed at 55-degrees-C in both MCA and PA. Ca2+ was much effective on MCA, but not on PA. Km value of PA to casein was 0.27%. Some metal ions affected both activities, and inhibition of NEM and SDS were considerable. Decomposition of alpha(s1)-casein was lower, and that of beta-casein was higher by the A. ustus enzyme than by chymosin, but no considerable difference in their activities was observed toward kappa-casein.