Substrate Specificity of the Milk-clotting Protease from Mucor pusillus Determined on the Oxidized B-chain of Insulin

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Abstract
The specificity of the milk-clotting protease from the fungus Mucor pusillus has been determined on the oxidized B- chain of insulin. After digestion of the oxidized B chain of insulin with M. pusillus protease (1,000/1 ratio of sub- strafe to enzyme) for 20 hours at pH 4 and 37 C, the peptides formed were separated by high voltage eleetrophoresis- chromatography, eluted from the chroma- togram and the amino acid composition of each peptide was determined after hy- drolysis with 6 ~ HC1. Hydrolysis of the oxidized B-chain of insulin by M. pusiIlus protease was observed at the bonds Phe 1 -- Yal 2, Leu 11 -- Val 12, Ala 14 -- Leu 15, Leu 15 -- Tyr 16, Tyr 16 ~ Leu 17, Leu 17 -- Val 18, Phe 24 -- Phe 25 and Phe 25 -- Tyr 26. Specificity of M. pusillus protease is compared with that of other milk-clotting enzymes.