Purification of 35K protease from the digestive juice of Bombyx mori

35K protease was purified from the digestive juice of Bombyx mori by a series of chromatography using Butyl-Toyopearl, Sephadex G-50 and DEAE-Sephacel. The purified protease gave a single protein band with a molecular mass of 35,000 on SDS-PAGE and its pi was 9. 1. It had optimal activity at pH11 and in the temperature under 40°C. The enzyme activity was almost completely lost at 60°C and at pH4. It was slightly inhibited by Cu²⁺and Mn²⁺, and strongly by diisopropylfluorophosphate, phenylmethylsulfonylfluoride and chymostatin, suggesting that the enzyme may be a chymotrypsin-like protease.