Production and purification of protease from a Bacillus subtilis that can deproteinize crustacean wastes☆

A protease-producing microorganism was isolated in northern Taiwan and identified as a strain of Bacillus subtilis. B. subtilis Y-108 thus isolated can be used for deproteinization of crustacean wastes in the preparation of chitin. For deproteinization tests, liquid phase fermentation of untreated shrimp shell, crab shell, and lobster shell wastes with this microbe showed protein removal of 88, 67, and 83%, respectively. In contrast, the protein removal of the acid treated wastes was 76, 62, 56%, respectively. The optimized conditions for protease production was found when the culture was shaken at 30°C for 3 days in 100 ml of medium (phosphate buffer adjusted to pH 6.0) containing 7% shrimp and crab shell powder (SCSP), 0.1% K2HPO4, 0.05% MgSO4, 1.0% arabinose, 1.5% NaNO3, and 1.5% CaCl2. Under such conditions, the protease of B. subtilis Y-108 attained the highest activity. It was as high as 20.2 U/ml. The protease was purified in a three-step procedure involving ammonium sulfate precipitation, DEAE-Sepharose CL-6B ionic exchange chromatography, and Sephacryl S-200 gel permeation chromatography. The enzyme was shown to have a relative molecular weight of 44 kDa by SDS polyacrylamide gel electrophoresis. The protease was most active at pH 8.0 and 50°C with casein as substrate. The protease was activated by Mn+2, Fe+2, Zn+2, Mg+2, Co+2, but inhibited completely by Hg+2. The protease was also inhibited by metal-chelating agent such as EDTA, sulfhydryl reagents as β-mercaptoethanol, and by cysteine hydrochloride, histidine, glycerol. The EDTA was the most effective inhibitor that caused complete inhibition of protease. It was concluded that this enzyme is a metal-chelator-sensitive neutral protease.