Production and purification of protease from a Bacilus suilis that can deproteinize crustacean wastes

摘要

A protease-producing microorganism was isolated in northern Taiwan and identified as a strain of Bacillus subtilis, B. subtilis Y-1408 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 7% shrimp and crab shell powder(SPSP), 0.1%, K_2HPO_4, 0.05% MgSO_4, 1.0 ARABINOSE, 1.5% NaNO_3 and 1.5% CaCl_2. 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 DSS polyacrylamide gel electrophoresis. The protease was most active at pH 8.0 ad 50 deg C with casein as substrate. The protease was activated by Mn ~(2+), Fe~()2+_, Zn~(2+), Co~(2+), but inhibited completely by Hg~(2+). The protease was also inhibited by metal-chelating agent such as EDTA, sulfhydryl reagents as #beta#-mercaptoethanol, and by cysteine hydrochloride, histidine, glycerol, The EDTA was the most effective inhibitor that caused completed inhibition of protease. It was concluded that this enzyme is a metal-chelator -sensitive neutral protease