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

A protease-producing microorganisrn was isolated in northern Taiwan and ide ntified as a strain of Bacillus subtilis. B. subtilis Y-108 thus isolated c an 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 opti mized conditions for protease production was found when the culture was sha ken at 30 degrees C for 3 days in 100 ml of medium (phosphate buffer adjust ed to pH 6.0) containing 7% shrimp and crab shell powder (SCSP), 0.1% K2HPO 4, 0.05% MgSO4, 1.0% arabinose, 1.5% NaNO3, and 1.5% CaCl2. Under such cond itions, 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 proced ure involving ammonium sulfate precipitation, DEAE-Sepharose CL-6B ionic ex change chromatography, and Sephacryl S-200 gel permeation chromatography. T he enzyme was shown to have a relative molecular weight of 44 kDa by SDS po lyacrylamide gel electrophoresis. The protease was most active at pH 8.0 an d 50 degrees 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 wa s also inhibited by metal-chelating agent such as EDTA, sulfhydryl reagents as beta-mercaptoethanol, and by cysteine hydrochloride, histidine, glycero l. The EDTA was the most effective inhibitor that caused complete inhibitio n of protease. It was concluded that this enzyme is a metal-chelator-sensit ive neutral protease. (C) 2000 Elsevier Science Inc. All rights reserved.