PURIFICATION AND CHARACTERIZATION OF EXTRACELLULAR

The research of Bacillus as a kind of probiotic for aquatic animals is increasing with the demand for environment friendly aquaculture, like biocontrol when the treatment is antagonistic to pathogens or bioremediation when water quality is improved.The Bacillus lichenif ormis srain De isolated from white shrimp( Litopanaeus vannamie)cultural ponds can produce many kinds of extracelluar enzyme, moreover, the activity of extracelluar protease is very high. Therefor, the study was carried out to collect itps extracellular protease and the physicochemical characters of the enzyme were investigated. The extracellular protease of the strain De was collected followed the semiperable membrane method and was purified followed a four-step purification procedure, including ammounium sulfate precipitation, Sephadex G- 100 gel filtration chromatography, anion- exchange chromatography on DEAE- cellulose and polyacrylamide gel electrophoresis. The activities of extracelluar protease was tested by theAzocasein digestion method.As results showed that extracellular protease secreted by strain De obtained a higher enzymatic activity when it was cultivated 12-18h at 30℃and pH 7.4- 8.2. Three predominant protein of the extracellular products BLP1、BLP2、BLP3 were obtained,whose molecular mass measured by polyacrylamide gel electrophoresis respectively were 66.2KD 31.0KD and rough 20.1KD. The protein content of purified extracellular protease was 0.773Lg/mL, comparatively, its＇ ppurified protein recovery rate was 11. 66%.Moreover, the optimum pH and temperature of the extracellular protease was found to be 7.8 and 45-65℃. The extracellular protease is thermophilic, since full biological activity is retained after heating at 100 e for 30min. Enzyme testing for inhibit ion of the extracellular protease indicated that only EDTA、Cu2+、Co2+and Mg2+ can inhibite enzymatic activity, but PMSF、Fe3+、Mn2+、Ba2+、Ca2+ can not, and Zn2+ can inhibite enzymatic activity to some extent.