Bacillus cereus TKU006所生產幾丁質酶及蛋白酶之純化與定性

Abstract

The protease-chitinase-producing by Bacillus cereus TKU006 was isolated from soil of northern of Taiwan. Shrimp shell powder (SSP) was used to be the carbon souse. The optimized culture condition for protease and chitinase production was composed of 2% SSP, 0.1 % K2HPO4 , 0.05 % MgSO4．7H2O, there have the maximum activated shaken in 250 mL Erlenmyer flask containing 25mL of above liquid medium at 25 ℃ and pH 7 for two days. The protease and chitinase of Bacillus cereus TKU006 were purified by ammonium sulfate precipitation, ionic exchange of DEAE-sepharose, CM-Sepharose, Phenyl-Sepharose, and sephadex S-200 and sephadex S-100 gel filtration . The overall activity yield of the purification protease were 0.07％,with specific protease activities of 0.03(U/mg). And, the overall activity yield of the purification chitinase were 11％,with specific chitinase activities of 0.62(U/mg). After purified by sephadex S-100, the protease was almost lost activity. The optimum pH, optimum temperature, pH stability, and thermal stability of protease were pH 9, 50℃, pH 3~11 and ＜50℃. The optimum pH, optimum temperature, pH stability, and thermal stability of chitinase were pH 5, 40℃, pH 3~11 and ＜60℃.The chitinase was inactivated by Fe2+ and Mn2+；the protease was inhibited completely by EDTA. The chitinase was inactivated by acetone, DMF and isoamyl alcohol； the protease in the presence of water-insoluble organic solvent such as ethyl alcohol, n-butyl alcohol, DMF, isopropyl alcohol and ethyl acetate, it retained only 20% of its activity. The molecular weight of the protease and the chitinase determined by SDS-PAGE and gel chromatography was approximately 39 kDa and 35 kDa, respectively.