| Abstract: | 由台灣北部土壤篩選到Bacillus cereus TKU006這株以蝦殼粉（shrimp shell powder；簡稱SSP）為主要碳源之蛋白酶和幾丁質酶生產菌。B. cereus TKU006生產蛋白酶和幾丁質酶之較適培養條件為2 %SSP、0.1 %K2HPO4、0.05 %MgSO4．7H2O，在25℃、pH 7之25 mL液體培養基，於250mL的三角錐型瓶中震盪培養2天，得最大之蛋白酶和幾丁質酶活性。B. cereus TKU006發酵上清液經硫酸銨沉澱、DEAE Sepharose、CM Sepharose、Phenyl-Sepharose、Sephacryl S-200和Sephacryl S-100可分離純化出蛋白酶和幾丁質酶。蛋白酶之回收率與比活性為0.07%與0.03 U/mg，而幾丁質酶的為11 %與0.62 U/mg。蛋白酶於經Sephacryl S-100純化步驟，活性幾已消失殆盡。蛋白質酶最適反應溫度與pH為50℃、9；熱穩定性及pH安定性分別為<50℃與pH 3~11。幾丁質酶最適反應溫度與pH為40℃、5；熱穩定性及pH安定性分別為<60℃與pH 3~11。幾丁質酶活性受Fe2+和Mn2+所抑制；蛋白酶活性受EDTA所抑制。幾丁質酶在丙酮、DMF或異戊醇存在下完全失活；蛋白酶在乙醇、正丁醇、二甲基甲醯胺(DMF)、異丙醇或乙酸乙酯存在下，只剩約20 %的殘餘活性。利用SDS-PAGE和膠體過濾層析法測得蛋白酶及幾丁質酶的分子量分別為39 kDa及35 kDa。
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. |
