| Abstract: | 本研究係以TKU001這株真菌細胞壁分解菌發酵蝦殼粉（shrimp shell powder；簡稱SSP）所得，探討所生產TKU001蛋白酶之純化分離及定性。TKU001生產蛋白酶之較適培養基為0.5%SSP、0.1%K2HPO4、0.05%MgSO4‧7H2O之50 mL液態培養基（pH 6）。於含50 mL較適培養基的250 mL三角錐形瓶中，於37℃振盪（150 rpm）培養3天後，可得較佳之蛋白酶活性（0.11 U/mL）。
所得發酵液經DEAE Sepharose CL-6B、Sephacryl S-200等層析分離步驟，可純化出二種蛋白酶F1及F2。利用SDS-PAGE測得F1及F2蛋白酶的分子量分別為41 kDa及75 kDa。F1之最適反應溫度為60℃、最適反應pH 8、熱安定性<60℃、pH安定性6~9；F2蛋白酶最適反應溫度為60℃、最適反應pH 7、熱安定性<50℃、pH安定性7~9。蛋白酶抑制劑及金屬離子對酵素活性的影響，分別受到EDTA和Fe2+、Cu2+ 、Mn2+的抑制，但F2蛋白酶不受Mn2+離子的抑制。界面活性劑對酵素的影響方面，F1及F2蛋白酶皆受到2 mM SDS作用而抑制。有機溶劑影響對F1蛋白酶之活性，在25%乙醚、甲苯、乙晴等有機溶劑存在下蛋白酶仍有70%以上之相對活性；F2蛋白酶之活性，在乙醚、甲苯、乙晴、丙酮等有機溶劑存在下蛋白酶仍有70%以上之相對活性。
The protease producing bacterium, TKU001 strain, was isolated from the cell wall screened via hydrolysising the fungi, and an experiment is with the fermented shrimp shell powder（Abbreviated as SSP）. TKU001 optimized culture was composed of 0.5% SSP, 0.1% K2HPO4, 0.05% MgSO4‧7H2O condition at pH 7. The bacterium was incubated in 250 mL Erlenmeyer flask containing 50 mL of above liquid medium and kept shaking at 37℃ for three day（150 rpm）.
The protease of Bacteria strain TKU001 was produced under the optimized culture condition. The supernatant further purification and separation procedures of protease were by DEAE-Sepharose and Sephacryl S-200 gel chromatography. The purification two kinds of proteases（F1&F2）. The apparent molecular mass based on SDS-PAGE was estimated 41 kDa from F1 protease；the estimate 75 kDa from F2 protease. The optimum temperature and pH of the F1 protease were 60℃ and 8 respectively, and the F1 protease was stable at<60℃ and pH 6~9. The optimum temperature and pH of the F2 protease were 60 ℃ and 7 respectively, and the F2 protease was stable at<50℃ and pH 7~9. The inhibitor and metal ions, separately receives was inactivated by EDTA and Fe2+, Cu2+, Mn2+ suppression, but F2 protease not Mn2+ suppression. Effect of surfactants on the activities of protease, was inactivated by SDS. In the presence of organic solvent such as Ethyl Ether, Toluene, Acetontitrile, the F1 protease retained more than 70% of its activity. In the presence of organic solvent such as Ethyl ether, toluene, aacetontitrile, acetone, the F2 protease retained more than 70% of its activity. |
