施博書(Po-Shu Shih)吳蕙芬(Whei-Fen Wu)2025-09-232025-09-232025https://scholars.lib.ntu.edu.tw/handle/123456789/732253由土壤環境中所分離出之菌株Bacillus amyloliquefaciens B-01，選殖出一段脂肪酶基因（lipA^+），並轉殖到E. coli中表現，得到具有分泌脂肪酶能力的轉殖菌株。利用IPTG誘導表達重組蛋白質，並進行蛋白質純化，得到分子量大小約為25 kDa的LipA脂肪酶，用以進行分解脂質能力測試。利用受質特異性，得知此脂肪酶相對於短鏈酯質更偏好長鏈酯質，尤其是在受質為p-nitrophenyl laurate（p-NPC12）時，其水解能力達最佳。此脂肪酶之最適反應溫度為35℃至45℃、最適pH值為10；且在溫度35℃及pH值為9時具有良好之穩定性。添加Mg^(2+)和Cu^(2+)離子，均可增強此酵素之活性。添加EDTA、DTT或Tween 20，不影響酵素之活性。另外，於多數有機溶劑存在下，亦不影響酵素之活性。因此，LipA為一極具潛能之鹼性酵素，可應用於工業發展之用途。A lipase gene lipA^+ in a soil isolated bacterium, Bacillus amyloliquefaciens B-01, was firstly identified by the bio-informatic analyses. Then, this lipase gene lipA^+ was cloned into the plasmid pET21a, and the resultant plasmid was transformed into E. coli BL21. Subsequently, after an IPTG induction, the LipA protein was largely expressed in the bacterial cells. By the Nickle column method, the LipA protein was purified with a molecular weight about 25 kDa. Besides, the LipA lipase prefers p-nitrophenyl laurate as substrates. The optimal temperature and pH value for LipA lipase activity is 35-45℃ and pH 10, respectively. Also, the LipA lipase is stable under 35℃ and pH 9. When the addition with a final concentration of 1 mM Mg^(2+) and Cu^(2+), the LipA enzyme activity was increased. Besides, the addition of EDTA, DTT or Tween 20, all have no effects on the enzyme activity. Moreover, the addition of organic solvents, most of them also have no effects on the enzyme activity. Taken together, LipA, as an alkali lipase, has the potential for the industrial application.液化澱粉芽孢桿菌純化脂肪酶酵素活性分析Bacillus amyloliquefaciensPurification of LipA lipaseAnalysis of LipA enzyme activityjournal article10.6578/TJACFS.202503_63(1).0004