Bacillus amyloliquefaciens之LipA分離純化及性質分析

Abstract

微生物所分泌的脂肪酶主要應用於食品工業，清潔劑工業及其他化學工業上，且其分離及純化較動物性脂肪酶及植物性脂肪酶更為容易，因此微生物脂肪酶的生產及研究有其重要性。而其中Bacillus spp.脂肪酶由於有耐熱性或在鹼性環境下酵素作用不受影響，因此特別受到重視，而且其脂肪酶的分子量大小是所有微生物脂肪酶中最小的一群，有利於在生物科技上的應用。然而由於在大規模量產微生物脂肪酶方面，原菌株通常無法生成大量的酵素，因此需透過選殖一個能夠生產大量脂肪酶，且具有特殊性質的轉化株，才能有效應用於工業上。雖然已有許多Bacillus spp.的脂肪酶基因已被定序，也進一步的表現於異源寄主中，但對於Bacillus amyloliquefaciens脂肪酶的研究仍不充分，我們從土壤環境中分離而得的菌株Bacillus amyloliquefaciens中選殖出一段脂肪酶基因，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，並測其分解脂質能力。利用IPTG誘導蛋白質生產並進一步的純化，得到分子量大小約為25 kDa的LipA脂肪酶。此脂肪酶最適反應溫度為35℃、最適pH值為10；此酵素在溫度35℃下酵素活性穩定、且在pH值為9-10時具有良好的pH值穩定性。由受質特異性測試可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。10 mM Zn2+、Fe2+、Cu2+、Ca2+、SDS、Triton-X-100 均會強烈的抑制此酵素活性。另外，在10% 有機溶劑存在下，DMSO、Isopropanol均可些微提高酵素活性，但到了有機溶劑濃度為25%時，大部分的有機溶劑會開始抑制酵素活性。以薄層層析法初步分析LipA脂肪酶水解橄欖油後產物的生成，從結果可知其隨著時間的拉長，產物fatty acid及diglyceride的生成量上升，表示此脂肪酶可應用於生質柴油的生產及油脂的改造。以酸鹼滴定法測定原菌株Bacillus amyloliquefaciens、轉殖菌株及LipA脂肪酶對橄欖油及大豆油的降解率，首先在橄欖油方面，在反應96小時後其降解率分別為38.82%、45.33%、53.46%；而三者對大豆油的降解率則分別為35.46%、43.14%、62.2%，表示三者均有能力水解橄欖油，但其作用效率不同。而此油類降解的測定可以進一步應用於廢油回收處理。

Microbial lipases are mainly applied in food industry, detergents and the other chemical industry. Because the isolation and purification of lipases from microorganisms is easier than those of animals and plants, the mass production and study of lipases from microorganisms are really important. Lipases of Bacillus spp. are heat and alkaline tolerant, so the lipase activity under heat and alkaline conditions can remain stable. Besides, lipases from the Bacillus spp. belong to those of the smallest molecular weight, and this make it a benefit for application in biotechnology. However, the original bacterial strain usually can not produce large amount of lipases, so it is better to select a transformant carrying a lipase gene in a plasmid which can produce large amount of lipases with special and specific characteristics for effective application in industry. Although many lipase genes form Bacillus spp. have been sequenced, studies on lipases of Bacillus amyloliquefaciens are still not comprehensive. A strain of Bacillus amyloliquefaciens was isolated from the environment and its lipase gene was selected, and cloned into pET21a, for expressing in E.coli by an induction with IPTG. Through the purification of the lipase protein (LipA) by the nickle column, a protein whose molecular weight is about 25kDa was obtained. The optimal temperature and pH value for LipA lipase activity are 35℃ and pH-10, respectively. Also, the LipA lipase is stable under 35℃ and pH value 9-10. The LipA lipase prefers p-nitrophenyl laurate as its substrate. The buffer containing a final concentration of 10 mM Zn2+, Fe2+, Cu2+, Ca2+, SDS or Triton-X-100 would strongly suppress the enzymatic activity. 10% DMSO and isopropanol would slightly enhance the enzyme activity, but 25% of most organic solvent could inhibit LipA activity. The results of the thin-layer-chromatography (TLC) showed that the longer the incubation time, the more degradation product of olive oil, including fatty acid and diglyceride. The degradation rate of olive oil after 96 hrs by B. amyloliquefaciens, the transformant expressing and LipA lipase was 38.8%, 45.3% and 53.5%, respectively, whereas for soybean oil, it was about 35.5%, 43% and 62%, respectively. Taken together, the above results revealed that LipA lipase has the potential to be applied in biodiesel production, recycled oil treatment and oil modification.