本文選題：脂肪酶 + 畢赤酵母異源表達　； 參考：《天津科技大學(xué)》2016年碩士論文

【摘要】：脂肪酶是一類可催化甘油三酯水解生成甘油及長鏈脂肪酸的水解酶。來源于耶氏解酯酵母的胞外脂肪酶LipY2,由于其在較低pH下仍具有較高的穩(wěn)定性且活性不受膽鹽的抑制,因此可作為口服類替代藥物治療胰腺炎引起的胰酶分泌不足等疾病。然而該酶在口服過程中,常因其易被胰蛋白酶降解而降低療效。故本研究擬通過蛋白質(zhì)工程手段對LipY2進行分子改造,以提高其對胰蛋白酶的抗性。本研究將肪酶基因lipY2克隆至分泌型質(zhì)粒pPIC9K上,并在畢赤酵母GS115中實現(xiàn)了異源表達。搖瓶發(fā)酵條件下,甲醇誘導(dǎo)培養(yǎng)60 h后重組脂肪酶LipY2的表達量最高達到1033 U/mL。同時根據(jù)脂肪酶LipY2的胰蛋白酶酶解質(zhì)譜分析以及脂肪酶的三維結(jié)構(gòu)分析,利用序列比對及同源建模相結(jié)合的手段,確定了 LiPY2中易被胰蛋白酶降解的位點。并針對這些位點,對lipY2基因進行了定點改造,構(gòu)建了 7個突變體,分別為M36、M63、M215以及組合突變體M36-63、M36-215、M63-215和M36-63-215。分別研究了不同位點的改變,對LiPY2的酶活、抗胰蛋白酶水解穩(wěn)定性及酸穩(wěn)定性等酶學(xué)性質(zhì)的影響,結(jié)果如下:與LipY2相比,M36(突變位點為K36、K39)的最適pH下降了一個單位,在相同濃度胰蛋白酶處理下的半衰期提高了 8.1%;M63(突變位點為R63)的最適pH也下降了一個單位,在相同濃度胰蛋白酶處理下的半衰期提高了 16.6%;M36-63(突變位點為K36、K39、R63)最適pH同樣下降了一個單位,在相同濃度胰蛋白酶處理下的半衰期提高了 22.3%。突變體M36、M63和M36-63的酶活力與LipY2基本相同,但在較低pH 1～3范圍內(nèi)的穩(wěn)定性以及對胰蛋白酶的抗性都有所提高,尤其以M36-63的效果最為顯著。而M215(突變位點為K215、K218、R220)及 M36-215(突變位點為 K36、K39、K215、K218、R220)、M63-215 突變位點為(R63、K215、K218、R220)和 M36-63-215(突變位點為 K36、K39、R63、K215、K218、R220)的酶活力卻非常低。
[Abstract]:Lipase is a kind of hydrolase which can catalyze the hydrolysis of triglycerides to glycerol and long chain fatty acids. LipY2, an extracellular lipase derived from yeast-lyase yeast, can be used as an oral substitute for the treatment of pancreatitis due to its high stability and activity not inhibited by bile salt. However, during oral administration, the enzyme often reduces its efficacy because of its easy degradation by trypsin. Therefore, in order to improve the resistance to trypsin, LipY2 was modified by protein engineering. In this study, lipY2 was cloned into secretory plasmid pPIC9K and heterologous expression was achieved in Pichia pastoris GS115. Under the condition of shaking flask fermentation, the expression of lipase LipY2 was up to 1033 U / mL after 60 h of methanol induction and culture. On the basis of lipase lipY2 trypsin mass spectrometry and lipase three-dimensional structure analysis, sequence alignment and homology modeling were used to determine the sites of LipY2 easily degraded by trypsin. Aiming at these sites, lipY2 gene was modified by site-directed transformation, and seven mutants, M36-633-215 and M36-63-215M36-63-215 and M36-63-215respectively, were constructed. The effects of different sites on the enzyme activity, hydrolysis stability and acid stability of LiPY2 were studied. The results were as follows: compared with LipY2, the optimum pH of M36 (mutant site K36 K39) was reduced by one unit. At the same concentration of trypsin, the optimal pH value of M63 (mutant site R63) was increased by one unit, and the half life increased by 8. 1% at the same concentration of trypsin. At the same concentration of trypsin, the half-life increased by 16.6C M36-63 (the mutation site was K36K39R63) and the optimum pH decreased by one unit, and the half-life increased by 22.3g under the same concentration of trypsin. The enzyme activities of M36-63 and M36-63 were basically the same as those of LipY2, but the stability and trypsin resistance of M36-63 were increased in the range of lower pH 1 ~ (3), especially the effect of M36-63. The enzyme activities of M215 (K215K218R220) and M36-215 (K36K39K215K218K220M23-215) and M36-63-215 (K36K39K39K215K215K218R220) and M36-63-215M36-63-215 (K36K39K39K215K215K218R220) were very low.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q55

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