耶氏解酯酵母脂肪酶基因lipY2的高效异源表达及分子改造

发布时间：2018-06-06 15:28

  本文选题：脂肪酶 + 毕赤酵母异源表达　； 参考：《天津科技大学》2016年硕士论文

【摘要】：脂肪酶是一类可催化甘油三酯水解生成甘油及长链脂肪酸的水解酶。来源于耶氏解酯酵母的胞外脂肪酶LipY2,由于其在较低pH下仍具有较高的稳定性且活性不受胆盐的抑制,因此可作为口服类替代药物治疗胰腺炎引起的胰酶分泌不足等疾病。然而该酶在口服过程中,常因其易被胰蛋白酶降解而降低疗效。故本研究拟通过蛋白质工程手段对LipY2进行分子改造,以提高其对胰蛋白酶的抗性。本研究将肪酶基因lipY2克隆至分泌型质粒pPIC9K上,并在毕赤酵母GS115中实现了异源表达。摇瓶发酵条件下,甲醇诱导培养60 h后重组脂肪酶LipY2的表达量最高达到1033 U/mL。同时根据脂肪酶LipY2的胰蛋白酶酶解质谱分析以及脂肪酶的三维结构分析,利用序列比对及同源建模相结合的手段,确定了 LiPY2中易被胰蛋白酶降解的位点。并针对这些位点,对lipY2基因进行了定点改造,构建了 7个突变体,分别为M36、M63、M215以及组合突变体M36-63、M36-215、M63-215和M36-63-215。分别研究了不同位点的改变,对LiPY2的酶活、抗胰蛋白酶水解稳定性及酸稳定性等酶学性质的影响,结果如下:与LipY2相比,M36(突变位点为K36、K39)的最适pH下降了一个单位,在相同浓度胰蛋白酶处理下的半衰期提高了 8.1%;M63(突变位点为R63)的最适pH也下降了一个单位,在相同浓度胰蛋白酶处理下的半衰期提高了 16.6%;M36-63(突变位点为K36、K39、R63)最适pH同样下降了一个单位,在相同浓度胰蛋白酶处理下的半衰期提高了 22.3%。突变体M36、M63和M36-63的酶活力与LipY2基本相同,但在较低pH 1～3范围内的稳定性以及对胰蛋白酶的抗性都有所提高,尤其以M36-63的效果最为显著。而M215(突变位点为K215、K218、R220)及 M36-215(突变位点为 K36、K39、K215、K218、R220)、M63-215 突变位点为(R63、K215、K218、R220)和 M36-63-215(突变位点为 K36、K39、R63、K215、K218、R220)的酶活力却非常低。
[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.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q55

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