理性设计提高华根霉脂肪酶耐热性及其前肽功能研究

发布时间：2018-04-25 19:24

  本文选题：华根霉脂肪酶 + 理性设计　； 参考：《江南大学》2014年硕士论文

【摘要】：脂肪酶是继蛋白酶和淀粉酶之后的第三大酶制剂，而根霉脂肪酶作为微生物脂肪酶的一种重要来源，因其高效的催化效率等特点，已有多种根霉脂肪酶制成商品化酶制剂，在食品、医药、化工及环保等多个行业表现出广阔的应用前景。华根霉脂肪酶是本实验室具有自主知识产权的脂肪酶，前期在酶的稳定性和外源表达量等方面已经取得较好的研究成果，扩大了其工业应用范围。 为了获得耐热性更高的华根霉脂肪酶，，同时研究其蛋白结构与功能的关系，本文一方面通过理性设计的思路，运用定点突变方法提高华根霉脂肪酶的耐热性；另一方面在毕赤酵母表达系统中研究了华根霉脂肪酶前肽对脂肪酶折叠和分泌的影响，主要研究内容包括： （1）获得了耐热性提高的突变酶。通过同源建模和序列比较，运用重叠延伸PCR方法对华根霉脂肪酶基因proRCL进行定点突变，构建了突变脂肪酶D190V。毕赤酵母表达的突变酶D190V与野生型酶r27RCL相比，D190V的最适温度比r27RCL高5℃，65℃下的半衰期提高了一倍，Km值下降了23.3%。通过结构分析，定点突变D190V提高该酶稳定性的主要原因可能在于提高了突变位点所在的α螺旋的稳定性以及增强了稳定蛋白质结构的氢键作用力。 （2）考察了脂肪酶前肽对脂肪酶体外折叠的辅助作用，比较了全长前肽的脂肪酶proRCLCNQ、携带截短前肽的脂肪酶r27RCLC和不含前肽的成熟肽脂肪酶mRCLC在28℃、4种GSH和GSSG浓度下的体外复性差异。不添加GSH和GSSG时，变性的脂肪酶可以自发折叠成活性的酶蛋白，而前肽有助于该过程的进行；当GSH/GSSG浓度比从0提高到2:1，复性6h的数据显示，proRCLCNQ、r27RCLC和mRCLC的复性率分别由13.6%、23.7%、8.7%提高至19.7%、74.5%、40.9%。复性率提高幅度由高到低顺序为：r27RCLC mRCLC proRCLCNQ，说明在不同氧化还原剂浓度下，r27RCLC上的截短前肽可以促进脂肪酶的自发折叠，而在较高还原剂浓度下proRCLCNQ上的全长前肽未能促进酶的折叠。前肽N端的1-67氨基酸对脂肪酶的折叠不是必需的，而68-94位氨基酸则对酶的折叠有明显的促进作用。 （3）考察了前肽序列对脂肪酶表达分泌的影响，比较了不同截短前肽序列的proRCLC基因在毕赤酵母中表达分泌差异。华根霉脂肪酶前肽N端的1-16位氨基酸对酶蛋白的表达分泌不是必需的，截去后反而提高了目标蛋白的表达和分泌。截去1-67位氨基酸，脂肪酶不能表达。截去1-35或者1-47位氨基酸的结果相似，不仅影响细胞的生长、降低目标蛋白的表达量和胞外的分泌，还造成胞外的杂蛋白量明显增多。
[Abstract]:Lipase is the third major enzyme preparation after protease and amylase. As an important source of microbial lipase, Rhizopus lipase has been made into commercial enzyme preparation due to its high catalytic efficiency. In food, medicine, chemical and environmental protection and other industries show broad application prospects. Rhizopus chinensis lipase is a lipase with independent intellectual property rights in our laboratory. Good research results have been obtained in the field of enzyme stability and exogenous expression in the early stage, which has expanded its industrial application scope. In order to obtain Rhizopus chinensis lipase with higher heat resistance and to study the relationship between its protein structure and function, on the one hand, the heat resistance of Rhizopus chinensis lipase was improved by site-directed mutagenesis through rational design. On the other hand, the effects of lipase prepeptide of Rhizopus chinensis on lipase folding and secretion were studied in Pichia pastoris. A mutant enzyme with improved heat resistance was obtained. By homologous modeling and sequence comparison, the lipase gene proRCL of Rhizopus chinensis was mutated by overlapping extended PCR method, and the mutant lipase D190V was constructed. The optimum temperature of the mutant D190V expressed by Pichia pastoris was more than that of the wild type enzyme r27RCL. The optimum temperature of D190V was 5 鈩

本文编号：1802629