华根霉Rhizopus chinensis CCTCC M201021脂肪酶基因的分子改造及酶学特性的探究

Molecular modification and enzymatic characteristics discovery of lipase from Rhizopus Chinensis CCTCC M201021

本文对华根霉Rhizopus chinensis CCTCC M201021脂肪酶(r27RCL)的氨基酸序列(A149C、V180C和F196C)进行定点突变。以未突变的野生型脂肪酶r27RCL作为对照,借助pPIC9K载体表达系统将野生脂肪酶及突变体在GS115中实现异源表达,并研究野生型脂肪酶r27RCL及突变体的酶学性质。结果表明,三个单突变体均不能与氨基酸残基C177形成二硫键,但是突变体r27RCL-V180C最大催化效率是野生型脂肪酶r27RCL的1.43倍,与不同碳链长度的p-NP(p-硝基苯酚)底物亲和力也相对提升,酶最适反应温度降低5℃。突变体r27RCL-A149C和r27RCLF196C较野生型具有更宽的p H作用范围。本文研究为探究华根霉来源的脂肪酶蛋白空间结构及酶基因改造提供了理论依据。

The amino acid sequences of Rhizopus chinensis CCTCC M201021 lipase r27RCL1 were sequenced by sitedirected mutations of A149C,V180C and F196C. Using wild lipase r27RCL as control,wild lipase and mutant were expressed in GS115 by p PIC9K vector expression system,and the enzymatic properties of wild lipase r27RCL and mutant were studied. The results showed that none of the three single mutants could form disulfide bonds with amino acid residues C177,but the maximum catalytic efficiency of the mutant r27RCL-V180C was 1. 43 times that of wild type lipase r27RCL,and its affinity to p-NPP-nitrophenol substrates with different carbon chain lengths was also relatively enhanced.The optimum reaction temperature of enzyme was reduced by 5 ℃. Both r27RCL-A149C and r27RCL-F196C had a wider range of p H than the wild type. This study provided a theoretical basis for exploring the spatial structure of lipase protein and enzyme gene modification of Rhizopus chinensis.