D190V点突变提高华根霉Rhizopus chinensis CCTCCM 201021脂肪酶的最适温度和热稳定性  

Improved optimum temperature and thermostability of the lipase from Rhizopus chinensis CCTCC M201021 by site-directed mutagenesis of D190V

作　　者：吴厚军[1] 喻晓蔚[1] 沙冲[1] 徐岩[1] 

机构地区：[1]工业生物技术教育部重点实验室江南大学酿酒科学与酶技术中心,江苏无锡214122

出　　处：《微生物学通报》2013年第11期1955-1961,共7页Microbiology China

基　　金：国家973计划项目(No.2011CB710800);国家863计划项目(No.2012AA022207;2011AA02A209;2011AA02A210);中央高校基本科研业务费专项资金项目(No.JUSRP11014);国家自然科学基金项目(No.20802027)

摘　　要：【目的】对来源于Rhizopus chinensis CCTCC M201021的脂肪酶进行了D190V定点突变,提高该酶的最适温度和热稳定性。【方法】对毕赤酵母表达的突变酶D190V与野生型酶r27RCL进行酶学性质比较。【结果】D190V的最适温度比r27RCL高5°C,65°C下的半衰期提高了一倍,在其他性质方面,突变酶D190V与r27RCL基本相似。【结论】通过结构分析表明,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。[Objective] D190V mutation was introduced into the lipase from Rhizopus chinensis CCTCC M201021 by site-directed mutagenesis to improve its optimum temperature and the thermostability. [Methods] The mutant lipase D190V and wild-type lipase r27RCL were expressed in Pichia pastoris and the enzymatic properties were characterized. [Results] The optimum temperature of D190V was 5 ℃higher than that of the wild-type, and the half-life （T1/2） of D190V at 65 ℃ exceeded that of r27RCL by 1-fold, other enzymatic properties were similar to r27RCL. [Conclusion] According to the analysis of structures, the reason of improved thermostability for the variant by only an amino acid substitution D190V was probably due to the improved stability of the α-helix located and the strengthened hydrogen bonding force in the protein structure.

关 键 词：定点突变 华根霉脂肪酶 最适温度 热稳定性 

分 类 号：Q936[生物学—微生物学]