D190V点突变提高华根霉Rhizopus chinensis CCTCC M201021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

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。

Abstract:

[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 °C higher than that of the wild-type, and the half-life (T1/2) of D190V at 65 °C 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.