定点突变引入二硫键提高华根霉脂肪酶热稳定性的研究

Improving thermostability of lipase from Rhizopus chinensis via introducing novel disulfide bond by site-directed mutagenesis

拟对来源于华根霉(Rhizopus chinensis)的脂肪酶r27RCL进行二硫键的构建,从而提高该酶的热稳定性。利用二硫键构建软件Disulfide by Design 2. 0预测突变位点(T201),对野生型脂肪酶r27RCL进行定点突变。借助于毕赤酵母表达系统,对脂肪酶野生型r27RCL及突变体r27RCLT201C进行异源表达,并对其酶学性质进行研究和比较。热稳定性实验显示突变体r27RCL-T201C在60℃处理25 min后剩余酶活较野生型提高了17. 6%。此外,突变体的pH稳定性也较野生型有所提高。对脂肪酶r27RCL进行二硫键的构建有助于提高该酶热稳定性,使其能更有效地应用于工业生产。

In order to improve themostability of Rhizopus chinensis lipase （r27RCL）, novel disulfide bond was introduced by site-directed mutagenesis. Novel disulfide bond was predicted and designed by web-based tool （Disulfide by Design 2.0）, and a single mutation T201C was introduced by site-directed mutagenesis. The r27RCL and r27RCL-T201C PCR products were linked with pPIC9K vector and then transformed into GS115 competent cell of P. pastoris for heterologous expression. Enzymatic properties of r27RCL and r27RCL-T201C were assayed and compared. The thermstability test showed that the residual enzyme activity of mutant r27RCL-T201C treated with 25 min at 60 ℃ was 17.6% higher than that of wild type. In addition, the lipase mutant r27RCL-T201C exhibited enhanced pH stability compared to that of r27RCL. The results showed that the novel disulfide bond introduction was an effective and efficient strategy for improving thermostability of lipase from Rhizopus ehinensis, and this lipase mutant would be a potential candidate for industrial use.