理性设计盐桥构建伯克霍尔德菌脂肪酶热稳定突变体

Computer-aid screening of thermostable lipase LipA from Burkholderia sp. ZYB002

【目的】借助蛋白质工程技术提高伯克霍尔德菌ZYB002脂肪酶LipA的热稳定性,以期更好地将其应用于工业生产中。【方法】利用YASARA、Fold X、Rosetta、Gromacs等生物信息学软件,构建1个脂肪酶Lip A的热稳定性提高的微型突变体电子文库;通过对突变体的结构信息和自由能变化进行评估,筛选出潜在的有价值的突变体。继而利用基因定点突变技术,构建上述候选突变体的突变基因文库,通过实验筛选出热稳定性提高的脂肪酶LipA突变体。【结果】利用上述方法,从构建的20个脂肪酶LipA突变体中,筛选到热稳定性有显著提高的3个突变体LipA-Asn^(125)Asp、LipA-Asn^(125)Glu和LipA-Gln^(262)Glu。经55°C处理12 min后,上述3个突变体的T1250值较野生型分别提高4.0°C、5.5°C和4.4°C;在55°C下的半衰期较野生型分别提高了2.2倍、3.8倍和2.6倍。【结论】利用生物信息学软件,构建脂肪酶LipA突变体电子文库,结合蛋白质的结构信息,可以快速筛选到热稳定性提高的脂肪酶LipA突变体。

[Objective] We improved the thermostability of lipase LipA from Burkholderia cecapia ZYB002 using protein engineering technology. [Methods] Lipase LipA mutant library was designed and screened using the following software, YASARA, FoldX, Rosetta, and Gromacs. We screened 27 thermostable lipase LipA mutants displaying the salt bridge effect among the resulting library of 341 variants, and then further screened using the site-directed mutagenesis technology. [Results] Three mutants LipA-Asn^125Asp, LipA-Asn125Glu and LipA-Gln^262Glu displayed improved thermostability. The T50^12 value of LipA-Asn^125Asp, LipA-Asn^125Glu and LipA-Gln262Glu increased by 4.0 ℃, 5.5 ℃ and 4.4 ℃, respectively. The half-life ofLipA-Asnl25Asp, LipA-Asn^125Glu and LipA-Gln^262Glu at 55 ℃ increased by 2.23-fold, 3.8-fold and 2.6-fold, respectively. [Conclusion] It is feasible to screen thermostable mutant from the computationally designed library.