理性设计定点突变提高Streptomyces kathirae SC-1酪氨酸酶热稳定性

摘要

利用SWISS-MODEL在线服务器以栗色浑圆链霉菌(Streptomyces castaneoglobisporus)酪氨酸酶(PDB登录号:2AHL)为模板对S.kathirae酪氨酸酶的3-D结构进行同源模拟.使用在线预测软件PoPMuSiC-2.1计算酪氨酸酶每个突变氨基酸的去折叠自由能变化(ΔΔG)来辅助设计提高酪氨酸酶的稳定性,利用定点突变构建突变体,并且在大肠杆菌中对野生型酪氨酸酶及突变体进行表达,最终获得了两个热稳定性提高的突变体R95Y、G123W.在此基础上进行复合突变,获得了一个热稳定性显著提高的突变体R95Y/G123W.该突变体在60℃的半衰期提高了2.43倍,最适反应温度由45℃提高到50℃,本研究所用的基于蛋白质结构的理性设计提高稳定性的策略也可以应用于其他工业酶类,显示了良好的应用前景.%Homology modeling of tyrosinase was performed using the SWISS-MODEL based on the known crystal structure of tyrosinase from S. castaneoglobisporus (PDB No. 2AHL). The POPMuSiC algorithm was applied to predict the folding free energy change(ΔΔG)of amino acid substitution. Site-directed mutagenesis was used to construct mutants at Arg95Tyr and Gly123Trp. The mutant and wild-type enzymes were expressed in Escherichia coli(DE3). As compared to the wild-type tyrosinase,all the mutant enzymes showed improved thermal properties. The mutant with combined substitution(Arg95Tyr/Gly123Trp) showed the most pronounced shifts in temperature optima,which was about 5 ℃ upward,and the half-life for thermal inactivation at 60 ℃ was increased by 1.5 times.The structure-based rational design strategies in this study may also provide further insight into the thermostability of other industrial enzymes and suggest further potential industrial applications.