鞘糖脂内切糖苷酶的半理性设计

Semi-rational design of endoglycoceramidase

[目的]对鞘糖脂内切糖苷酶EGCaseⅡ进行半理性设计,获得高水解活性突变体。[方法]用半理性设计方法进行突变库设计,利用HPLC对突变库进行筛选,随后对阳性突变体进行动力学及底物谱表征,并利用结构建模对活性提高的分子机制进行解析。[结果]获得了对鞘糖脂GM1、GM3水解活性提高的突变体S63G/D311E、I276L/D311V,活性分别提高为野生型的25.3倍、11.8倍。酶动力学表征显示,S63G/D311E的K_M由0.17 mmol/L降低到0.06 mmol/L,kcat由5.5 min^(-1)增大到50.3 min^(-1)。酶-底物复合物模式结构分析表明,D311E、D311V、I276L这几种突变更有利于酶与底物结合,从而提高酶活性。[结论]通过半理性设计成功获得对GM1和GM3水解活性分别提高25.3倍和11.8倍的EGCaseⅡ突变体。

[Objective] The mutants of endoglycoceramidaseⅡ with higher hydrolytic activity were obtained by semi-rational design. [Methods] The mutagenesis libraries were constructed by semi-rational design. The libraries were screened by High Performance Liquid Chromatography. The catalytic kinetics and substrate spectra of positive mutants were determined. The molecular mechanism for activity improvement was analyzed by molecular modelling. [Results]Several mutants were obtained with significantly improved activity for glycosphingolipid GM1 and GM3. S63 G/D311 E was the best mutant for GM1,which had 25.3 times higher activity than the wild-type enzyme. I276 L/D311 V was the best mutant for GM3,which had 11. 8 times higher activity than the wild-type enzyme. Kinetic analysis for S63 G/D311 E showed that its K_Mvalue decreased from 0. 17 mmol/L to 0. 06 mmol/L while the kcatvalue increased from 5. 5 min-1 to 50. 3 min-1. The analysis of enzyme-substrate complex modelled structure indicated that D311 E,D311 V,I276 L might result in better enzyme-substrate recognition and more favorable catalysis. [Conclusion]Through semi-rational design,the mutants of endoglycoceramidaseⅡwith significantly higher hydrolytic activity were created successfully for GM1 and GM3,which had 25. 3 times and 11. 8 times higher activity than the wild-type enzyme,respectively.