绿脓杆菌芳香硫酸酯酶序列与活性关系的初步表征

Preliminary characterization of sequence-activity relationship of Pseudomonas Aeruginosa arylsulfatase

目的:通过定点突变初步表征绿脓杆菌芳香硫酸酯酶(Pseudomonas Aeruginosa arylsulfatase,PAAS)序列-活性关系,识别通过迭代饱和突变提高突变体比活性的合适位点。方法:分析PAAS活性中心三维构象初步识别催化相关位点;定点突变获得突变体,重组表达后Ni2+-NTA亲和纯化,表征其酶学性质。结果:与野生型相比,R55、M72、G138、D317、K375等位点用常见氨基酸替换/突变都显著降低其催化活性;R55、D317和K375等突变显著降低底物选择性和/或热稳定性,而M72和G138突变主要改变其催化活性。结论:M72和G138适合作为通过迭代饱和突变实施定向进化的候选位点。

Objective：To investigate the preliminary sequence-activity relationship of Pseudomonas Aeruginosa arylsulfatase（PAAS） by site-directed mutagenesis and to recognize residues suitable for iterative-saturation-mutagenesis. Methods ： Candidates of residues involved in catalysis were recognized by analysis of conformation of PAAS active site ; mutants were generated by site-directed mutagenesis and characterized after recombinant expression and Ni2＋-NTA affinity.purification. Results：In comparison to the wild type,the mutagenesis of R55, M72, G138, D317 and K375 caused the significant decrease of catalytic efficacy;the mutagenesis of R55, D317 and K375 reduced substrate preference for 4-nitrophenylsulfate and/or thermostability, but the mutagenesis of M72 and G138 caused primarily the decrease of activity. Conclusion：M72 and G138 are suitable candidate sites for directed evolution by iterative-satura- tion-mutagenesis to improve mutant activity.