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

摘要:

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。

Abstract:

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 muta－genesis 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.