红纹黄单胞菌α-氨基酸酯水解酶的克隆、序列分析及热稳定性提高

摘要

[目的]克隆红纹黄单胞菌α-氨基酸酯水解酶基因全序列,对序列进行生物信息学分析,并提高酶的热稳定性.[方法]利用多聚酶链式反应(PCR)克隆α-氨基酸酯水解酶基因全序列;应用生物信息学软件对获得的基因序列及编码的蛋白序列进行分析；通过同源建模,预测红纹黄单胞菌α-氨基酸酯水解酶的三维结构；通过定点突变替换氨基酸序列中高度柔性的位点,提高该酶的热稳定性.[结果]从红纹黄单胞菌(Xanthomonas rubrillineans)中扩增得到α-氨基酸酯水解酶基因aeh (GenBank登录号JF744990),核苷酸序列长度1 917 bp,编码638个氨基酸.序列比对和同源性分析显示,该酶与白纹黄单胞菌Xanthomonas albilineans str.GPE PC73的肽酶及地毯草黄单胞菌Xanthomonas axonopodis pv.citri str.306的戊二酰-7-氨基头孢烷酸酰化酶氨基酸序列相似性最高,分别为91％和83％,系统进化分析表明,该酶与白纹黄单胞菌Xanthomonas albilineans str.GPE PC73的肽酶亲缘性最高.基于预测的三维模型,对高度柔性的位点进行饱和突变,从282株突变体中筛选得到3株T50较野生型高5℃以上的突变体.[结论]对红纹黄单胞菌AEH的氨基酸序列分析有助于探索同源蛋白的进化过程.对高度柔性位点进行饱和突变的策略可以用于提高热稳定性.%[Objective] The study aimed to clone a-amino acid ester hydrolase gene from Xanthomonas rubrillineans, to perform bioinformatics analysis and increase the thermostability of the enzyme.[Methods] The full length of aeh was cloned by polymerase chain reaction (PCR).The gene sequence obtained and the putative amino acid sequence were analyzed by bioinformatics software, and the three-dimensional structure of X.rubrillineans AEH was predicted by homology modeling.In order to improve thermostability, sites displaying high degree of flexibility were replaced through site-directed mutagenesis.[Results] Aeh was obtained by PCR from X.rubrillineans (GenBank accession: JF744990).The nucleotide sequence is 1 917 bp length, encoding a polypeptide of 638 amino acids and shares 91% and 83% identity to peptidase from X.albilineans str.GPE PC73 and GL-7-ACA acylase from X.axonopodis pv.citri str.306 respectively.Based on the predicted model, sites displaying high degree of flexibility were replaced through saturated mutagenesis and 3 variants with 5 ℃ higher T50 than wild type were distinguished from 282 variants by screening.[Conclusion] The sequence analysis of X.rubrillineans AEH was benefit for exploration of evolution history.The strategy of replacing highly flexible residues by saturated mutagenesis can be used for enhancing thermostability.