红纹黄单胞菌a-氨基酸酯水解酶的克隆、序列分析及热稳定性提高

Cloning, bioinformatics analysis and improving thermostability of a-amino acid ester hydrolase from Xanthomonas rubrillineans

摘要:

【目的】克隆红纹黄单胞菌a-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a-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a-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a-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 °C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。

Abstract:

[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 °C 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.