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

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.