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.

Key words:Alpha-amino acid ester hydrolase, Xanthomonas rubrillineans, Sequence analysis, Thermostability, Site-directed saturated mutagenesis