文献类型：期刊文献

英文题名：A novel esterase from a marine mud metagenomic library for biocatalytic synthesis of short-chain flavor esters

作者：Gao, Wenyuan[1];Wu, Kai[1];Chen, Lifeng[1];Fan, Haiyang[1];Zhao, Zhiqiang[1];Gao, Bei[1];Wang, Hualei[1];Wei, Dongzhi[1]

机构：[1]E China Univ Sci & Technol, New World Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2016

卷号：15

外文期刊名：MICROBIAL CELL FACTORIES

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000370337000001)】；

基金：This work was supported by the National Natural Science Foundation of China (No. 21406068/B060804), the Fundamental Research Funds for the Central Universities, National Basic Research Program of China (No. 2012CB721103), and the National High Technology Research and Development Program of China (863, Program NO. 2012AA022206).

语种：英文

外文关键词：Metagenomic library; Functional screening; Esterase; Transesterification; Short-chain flavor esters; High substrate loading

摘要：Background: Marine mud is an abundant and largely unexplored source of enzymes with unique properties that may be useful for industrial and biotechnological purposes. However, since most microbes cannot be cultured in the laboratory, a cultivation-independent metagenomic approach would be advantageous for the identification of novel enzymes. Therefore, with the objective of screening novel lipolytic enzymes, a metagenomic library was constructed using the total genomic DNA extracted from marine mud. Results: Based on functional heterologous expression, 34 clones that showed lipolytic activity were isolated. The five clones with the largest halos were identified, and the corresponding genes were successfully overexpressed in Escherichia coli. Molecular analysis revealed that these encoded proteins showed 48-79 % similarity with other proteins in the GenBank database. Multiple sequence alignment and phylogenetic tree analysis classified these five protein sequences as new members of known families of bacterial lipolytic enzymes. Among them, EST4, which has 316 amino acids with a predicted molecular weight of 33.8 kDa, was further studied in detail due to its strong hydrolytic activity. Characterization of EST4 indicated that it is an alkaline esterase that exhibits highest hydrolytic activity towards p-nitrophenyl butyrate (specific activity: 1389 U mg(-1)) at 45 degrees C and pH 8.0. The half-life of EST4 is 55 and 46 h at 40 and 45 degrees C, respectively, indicating a relatively high thermostability. EST4 also showed remarkable stability in organic solvents, retaining 90 % of its initial activity when incubated for 12 h in the presence of hydrophobic alkanes. Furthermore, EST4 was used as an efficient whole-cell biocatalyst for the synthesis of short-chain flavor esters, showing high conversion rate and good tolerance for high substrate concentrations (up to 3.0 M). These results demonstrate a promising potential for industrial scaling-up to produce short-chain flavor esters at high substrate concentrations in non-aqueous media. Conclusions: This manuscript reports unprecedented alcohol tolerance and conversion of an esterase biocatalyst identified from a marine mud metagenomic library. The high organic solvent tolerance and thermostability of EST4 suggest that it has great potential as a biocatalyst.