Abstract
BackgroundMarine 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.ResultsBased 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 °C and pH 8.0. The half-life of EST4 is 55 and 46 h at 40 and 45 °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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0435-5) contains supplementary material, which is available to authorized users.
Highlights
Marine mud is an abundant and largely unexplored source of enzymes with unique properties that may be useful for industrial and biotechnological purposes
The marine mud metagenomic library revealed more than 40,000 fosmid clones and represented about 1.6 Gb of the microbial community DNA
This restriction analysis suggests that the metagenomic library is of high quality and diversity
Summary
Marine mud is an abundant and largely unexplored source of enzymes with unique properties that may be useful for industrial and biotechnological purposes. Since most microbes cannot be cultured in the laboratory, a cultivation-independent metagenomic approach would be advantageous for the identification of novel enzymes. With the objective of screening novel lipolytic enzymes, a metagenomic library was constructed using the total genomic DNA extracted from marine mud. The identification of novel biocatalysts from microorganisms is limited by the fact that only 1 % of microorganisms can be cultured using conventional laboratory methods [6]. There is great interest in further metagenomicbased searches for novel enzymes from different sources and with greater industrial applicability
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