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

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.

Immobilization of a Novel ESTBAS Esterase from Bacillus altitudinis onto an Epoxy Resin: Characterization and Regioselective Synthesis of Chloramphenicol Palmitate

“…Lx-Est BAS ∆SP retained over 85% of its initial catalytic activity after storage for six weeks (4 • C), whereas the free enzyme lost~80% of its activity after only two weeks and lost almost all its activity in less than three weeks. These results clearly indicated that enzyme immobilization significantly increased storage stability, in line with another report [37].…”

A Novel Cold Active Esterase from a Deep Sea Sponge Stelletta normani Metagenomic Library

“…The CAP domain is highlighted in red, the catalytic triad Ser144, Glu238, and His268 is highlighted in magenta, the oxyanion hole comprising of residues Gly76 and Gly77 is highlighted in yellow, the catalytic domain is highlighted in light blue. an important role in marine carbon degradation and cycling, and given that a functional metagenomics based approach had previously been successfully employed to identify the E40 esterase from sediment from the South China Sea (Li et al, 2012;Li P. Y. et al, 2015), and that marine sponge and sediment metagenomics libraries had resulted in the discovery of a variety of novel lipases including the recent reports of a cold-active salt tolerant esterase from artic sediment (De Santi et al, 2016); and a high organic solvent tolerant and thermostable esterase from marine mud (Gao et al, 2016); we targeted the metagenome of the deep sea sponge S. normani in an attempt to increase the repertoire of marine derived halophilic esterases. To this end we succeeded in identifying a gene encoding a novel psychrophilic esterase (7N9) from the hormone sensitive lipase (Hsl) family IV following the functional screening of a S. normani metagenomic library.…”

Integrative computational approach for genome-based study of microbial lipid-degrading enzymes

“…The properties of microbial lipases and their industrial applications have been reviewed by Verma et al (2012). A large set of microbial lipases has been successfully identified using metagenomics, which is a cultivation-independent approach, and targets samples from extreme environments (Kim et al 2004;Barriuso et al 2013;Gao et al 2016). …”

“…Next-generation sequencing technologies have allowed the sequencing of entire microbial genomes and metagenomics from particular environments, such as activated sludge and marine mud (Kim et al 2004;Ranjan et al 2005;Liaw et al 2010;Barriuso et al 2013;Gao et al 2016). These molecular genomics applications have permitted the analysis of sequencing data to identify target enzymes with specific functionalities for industrial interest.…”

“…Lipases prefer to catalyse the hydrolysis of water-insoluble substrates with long-chain fatty acyl chains (C10-carbon atoms), such as triacylglycerides, phospholipids and cholesteryl esters, whereas esterases preferentially hydrolyse water-soluble or emulsified esters of short-chain carboxylic acids (\10-carbon atoms) (Arpigny and Jaeger 1999;Wong and Schotz 2002;Gao et al 2016). These enzymes do not require cofactors and are stable in organic solvents, which facilitate their catalysis of several biochemical reactions, including hydrolysis, esterification, transesterification, interesterification, kinetic resolution of racemic acids and alcohols, regioselective acylation of steroids and the synthesis of peptides and other chemicals.…”