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A novel esterase from a soil metagenomic library displaying a broad substrate range.

ABSTRACT: A novel esterase gene was isolated from a soil metagenomic library. The gene encoded a protein of 520 amino acids which contained a 21 aa signal peptide. Primary structure analysis of the protein sequence revealed that it contained a conserved active site motif (SxSxG) and a structural motif (CS-D-HC). Then the esterase gene was cloned and expressed in Escherichia coli BL21(DE3). SDS-PAGE analysis of the purified esterase showed that it was expressed in a highly soluble form and its molecular mass was estimated to be 55 kDa. Characterization of the esterase revealed that it exhibited high activity toward p-nitrophenyl esters with short acyl chains and especially p-nitrophenyl acetate, suggesting that it was a typical carboxylesterase rather than a lipase. With p-nitrophenyl acetate as substrate, the enzyme showed its optimal activity at pH 7.0 and 30 °C, and it was stable at a broad pH range from 4.5 to 10.0 and temperature not higher than 50 °C. Furthermore, the enzyme showed different substrate specificity from known esterase, it was not only hydrolyzing against p-nitrophenyl esters, but also hydrolyzing all hydroxybenzoic esters and hydroxycinnamic ester assayed. As it was an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase, chlorogenate esterase and tannase activities, it could serve as a valuable candidate for applications in biotechnology.

SUBMITTER: Yao J

PROVIDER: S-EPMC7936011 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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A novel esterase from a soil metagenomic library displaying a broad substrate range.

Yao Jian J Gui Lun L Yin Shaocheng S

AMB Express 20210305 1

A novel esterase gene was isolated from a soil metagenomic library. The gene encoded a protein of 520 amino acids which contained a 21 aa signal peptide. Primary structure analysis of the protein sequence revealed that it contained a conserved active site motif (SxSxG) and a structural motif (CS-D-HC). Then the esterase gene was cloned and expressed in Escherichia coli BL21(DE3). SDS-PAGE analysis of the purified esterase showed that it was expressed in a highly soluble form and its molecular ma ...[more]

PMID: 33666762

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Project description: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.

Dataset Information

A novel esterase from a soil metagenomic library displaying a broad substrate range.

Publications

A novel esterase from a soil metagenomic library displaying a broad substrate range.

Similar Datasets

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