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Characterization of ML-005, a Novel Metaproteomics-Derived Esterase.

ABSTRACT: A novel gene encoding for a lipolytic enzyme, designated ML-005, was recently identified using a functional metaproteomics approach. We heterologously expressed this protein in Escherichia coli and biochemically characterized it. ML-005 exhibited lipolytic activity toward short-chained substrates with the preferred substrate being p-nitrophenyl-butyrate, suggesting that ML-005 is an esterase. According to homology analysis and site-directed mutagenesis, the catalytic triad of the enzyme was identified as Ser-99, Asp-164, and His-191. Its optimal pH was determined to be at pH 8. Optimal activity was observed at 45°C. It also exhibited temperature, pH and salt tolerance. Residual relative activity after incubating at 50-60°C for 360 min was above 80% of its initial activity. It showed tolerance over a broad range of pH (5-12) and retained most of its initial activity. Furthermore, incubating ML-005 in 1 - 5M NaCl solution had negligible effect on its activity. DTT, EDTA, and ß-mercaptoethanol had no significant effect on ML-005's activity. However, addition of PMSF led to almost complete inactivation consistent with ML-005 being a serine hydrolase. ML-005 remains stable in the presence of a range of metal ions, but addition of Cu2+ significantly reduces its relative activity. Organic solvents have an inhibitory effect on ML-005, but it retained 21% of activity in 10% methanol. SDS had the most pronounced inhibitory effect on ML-005 among all detergents tested and completely inactivated it. Furthermore, the Vmax of ML-005 was determined to be 59.8 ?M/min along with a Km of 137.9 ?M. The kcat of ML-005 is 26 s-1 and kcat/Km is 1.88 × 105 M-1 s-1.

SUBMITTER: Sukul P

PROVIDER: S-EPMC6119806 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Pyrethroids and pyrethrins are widely used insecticides. Extensive applications not only result in pest resistance to these insecticides, but also may lead to environmental issues and human exposure. Numerous studies have shown that very high exposure to pyrethroids might cause potential problems to man and aquatic organisms. Therefore, it is important to develop a rapid and efficient disposal process to eliminate or minimize contamination of surface water, groundwater and agricultural products by pyrethroid insecticides. Bioremediation is considered to be a reliable and cost-effective technique for pesticides abatement and a major factor determining the fate of pyrethroid pesticides in the environment, and suitable esterase is expected to be useful for potential application for detoxification of pyrethroid residues. Soil is a complex environment considered as one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms in nature are inaccessible as they are uncultivable in the laboratory. Metagenomic approaches provide a powerful tool for accessing novel valuable genetic resources (novel enzymes) and developing various biotechnological applications. RESULTS: The pyrethroid pesticides residues on foods and the environmental contamination are a public safety concern. Pretreatment with pyrethroid-hydrolyzing esterase has the potential to alleviate the conditions. To this end, a pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, sequence analysis of the DNA responsible for the pye3 gene revealed an open reading frame of 819 bp encoding for a protein of 272 amino acid residues. Extensive multiple sequence alignments of the deduced amino acid of Pye3 with the most homologous carboxylesterases revealed moderate identity (45-49%). The recombinant Pye3 was heterologously expressed in E. coli BL21(DE3), purified and characterized. The molecular mass of the native enzyme was approximately 31 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of the Pye3 indicated molecular mass of 31 kDa and 31.5 kDa, respectively, suggesting that the Pye3 is a monomer. The purified Pye3 not only degraded all pyrethroid pesticides tested, but also hydrolyzed rho-nitrophenyl esters of medium-short chain fatty acids, indicating that the Pye3 is an esterase with broader specificity. The Km values for trans-Permethrin and cis-permethrin are 0.10 muM and 0.18 muM, respectively, and these catalytic properties were superior to carboxylesterases from resistant insects and mammals. The catalytic activity of the Pye3 was strongly inhibited by Hg2+, Ag+, rho-chloromercuribenzoate, whereas less pronounced effect was observed in the presence of divalent cations, the chelating agent EDTA and phenanthroline. CONCLUSION: A novel pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, the broader substrate specificities and higher activity of the pyrethroid-hydrolyzing esterase (Pye3) make it an ideal candidate for in situ for detoxification of pyrethroids where they cause environmental contamination problems. Consequently, metagenomic DNA clone library offers possibilities to discover novel bio-molecules through the expression of genes from uncultivated bacteria.

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Characterization of ML-005, a Novel Metaproteomics-Derived Esterase.

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