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Cloning and biochemical characterization of a novel carbendazim (methyl-1H-benzimidazol-2-ylcarbamate)-hydrolyzing esterase from the newly isolated Nocardioides sp. strain SG-4G and its potential for use in enzymatic bioremediation.

ABSTRACT: A highly efficient carbendazim (methyl-1H-benzimidazol-2-ylcarbamate, or MBC)-mineralizing bacterium was isolated from enrichment cultures originating from MBC-contaminated soil samples. This bacterium, Nocardioides sp. strain SG-4G, hydrolyzed MBC to 2-aminobenzimidazole, which in turn was converted to the previously unknown metabolite 2-hydroxybenzimidazole. The initial steps of this novel metabolic pathway were confirmed by growth and enzyme assays and liquid chromatography-mass spectrometry (LC-MS) studies. The enzyme responsible for carrying out the first step was purified and subjected to N-terminal and internal peptide sequencing. The cognate gene, named mheI (for MBC-hydrolyzing enzyme), was cloned using a reverse genetics approach. The MheI enzyme was found to be a serine hydrolase of 242 amino acid residues. Its nearest known relative is an uncharacterized hypothetical protein with only 40% amino acid identity to it. Codon optimized mheI was heterologously expressed in Escherichia coli, and the His-tagged enzyme was purified and biochemically characterized. The enzyme has a K(m) and k(cat) of 6.1 muM and 170 min(-1), respectively, for MBC. Radiation-killed, freeze-dried SG-4G cells showed strong and stable MBC detoxification activity suitable for use in enzymatic bioremediation applications.

SUBMITTER: Pandey G

PROVIDER: S-EPMC2863442 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Cloning and biochemical characterization of a novel carbendazim (methyl-1H-benzimidazol-2-ylcarbamate)-hydrolyzing esterase from the newly isolated Nocardioides sp. strain SG-4G and its potential for use in enzymatic bioremediation.

Pandey Gunjan G Dorrian Susan J SJ Russell Robyn J RJ Brearley Clint C Kotsonis Steven S Oakeshott John G JG

Applied and environmental microbiology 20100312 9

A highly efficient carbendazim (methyl-1H-benzimidazol-2-ylcarbamate, or MBC)-mineralizing bacterium was isolated from enrichment cultures originating from MBC-contaminated soil samples. This bacterium, Nocardioides sp. strain SG-4G, hydrolyzed MBC to 2-aminobenzimidazole, which in turn was converted to the previously unknown metabolite 2-hydroxybenzimidazole. The initial steps of this novel metabolic pathway were confirmed by growth and enzyme assays and liquid chromatography-mass spectrometry ...[more]

PMID: 20228105

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Project description:BackgroundPyrethroids 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.ResultsThe 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.ConclusionA 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.

Project description:BackgroundMicrobes are rich sources of enzymes and esterases are one of the most important classes of enzymes because of their potential for application in the field of food, agriculture, pharmaceuticals and bioremediation. Due to limitations in their cultivation, only a small fraction of the complex microbial communities can be cultured from natural habitats. Thus to explore the catalytic potential of uncultured organisms, the metagenomic approach has turned out to be an effective alternative method for direct mining of enzymes of interest. Based on activity-based screening method, an esterase-positive clone was obtained from metagenomic libraries.ResultsFunctional screening of a soil metagenomic fosmid library, followed by transposon mutagenesis led to the identification of a 1179 bp esterase gene, estM2, that encodes a 392 amino acids long protein (EstM2) with a translated molecular weight of 43.12 kDa. Overproduction, purification and biochemical characterization of the recombinant protein demonstrated carboxylesterase activity towards short-chain fatty acyl esters with optimal activity for p-nitrophenyl butyrate at pH 8.0 and 37 °C. Amino acid sequence analysis and subsequent phylogenetic analysis suggested that EstM2 belongs to the family VIII esterases that bear modest similarities to class C β-lactamases. EstM2 possessed the conserved S-x-x-K motif of class C β-lactamases but did not exhibit β-lactamase activity. Guided by molecular docking analysis, EstM2 was shown to hydrolyze a wide range of di- and monoesters of alkyl-, aryl- and benzyl-substituted phthalates. Thus, EstM2 displays an atypical hydrolytic potential of biotechnological significance within family VIII esterases.ConclusionsThis study has led to the discovery of a new member of family VIII esterases. To the best of our knowledge, this is the first phthalate hydrolase (EstM2), isolated from a soil metagenomic library that belongs to a family possessing β-lactamase like catalytic triad. Based on its catalytic potential towards hydrolysis of both phthalate diesters and phthalate monoesters, this enzyme may find use to counter the growing pollution caused by phthalate-based plasticizers in diverse geological environment and in other aspects of biotechnological applications.

Project description:BackgroundPyrethroids are potentially harmful to human health and ecosystems. It is necessary to develop some efficient strategies to degrade pyrethroid residues. Biodegradation is generally considered as a safe, efficient, and inexpensive way to eliminate environmental contaminants. To date, although several pyrethroid-hydrolyzing esterases have been cloned, there has been no report about a pyrethroid hydrolase with high hydrolytic activity, good stability, and high productivity, indispensable enzymatic properties in practical biodegradation. Almost all pyrethroid hydrolases are intracellular enzymes, which require complex extraction protocols and present issues in terms of easy inactivation and low production.ResultsIn this study, random mutagenesis was performed on one pyrethroid-hydrolyzing esterase, Sys410, to enhance its activity and thermostability. Two beneficial mutations, A171V and D256N, were obtained by random mutagenesis and gave rise to the mutant M2. The mutant displayed ~1.5-fold improvement in the kcat/Km value and 2.46-fold higher catalytic activity. The optimal temperature was 10 °C higher than that of the wild-type enzyme (55 °C). The half-life at 40-65 °C was 3.3-310 times longer. It was surprising that M2 has a half-life of 12 h at 70 °C while Sys410 was completely inactivated at 70 °C. In addition, the desired gene was extracellularly expressed in a Pichia pastoris host system. The soluble expression level reached up to 689.7 mg/L. Remarkably, the enzyme could efficiently degrade various pyrethroids at moderate temperature for 15 min, exceeding a hydrolysis rate of 98%, which is the highest value ever reported.ConclusionsThis is the first report about random mutagenesis and secretory expression of pyrethroid-hydrolyzing esterase with high-level productivity and purity in P. pastoris. Broad substrate specificity, enhanced activity and thermostability make M2 an ideal candidate for the biodegradation of pyrethroid residues.

Project description:Hydrophobic interactions and hydrogen bonds are 2 types of noncovalent interactions that play distinct roles in the folding and structural stability of proteins. However, the specific roles of these interactions in hydrophobic or hydrophilic environments in α/β-hydrolases are not fully understood. A hyperthermophilic esterase EstE1 in a dimer maintains the C-terminal β8-α9 strand-helix via hydrophobic interactions (Phe276 and Leu299), constituting a closed dimer interface. Moreover, a mesophilic esterase rPPE in a monomer maintains the same strand-helix via a hydrogen bond (Tyr281 and Gln306). Unpaired polar residues (F276Y in EstE1 and Y281A/F and Q306A in rPPE) or reduced hydrophobic interactions (F276A/L299A in EstE1) between the β8-α9 strand-helix decrease thermal stability. EstE1 (F276Y/L299Q) and rPPE WT, both with the β8-α9 hydrogen bond, showed the same thermal stability as EstE1 WT and rPPE (Y281F/Q306L), which possess hydrophobic interactions instead. However, EstE1 (F276Y/L299Q) and rPPE WT exhibited higher enzymatic activity than EstE1 WT and rPPE (Y281F/Q306L), respectively. This suggests that α/β-hydrolases favor the β8-α9 hydrogen bond for catalytic activity in monomers or oligomers. Overall, these findings demonstrate how α/β-hydrolases modulate hydrophobic interactions and hydrogen bonds to adapt to different environments. Both types of interactions contribute equally to thermal stability, but the hydrogen bond is preferred for catalytic activity. IMPORTANCE Esterases hydrolyze short to medium-chain monoesters and contain a catalytic His on a loop between the C-terminal β8-strand and α9-helix. This study explores how hyperthermophilic esterase EstE1 and mesophilic esterase rPPE adapt to different temperatures by utilizing the β8-α9 hydrogen bonds or hydrophobic interactions differently. EstE1 forms a hydrophobic dimer interface, while rPPE forms a monomer stabilized by a hydrogen bond. The study demonstrates that these enzymes stabilize β8-α9 strand-helix differently but achieve similar thermal stability. While the β8-α9 hydrogen bond or hydrophobic interactions contribute equally to thermal stability, the hydrogen bond provides higher activity due to increased catalytic His loop flexibility in both EstE1 and rPPE. These findings reveal how enzymes adapt to extreme environments while maintaining their functions and have implications for engineering enzymes with desired activities and stabilities.

Project description:BACKGROUND:Pyrethroids are potentially harmful to living organisms and ecosystems. Thus, concerns have been raised about pyrethroid residues and their persistence in agricultural products. To date, although several pyrethroid-hydrolyzing enzymes have been cloned, very few reports are available on pyrethroid-hydrolyzing enzymes with cold adaptation, high hydrolytic activity and good reusability, indispensable properties in practical bioremediation of pyrethroid-contaminated vegetables. RESULTS:Here, a novel gene (est684) encoding pyrethroid-hydrolyzing esterase was isolated from the Mao-tofu metagenome for the first time. Est684 encoded a protein of 227 amino acids and was expressed in Escherichia coli BL21 (DE3) in soluble form. The optimum temperature was 18 °C. It maintained 46.1% of activity at 0 °C and over 50% of its maximal activity at 4-35 °C. With the goal of enhancing stability and recycling biocatalysts, we used mesoporous silica SBA-15 as a nanometer carrier for the efficient immobilization of Est684 by the absorption method. The best conditions were an esterase-to-silica ratio of 0.96 mg/g (w/w) and an adsorption time of 30 min at 10 °C. Under these conditions, the recovery of enzyme activity was 81.3%. A large improvement in the thermostability of Est684 was achieved. The half-life (T1/2) of the immobilized enzyme at 35 °C was 6 h, 4 times longer than the soluble enzyme. Interestingly, the immobilized Est684 had less loss in enzyme activity up to 12 consecutive cycles, and it retained nearly 54% of its activity after 28 cycles, indicating excellent operational stability. Another noteworthy characteristic was its high catalytic activity. It efficiently hydrolyzed cyhalothrin, cypermethrin, and fenvalreate in pyrethroid-contaminated cucumber within 5 min, reaching over 85% degradation efficiency after four cycles. CONCLUSIONS:A novel cold-adapted pyrethroid-hydrolyzing esterase was screened from the Mao-tofu metagenome. This report is the first on immobilizing pyrethroid-hydrolyzing enzyme on mesoporous silica. The immobilized enzyme with high hydrolytic activity and outstanding reusability has a remarkable potential for bioremediation of pyrethroid-contaminated vegetables, and it is proposed as an industrial enzyme.

Dataset Information

Cloning and biochemical characterization of a novel carbendazim (methyl-1H-benzimidazol-2-ylcarbamate)-hydrolyzing esterase from the newly isolated Nocardioides sp. strain SG-4G and its potential for use in enzymatic bioremediation.

Publications

Cloning and biochemical characterization of a novel carbendazim (methyl-1H-benzimidazol-2-ylcarbamate)-hydrolyzing esterase from the newly isolated Nocardioides sp. strain SG-4G and its potential for use in enzymatic bioremediation.

Similar Datasets

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