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A novel family VIII carboxylesterase derived from a leachate metagenome library exhibits promiscuous β-lactamase activity on nitrocefin

Abstract

The realization that majority of microbes are not amenable to cultivation as isolates under laboratory conditions has led to the culture-independent metagenomic approach as a novel technique for novel biocatalyst discovery. A leachate fosmid shotgun metagenome library was constructed and subsequently screened for esterolytic activities on a tributyrin agar medium. Nucleotide sequencing and translational analysis of an esterase-positive fosmid clone led to the identification of a 1,281 bp esterase gene (estC) encoding a protein (EstC) of 427 aa with translated molecular weight of 46.3 kDa. The EstC primary structure contained a signal leader peptide (29 aa), which could be cleaved to form a mature protein of 398 aa with molecular weight 43.3 kDa. Homology searches revealed that EstC belonged to the family VIII esterases, which exploit a serine residue within the S-x-x-K motif as a catalytic nucleophile. Substrate specificity studies showed that EstC prefers short to medium acyl chain length of p-nitrophenyl esters, a characteristic typical of “true” carboxylesterases. Moreover, EstC represents the first member of the family VIII esterases with a leader peptide and a detectable promiscuous β-lactam hydrolytic activity. Site-directed mutagenesis studies also revealed that in addition to Ser103 and Lys106 residues, the Tyr219 residue also plays a catalytic role in EstC. The organic solvent stability and the specificity towards esters of tertiary alcohols linalyl acetate (3,7-dimethyl-1,6-octadien-3-yl acetate) make EstC potentially useful in biocatalysis.

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Acknowledgments

The work was supported by the CSIR-YREF (Young Researcher Establishment Fund). The authors would also like to thank Mr Harris Tshwane Manchidi for the help with sample collection, Dr Edwin Mutlane for synthesizing the p-nitrobutyranilide substrate, and the members of the CSIR (Enzyme technologies group) for their useful comments and suggestions.

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Correspondence to Konanani Rashamuse.

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Rashamuse, K., Magomani, V., Ronneburg, T. et al. A novel family VIII carboxylesterase derived from a leachate metagenome library exhibits promiscuous β-lactamase activity on nitrocefin. Appl Microbiol Biotechnol 83, 491–500 (2009). https://doi.org/10.1007/s00253-009-1895-x

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Keywords

  • Metagenome library
  • Carboxylesterases
  • Promiscuous β-lactamase activity
  • Family VIII esterases