Discovery of a novel carboxylesterase through functional screening of a pre-enriched environmental library

Aims:  The aim of this study was to demonstrate the application of environmental sample pre-enrichment to access novel carboxylesterases from environmental genomes, along with subsequent heterologous expression and characterization of the discovered enzyme(s).
Methods and Results:  A positive recombinant clone (UVCL29), conferring an esterase phenotype was identified from a shotgun gene library. The complete sequence of the 3·0 kb DNA insert from the pUVCL29 recombinant plasmid was obtained using primer-walking strategies. Nucleotide sequence analysis revealed a complete 945 bp open reading frame (ORF1). Translational analysis of the ORF1 showed a protein of 314 amino acids (named EstAM) with a predicted molecular weight of 34 kDa. EstAM's primary structure showed a classical (–G–D–S–A–G–) motif, corresponding with the generally conserved (G–x–S–x–G) esterase signature motif. Identity searches indicated that EstAM has high sequence similarity with esterases from family IV. EstAM was successfully expressed in Escherichia coli in a biologically active form. Partial purification was achieved using a one-step Pro-PurTM IMAC column. Biochemical characterization revealed that EstAM has a temperature optimum of 40°C.
Conclusion:  Based on its substrate profile, EstAM was classified as a carboxylesterase because of its preference for short p -nitrophenyl ester substrates.
Significance and Impact of the Study:  This study is a demonstration of the successful application of environmental sample pre-enrichment technology in accessing novel esterases from a mining environment.