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  • Title: Discovery of a novel carboxylesterase through functional screening of a pre-enriched environmental library.
    Author: Rashamuse K, Ronneburg T, Hennessy F, Visser D, van Heerden E, Piater L, Litthauer D, Möller C, Brady D.
    Journal: J Appl Microbiol; 2009 May; 106(5):1532-9. PubMed ID: 19226392.
    Abstract:
    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 degrees 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.
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