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

作者：Rashamuse K; Ronneburg T; Hennessy F; Visser D; van Heerden E; Piater L; Litthauer D; Moeller C; Brady D^*

来源：Journal of Applied Microbiology, 2009, 106(5): 1532-1539.

DOI：10.1111/j.1365-2672.2008.04114.x

• carboxylesterase
• environmental library
• functional screening

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).
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.
Based on its substrate profile, EstAM was classified as a carboxylesterase because of its preference for short p-nitrophenyl ester substrates.
This study is a demonstration of the successful application of environmental sample pre-enrichment technology in accessing novel esterases from a mining environment.

• 出版日期2009-5