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

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°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.

Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil

“…Family IV carboxylesterases include hormone-sensitive lipases (HSLs). Enzymes in this family are similar to the mammalian HSL enzyme [1,38], which is involved in lipid metabolism and energy homeostasis. HSLs control release of fatty acids from triacylglycerols stored in adipose tissue.…”

“…Mammalian HSLs contain a catalytic domain and an associated unique regulatory module in the N-terminal domain [35]. Family IV carboxylesterases have four conserved sequence motifs: the oxyanion region GGGX, the pentapeptide -GDSAG-signature motif, and two C-terminal conserved motifs, -DPLR-and -HGF- [1,38,46]. The deduced amino acid sequence indicates that EstIM1 is similar to other esterases of family IV, as it also contains a GGGX (N-terminal oxyanion region) motif, the pentapeptide GXSXG (a nucleophilic elbow), a DPLR motif (at position 252-255), and an HGF motif (at position 282-284) (Fig.…”

“…AAC38151). Carboxylesterases in family IV hydrolyze acyl chains less than 10 carbon atoms in length [32], and can be either thermolabile [18] or thermostable [38,40]. We used a spectrophotometric assay to search for new genes encoding lipases/esterases in a metagenomic library, overexpressed one such gene, and characterized the gene product.…”

Accessing Carboxylesterase Diversity from Termite Hindgut Symbionts through Metagenomics

“…Functional screening of the recombinant esterase-positive clones in E. coli EPI100-T1 was performed on LB agar plates supplemented with isopropyl-␤ -D -thiogalactopyranoside (IPTG, 0.1 m M ), chloramphenicol (12.5 g ml -1 ), tributyrin 1% (v/v) and gum arabic 0.1% (w/v), followed by incubation at 37 ° C. Esterase-positive clones were identified by the presence of zone of clearance around the colony margins [Rashamuse et al, 2009a].…”

“…In principle, culture-independent metagenomics approaches allow for direct tapping of the entire genetic pool held within natural microbial populations, which cannot be comprehensively accessed using classical enrichment approaches [Nacke et al, 2011;Rondon et al, 2000]. The capacity of the culture-independent metagenomics approach has been demonstrated by the discovery of novel enzyme genes with potential industrial applications [Chandrasekharaiah et al, 2011;Hu et al, 2010;Rashamuse et al, 2009aRashamuse et al, , 2012.…”