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 beta-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.

Ruminant digestive tract microbes hydrolyse plant biomass, and the application of metagenomic techniques can provide good coverage of their glycosyl hydrolase enzymes. A metagenomic library of circa 70,000 fosmids was constructed from bacterial DNA isolated from bovine rumen and subsequently screened for cellulose hydrolysing activities on a CMC agar medium. Two clones were selected based on large clearance zones on the CMC agar plates. Following nucleotide sequencing, translational analysis and homology searches, two cellulase encoding genes (cel5A and cel5B) belonging to the glycosyl hydrolyse family 5 were identified. Both genes encoded pre-proteins of about 62 kDa, containing signal leader peptides which could be cleaved to form mature proteins of about 60 kDa. Biochemical characterisation revealed that both enzymes showed alkaline pH optima of 9.0 and the temperature optima of 65 °C. Substrate specificity profiling of the two enzymes using 1,4-β-D-cello- and xylo-oligosaccharides revealed preference for longer oligosaccharides (n ≥ 3) for both enzymes, suggesting that they are endo-cellulases/xylanases. The bifunctional properties of the two identified enzymes render them potentially useful in degrading the β-1,4 bonds of both the cellulose and hemicellulose polymers.

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.

A metagenome expression library was created from Trinervitermes trinervoides termite hindgut symbionts and subsequently screened for feruloyl esterase (FAE) activities, resulting in seven recombinant fosmids conferring feruloyl esterase phenotypes. The amino acid sequence lengths of the seven FAE encoding open reading frames (ORFs) ranged from 260 to 274 aa and encoded polypeptides of between 28.9 and 31.4 kDa. The highest sequence identity scores for the seven ORFs against the GenBank database were between 45 and 59 % to a number of carboxyl ester hydrolyses. The seven FAE primary structures contained sequence motifs that correspond well with a classical pentapeptide (G-x-S-x-G) serine hydrolyse signature motif which harbours the catalytic serine residue in other FAE families. Six of the seven fae genes were successfully expressed heterologously in Escherichia coli, and the purified enzymes exhibited temperature optima range of 40-70 °C and the pH optima of between 6.5 and 8.0. The k(cat)/K(M) ratios for the six characterised FAEs showed the following order of substrate preference: methyl sinapate > methyl ferulate > ethyl ferulate. All six FAEs showed poor conversion rates against methyl p-coumarate and methyl caffeate, both of which lacked the methoxy (O-CH₃) group substituent on the aromatic ring of the ester substrates, emphasising the requirement for at least one methoxy group on the aromatic ring of the hydroxycinnamic acid ester substrate for optimal FAE activity.