ABSTRACT
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-ß-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 ß-1,4 bonds of both the cellulose and hemicellulose polymers.
Subject(s)
Metagenome , Rumen/microbiology , Xylosidases/genetics , Xylosidases/metabolism , Animals , Cattle , Cloning, Molecular , Enzyme Stability , Gene Expression , Gene Library , Hydrogen-Ion Concentration , Molecular Sequence Data , Protein Sorting Signals , Sequence Analysis, DNA , Substrate Specificity , Temperature , Xylosidases/chemistry , Xylosidases/isolation & purificationABSTRACT
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.
Subject(s)
Bacteria/enzymology , Carboxylesterase/isolation & purification , Environmental Microbiology , Genomic Library , Amino Acid Sequence , Bacteria/genetics , Bacteria/metabolism , Base Sequence , Carboxylesterase/chemistry , Carboxylesterase/genetics , Carboxylesterase/metabolism , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Enzymologic/physiology , Molecular Sequence Data , PhylogenyABSTRACT
DnaJ-like proteins are defined by the presence of an approximately 73 amino acid region termed the J domain. This region bears similarity to the initial 73 amino acids of the Escherichia coli protein DnaJ. Although the structures of the J domains of E coli DnaJ and human heat shock protein 40 have been solved using nuclear magnetic resonance, no detailed analysis of the amino acid conservation among the J domains of the various DnaJ-like proteins has yet been attempted. A multiple alignment of 223 J domain sequences was performed, and the levels of amino acid conservation at each position were established. It was found that the levels of sequence conservation were particularly high in 'true' DnaJ homologues (ie, those that share full domain conservation with DnaJ) and decreased substantially in those J domains in DnaJ-like proteins that contained no additional similarity to DnaJ outside their J domain. Residues were also identified that could be important for stabilizing the J domain and for mediating the interaction with heat shock protein 70.