Abstract
An esterase-producing clone Aph2 was isolated from the Apharwat soil metagenomic library, a mountain peak in NW Himalayas. ORF 2 (Est Ac) of clone Aph2 corresponds to 271 aa protein and showed 26 % sequence similarity to carboxylesterase gene of Synechococcus sp. JA-2-3B. Est Ac contains nucleophilic Ser in S68-X-X-K71 motif of β-lactamases with Tyr Y103. The conserved sequences are common with family VIII carboxylesterase and class C β-lactamase sequences. Phylogenetic analysis revealed that Est Ac sequence is closely related to esterase than to β-lactamases. In silico 3D protein structure of Est Ac was generated using MODELLER software (9.10 version). Model was generated on the basis of carboxylesterase template (PDB:1CI8) of Est B (Burkholderia gladioli) and the stereochemical parameters of the model generated were satisfactory. Docking with diisopropyl-fluorophosphate confirmed catalytic activity of Ser68 present in S-X-X-K motif.
Highlights
Genes with wide industrial applications have been isolated from animals, plants and microbes with the largest source being the microbes (Wiseman 1995)
Metagenomic library of soil metagenome isolated from Apharwat mountain peak in the north western Himalayas was constructed and 10,000 clones were screened for esterase activity on the tributyrin plates
Multiple sequence alignment of sequences of family VIII esterases, class C b-lactamases with Est Ac was carried out using CLUSTALX software
Summary
Genes with wide industrial applications have been isolated from animals, plants and microbes with the largest source being the microbes (Wiseman 1995). Microbial genes are Multiple sequence alignment and phylogenetic analysis. Multiple sequence alignment was done using CLUSTALX (Larkin et al 2007). The sequences selected for multiple alignment were retrieved from NCBI. Class C b-lactamase included that from Candidatus kori (Yp_589716), Caulobacter sp.K31 (Yp_001682441) and family VIII carboxylesterase of that of Pseudomonas sp. (gi AAA25813.1), esterase A Streptomyces anulatus (gi CAA78842.1) and Est B Burkholderia gladioli (PDB:1CI8) sequences. Evolutionary relationship between Est Ac, family VIII carboxylesterase and class C b-lactamase was inferred using neighbour joining method conducted with CLc software. Functional ORF was submitted to GenBank under accession number JX068525.1
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