A novel cold active esterase derived from Colombian high Andean forest soil metagenome

• Published in: World journal of microbiology & biotechnology 2012, Vol.28 (1), p.361-370
• Main Authors: Jiménez, Diego Javier, Montaña, José Salvador, Álvarez, Diana, Baena, Sandra
• Format: Article
• Language: English
• Subjects: Acinetobacter - enzymology; Acinetobacter - genetics; Acinetobacter - isolation & purification; Actinobacteria; Altitude; Amino Acid Sequence; Amino acids; Analysis; Applied Microbiology; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Binding sites; Biochemistry; Biology; Biomedical and Life Sciences; Biotechnology; Biotechnology industry; Cloning; Cold; Cold Temperature; Colombia; Conserved Sequence; DNA, Bacterial - genetics; E coli; Ecosystem biology; Environmental Engineering/Biotechnology; Enzymes; Escherichia coli; Esterases - genetics; Esterases - isolation & purification; Esterases - metabolism; Forest soils; Genes; Genomic Library; Genomics; Kinetics; Life Sciences; Lipase - genetics; Lipase - isolation & purification; Lipase - metabolism; Metagenome; Microbial activity; Microbiology; Microorganisms; Molecular Sequence Data; Original Paper; Phylogeny; Proteins; Proteobacteria; Sequence Homology, Amino Acid; Soil Microbiology; Studies; Trees
• ISSN: 0959-3993; 1573-0972
• DOI: 10.1007/s11274-011-0828-x

In order to search new lipolytic enzymes and conduct bioprospecting of microbial communities from high Andean forest soil, a metagenomic library of approximately 20,000 clones was constructed in Escherichia coli using plasmid p-Bluescript II SK+. The library covered 80 Mb of the metagenomic DNA mainly from Proteobacteria , Actinobacteria and Acidobacteria . Two clones with lipolytic activity in tributyrin as a substrate were recovered. Clone BAA3G2 (pSK- est GX1) was selected and the entire 4.6 Kb insert sequence was determined. The sequence had a GC content of 70.6% and could be derived from an undescribed Actinobacteria genome. One open reading frame encoded a polypeptide of 210 amino acids (gene est GX1) with a molecular mass of 22.4 kDa that contained the pentapeptide G-P- S -G-G near the N-terminus essential for lipase activity and the putative catalytic triad was identified, also a putative ribosomal binding site located 18 bp upstream the est GX1 ATG start codon was identified. The phylogenetic analysis suggested that the protein belonged to a new lipase family. The secreted enzyme showed a preference for short length fatty acids, with specific activity against p -nitrophenyl-butyrate (0.142 U/mg of total protein), it was cold active with relative activity of 30% at 10°C and moderately thermo active with relative activity of 80% at 50°C and had a pH optimum of 8.0 at 40°C.