A novel cold active esterase derived from Colombian high Andean forest soil metagenome.
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-estGX1) 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 estGX1) 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 estGX1 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.
Unidad de Saneamiento y Biotecnología Ambiental, Department of Biology, Pontificia Universidad Javeriana, Carrera 7 No. 43-82, Building 53, PO Box 5671, Bogotá, D.C., Colombia.
Diego Javier
DJ
Jiménez
José Salvador
JS
Montaña
Diana
D
Alvarez
Sandra
S
Baena
D000150:Acinetobacter / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D000531:Altitude; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003080:Cold Temperature; D003105:Colombia; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004950:Esterases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015698:Genomic Library; D007700:Kinetics; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054892:Metagenome; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D014197:Trees
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004950:Esterases; D008049:Lipase