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

作者:Javier Jimenez Diego; Salvador Montana Jose; Alvarez Diana; Baena Sandra
来源:World Journal of Microbiology and Biotechnology, 2012, 28(1): 361-370.
DOI:10.1007/s11274-011-0828-x

摘要

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 10A degrees C and moderately thermo active with relative activity of 80% at 50A degrees C and had a pH optimum of 8.0 at 40A degrees C.

  • 出版日期2012-1