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

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Abstract

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.

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Acknowledgments

We thank I. Balzer for useful discussion, M. M. Zambrano for reviewing the manuscript and D. Borda for library screening. This research was supported by Department of Science, Technology and Innovation (COLCIENCIAS)—SENA (project number 6570-392-19990) and was done under MAVDT (contract number 15, 2008) for access to genetic resources and UAESPNN (research permit code DTNO-N-20/2007).

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  1. 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 Jiménez & Sandra Baena

  2. Department of Microbiology, Pontificia Universidad Javeriana, Carrera 7 No. 43-82, Building 53, PO Box 5671, Bogotá, D.C., Colombia

    José Salvador Montaña

  3. Unidad de Genética y Biología del Desarrollo, Department of Biology, Pontificia Universidad Javeriana, Carrera 7 No. 43-82, Building 53, PO Box 5671, Bogotá, D.C., Colombia

    Diana Álvarez

  4. Colombian Center for Genomic and Bioinformatics from Extreme Environments (GeBiX), Carrera 5 No. 66A-34, 113, PO Box 110231, Bogotá, D.C., Colombia

    Diego Javier Jiménez, José Salvador Montaña, Diana Álvarez & Sandra Baena

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  1. Diego Javier Jiménez
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  2. José Salvador Montaña
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Correspondence to José Salvador Montaña.

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Jiménez, D.J., Montaña, J.S., Álvarez, D. et al. A novel cold active esterase derived from Colombian high Andean forest soil metagenome. World J Microbiol Biotechnol 28, 361–370 (2012). https://doi.org/10.1007/s11274-011-0828-x

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