title: Identification of lipase encoding genes from Antarctic seawater bacteria using degenerate primers: expression of a cold-active lipase with high specific activity.
abstract: Cold-active enzymes are valuable catalysts showing high activity at low and moderate temperatures and low thermostability. Among cold-active enzymes, lipases offer a great potential in detergent, cosmetic, biofuel and food or feed industries. In this paper we describe the identification of novel lipase coding genes and the expression of a lipase with high activity at low temperatures. The genomic DNA from Antarctic seawater bacteria showing lipolytic activity at 4°C was used to amplify five DNA fragments that partially encode novel lipases using specifically designed COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP). All the fragments were found to have a high identity with an α/β-hydrolase domain-containing protein identified by the sequencing of the complete genome of Shewanella frigidimarina NCIMB 400. The complete sequence of one of the lipase-coding gene fragments, lipE13, was obtained by genome walking. Considering that the other fragments had a high identity to the putative lipase from S. frigidimarina NCIMB 400, the complete lipase genes were amplified using oligonucleotide primers designed based on the 5' and 3' regions of the coding sequence of the related protein. This strategy allowed the amplification of 3 lipase-encoding genes of which one was expressed in the periplasm using the Escherichia coli BL21(DE3)/pET-22b(+) expression system. The recombinant protein was obtained with activity toward p-nitrophenyl caproate showing a high specific activity between 15 and 25°C.
authors.affiliation: Centre for Biotechnology and Bioengineering (CeBiB), Department of Chemical Engineering and Biotechnology, University of Chile, Beauchef 850, Santiago, Chile; Department of Chemical and Bioprocesses Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Avenida Vicuña Mackenna 4860, Santiago, Chile.
authors.forename: Loreto P
authors.initials: LP
authors.lastname: Parra
authors.affiliation: Centre for Biotechnology and Bioengineering (CeBiB), Department of Chemical Engineering and Biotechnology, University of Chile, Beauchef 850, Santiago, Chile.
authors.forename: Giannina
authors.initials: G
authors.lastname: Espina
authors.affiliation: Centre for Biotechnology and Bioengineering (CeBiB), Department of Chemical Engineering and Biotechnology, University of Chile, Beauchef 850, Santiago, Chile.
authors.forename: Javier
authors.initials: J
authors.lastname: Devia
authors.affiliation: Centre for Biotechnology and Bioengineering (CeBiB), Department of Chemical Engineering and Biotechnology, University of Chile, Beauchef 850, Santiago, Chile.
authors.forename: Oriana
authors.initials: O
authors.lastname: Salazar
authors.affiliation: Centre for Biotechnology and Bioengineering (CeBiB), Department of Chemical Engineering and Biotechnology, University of Chile, Beauchef 850, Santiago, Chile.
authors.forename: Barbara
authors.initials: B
authors.lastname: Andrews
authors.affiliation: Centre for Biotechnology and Bioengineering (CeBiB), Department of Chemical Engineering and Biotechnology, University of Chile, Beauchef 850, Santiago, Chile. Electronic address: juasenjo@ing.uchile.cl.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Asenjo
mesh_terms: D000595:Amino Acid Sequence; D000864:Antarctic Regions; D002208:Caproates / Q000378:metabolism; D003080:Cold Temperature; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism; D005798:Genes, Bacterial; D008049:Lipase / Q000235:genetics*; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012623:Seawater / Q000382:microbiology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020592:Shewanella / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D002208:Caproates; D017931:DNA Primers; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D008049:Lipase
keywords: Cold-active; Degenerate primers; High activity; Lipase