Identification of lipase encoding genes from Antarctic seawater bacteria using degenerate primers: Expression of a cold-active lipase with high specific activity
| Autor: | Loreto P. Parra, Giannina Espina, Oriana Salazar, Barbara A. Andrews, Javier Devia, Juan A. Asenjo |
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| Rok vydání: | 2015 |
| Předmět: |
DNA
Bacterial Shewanella Recombinant Fusion Proteins Antarctic Regions Bioengineering medicine.disease_cause Applied Microbiology and Biotechnology Biochemistry Shewanella frigidimarina Substrate Specificity Complete sequence chemistry.chemical_compound Escherichia coli Primer walking medicine Seawater Amino Acid Sequence Lipase Caproates Gene Phylogeny DNA Primers Sequence Homology Amino Acid biology Molecular biology Cold Temperature genomic DNA chemistry Genes Bacterial biology.protein Sequence Alignment DNA Biotechnology |
| Zdroj: | Artículos CONICYT CONICYT Chile instacron:CONICYT |
| ISSN: | 0141-0229 |
| DOI: | 10.1016/j.enzmictec.2014.10.004 |
| Popis: | 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, lip E13, 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. |
| Databáze: | OpenAIRE |
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