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Identification of lipase encoding genes from Antarctic seawater bacteria using degenerate primers: Expression of a cold-active lipase with high specific activity

Paper ID Volume ID Publish Year Pages File Format Full-Text
16969 42627 2015 6 PDF Available
Title
Identification of lipase encoding genes from Antarctic seawater bacteria using degenerate primers: Expression of a cold-active lipase with high specific activity
Abstract

•Purified lipase E5 shows a high activity in the range 15–25 °C.•The enzyme showed an optimal activity at 25 °C at pH 8 and the retention of 35% of its maximum activity at 10 °C.•CODEHOP primers are a valuable contribution for the isolation of cold-active lipases.

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.

Keywords
Lipase; Cold-active; High activity; Degenerate primers
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Identification of lipase encoding genes from Antarctic seawater bacteria using degenerate primers: Expression of a cold-active lipase with high specific activity
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 68, January 2015, Pages 56–61
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us