13th ICB and 5th ICBMB , 2013-04-16

Title : ( Cloning, sequencing and expression in Escherichia coli of a lipase gene from Bacillus subtilis DR8806 )

Authors: Ahmad Asoodeh , Shirin Emtenani ,

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Background: Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are ubiquitous enzymes that catalyze the hydrolysis of fats and oils through acting on carboxylic ester bond. Lipases have potential applications in the detergent, food, leather, textile, oil and fat, cosmetic, paper, and pharmaceutical industries.The aim of this study was to perform the isolation, cloning and expression of a lipase from Bacillussubtilis DR8806 bearing the features of a biotechnologically important group of enzymes. Methods and Materials: In this study, the cloning vector pTZ57RT containing B. subtilis lipase gene was transformed into the Escherichia coli DH5α. The lipolytic gene was then subcloned and expressed in E.coli BL21 using the pET28a(+) expression system. The insertion of target fragment into expression vector was firstly confirmed by colony PCR using specific linker primers. Cloning of lipase gene into pET28a(+) vector was also confirmed by double digestion. The expression of the recombinant lipase was induced using different concentrations of IPTGat various incubation times and temperatures. The lipase gene expressed as an Nterminal His-tag fusion protein was purified using Ni-NTA affinity chromatography. Protein expression was investigated by SDS-PAGE and Coomassie brilliant blue staining. Results and Conclusions:Sequence analysis of lipase gene showed an open reading frame (ORF) of 639 nucleotides coding a polypeptide of 212 amino acids.The DNA sequence and deduced amino acid sequence of the lipase showed striking similarities to lipases from B. subtilis strains. High level expression of the lipase in E.coli BL21 under the control of strong T7 promoter was observed after 6 h treatment with 1 mM IPTG at 28◦C.The expressed lipase wasisolated and purified to homogeneity in a single chromatographic step.SDS-PAGE analysis revealed that the recombinant protein had a molecular weight of approximately 23 kDa.

Keywords

, Bacillus subtilis, Lipase, Gene
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@inproceedings{paperid:1034861,
author = {Asoodeh, Ahmad and Emtenani, Shirin},
title = {Cloning, sequencing and expression in Escherichia coli of a lipase gene from Bacillus subtilis DR8806},
booktitle = {13th ICB and 5th ICBMB},
year = {2013},
location = {یزد, IRAN},
keywords = {Bacillus subtilis; Lipase; Gene cloning;Expression},
}

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%0 Conference Proceedings
%T Cloning, sequencing and expression in Escherichia coli of a lipase gene from Bacillus subtilis DR8806
%A Asoodeh, Ahmad
%A Emtenani, Shirin
%J 13th ICB and 5th ICBMB
%D 2013

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