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

Title : ( Molecular Cloning and Expression of a Bacillus subtilis Alpha-Amylase Gene in E. coli BL21 )

Authors: Ahmad Asoodeh , Shamsi Emtenani ,

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Introduction: Alpha-amylases (α-1, 4-glucan-4-glucanohydrolases, EC 3.2.1.) are endo-acting enzymes that catalyze the hydrolysis of α1→4 glycosidic bonds of starch molecule, releasing glucose, maltose and maltotriose units. Constituting 50% of global enzyme market, alpha-amylases are of great importance in industry. Among different organisms, microbial amylases present widespread application in starch processing, baking, textile and pharmaceutical industries. In the present study, we sought to isolate and clone an alpha-amylase gene from Bacillus subtilis DR8806 in an expression host, E. coli BL21 DE3. Methods: Bacillus subtilis DR8806 previously isolated from Dig Rostam hot spring was utilized for genomic DNA extraction. Designing specific linker primers containing restriction sites for BamHІ and HindІІІ, the extracellular α-amylase was isolated. The isolated fragment was then inserted into pTZ57R/T cloning vector and transformed to E. coli DH5α for cloning and nucleotide sequencing. Subsequently, amylase encoding gene containing two sticky ends was heterologously expressed in E. coli BL21 DE3 using pET28a (+) expression system. Under the control of T7 promoter, the expression of recombinant protein was performed using different concentrations of IPTG as an inducer. The recombinant His-tagged protein was purified using single step Ni-NTA affinity chromatography and analyzed by SDS–PAGE. Results and conclusion: Using PCR technique, amylase coding region containing 1980 bp was isolated. The insertion of corresponding gene into cloning vector was confirmed through colony-PCR and restriction double digestion. Cloned with the correct open reading frame, alpha-amylase sequence shared high homology with alpha-amylase genes of Bacillus subtilis strains. Under optimized conditions, the recombinant construct was expressed using 1 mM IPTG at 28 °C for 6 hours. Analyzed on SDS-PAGE, recombinant product with an approximate molecular weight of 68 kD was obtained. Recombinant α-amylase from Bacillus subtilis was successfully cloned and expressed with high yield in comparison to wild-type enzyme.

Keywords

, Bacillus subtilis, Alph-amylase, Cloning,
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@inproceedings{paperid:1034862,
author = {Asoodeh, Ahmad and Emtenani, Shamsi},
title = {Molecular Cloning and Expression of a Bacillus subtilis Alpha-Amylase Gene in E. coli BL21},
booktitle = {13th ICB and 5th ICBMB},
year = {2013},
location = {یزد, IRAN},
keywords = {Bacillus subtilis; Alph-amylase; Cloning; Purification},
}

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%0 Conference Proceedings
%T Molecular Cloning and Expression of a Bacillus subtilis Alpha-Amylase Gene in E. coli BL21
%A Asoodeh, Ahmad
%A Emtenani, Shamsi
%J 13th ICB and 5th ICBMB
%D 2013

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