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Egypt. J. Exp. Biol. (Bot.). 2012; 8(1): 51-57


PCR CLONING, SEQUENCE AND OVEREXPRESSION OF THE XYLANASE A GENE FROM LOCALLY ISOLATED BACTERIUM, BACILLUS SUBTILIS ATF- 40

Atef Mohamed Ibrahim.

Abstract
A new bacterium, Bacillus subtilis strain-ATF40 was isolated from sadat city soil in Minoufiya governerate, Egypt. B. subtilis ATF-40 was characterized, identified morphologically, and biochemically and its 16s rRNA gene also sequenced and deposited in the GenBank JF312740. Xylanase A gene was cloned, sequenced and deposited in the GenBank JF312741. Xylanase A gene has a complete open reading frame 627 bp, with start codon ATG and stop codon TAA. It was amplified from chromosomal DNA of B. subtilis strain ATF- 40.The PCR product was cloned into pTZ57R/T using E. coli 107 and overexpressed in E. coli L21(DE3) using pMal-c2 vector. The positive clone was screened on xylan agar plates by Congo-red staining method.The pMal-c2 vector has maltose binding protein (MBP) gene with a molecular weight 42 KDa. The molecular weight of the fused protein is about 64.5 KDa, this is in agreement with the molecular weighs of MBP 42 KDa and the xylanase protein 22.5 KDa. The fused protein MBP-xylanase activity was also measured before cleavage with factor Xa protease and it is active.

Key words: Cloning, Xylanase, Bacillus subtilis ATF-40



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