Cloning and Over-expression of xynB Gene of Bacillus subtilis subsp. spizizenii W23 into Escherichia coli Origami Host Cells

Abstract

<p class="Els-Abstract-text">The <em>xyn</em>B gene of <em>Bacillus</em><em> subtilis</em> subsp. spizizenii W23 is predicted to encode a xylan 1,4-beta-xylosidase. Application of XynB enzymes in industries is wide. Production of this enzyme in its host cells is naturally restricted by repression process. It will give certain beneficial to over-expressed the enzymes in other host-cells under inducing promoter. This study aimed to clone the <em>xyn</em>B gene from <em>Bacillus</em><em> subtilis</em> subsp. spizizenii W23, to pMMB67EH plasmid, and to over-express the <em>xyn</em>B gene in <em>Escherichia coli </em>Origami as host cells. The <em>x</em><em>yn</em>B gene was successfully amplified by polymerase chain reaction (PCR) technique using a pair of primers flanking the gene sequence and chromosomal DNA of the W23 strain as a template. The <em>xyn</em>B gene inserted in recombinant plasmid was confirmed by PCR detection using primers pair’s specific for <em>xyn</em>B gene and for the vector, then continued by restriction analyses.  The result showed that transformants clone 9 and 10 bear the recombinant pMMB-<em>xyn</em>B plasmid. The xylanase activity of <em>xyn</em>B gene in <em>Escherichia coli</em> Origami clone 10 was detected by sodium-dodecyl-sulfate polyacrylamide gel analyses and with addition of isopropyl-β-D-thio-galactoside (IPTG) as an inducer. The protein seem to be over-expressed as intra- and extra-cellular protein detected on SDS-PAGE gel. Result from xylan degrading activity on Luria-Bertani-xylan-IPTG plate with addition of Congo Red, showed that the cells with pMMB-<em>xyn</em>B recombinant plasmid have clear zone around the colonies while the transformant bearing an empty plasmid showed no clear zone. It could be concluded that the <em>xyn</em>B gene of <em>Bacillus subtilis</em> subsp.spizizenii W23 has been successfully been cloned on pMMB67EH plasmid and over-expressed in the <em>Escherichia coli</em> Origami cells as intra- and extra-cellular protein, as observed on SDS-PAGE gel analysis. The protein has activity on xylan degradation.</p>