Cloning, Expression and Characterization of Xylanase (xyn-akky1) from Bacillus subtilis in Escherichia coli

Özet

In this study, Bacillus subtilis akky1 strain was isolated from the soil of beech forest in Akkuş City, Ordu Province, Turkey. akky1 strain was identified by 16S rRNA analysis. The full-length 16S rRNA sequence of akky1 strain showed the 100% similarity with Bacillus subtilis strain B7 (KC310823.1) . A 642 bp DNA fragment was obtained from genomic DNA using primers designed based on the gene sequence of Bacillus subtilis xylanase given in GenBank. The gene encoding xylanase was cloned into pET28b (+) plasmid vector, sequenced and expressed in Escherichia coli BL21 (DE3). The hexahistidine (6xHis) tagged fusion protein was purified using nickel affinity chromatography and the xylanase activity was measured. The molecular mass of the purified xylanase was approximately 26 kDa as estimated by SDS-PAGE. The xylanase had optimal activity at pH 6.0 and 60°C. The Km values of the recombinant enzyme towards beechwood was 3.33 mg/ml.