Expression of Recombinant Bacillus amyloliquefaciens Xylanase A in Escherichia coli and Potential Application in Xylan Hydrolysis

Xin Xu, Mingqi Liu, Xianjun Dai
2015 BioResources  
Bacillus amyloliquefaciens xylanase A (baxA), an endoxylanase (EC. 3.2.1.8) gene, was cloned through PCR using the genome of B. amyloliquefaciens as a template. The open reading frame of baxA was 642 bp, and the gene encoded a 213 amino acid protein with a predicted molecular mass of 23.3 kDa. reBaxA1 produced in Escherichia coli with the pET30a(+) vector (T7 lac promoter) formed inclusion body and did not show any xylanase activity. reBaxA2 produced in E. coli with the pCold TF vector (cspA
more » ... TF vector (cspA lac promoter) showed high xylanase activity; this enzyme was secreted into the culture medium and remained in the cell. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis showed that the molecular weights of reBaxA1 and reBaxA2 were approximately 28.5 and 77.2 kDa, respectively. The optimal activity of reBaxA2 occurred at 55 °C and pH 6.0. Moreover, the Michaelis-Menten constant (Km) and maximal activity (Vmax) of reBaxA2 were 4.98 mg•ml -1 and 12.79 μmol•min -1 •mL -1 , respectively. High-performance liquid chromatography analysis showed that reBaxA2 released xylooligosaccharides from birchwood, beechwood, and oat spelt xylans, with xylopentaose, xylotriose, and xylotetraose as major products, respectively.
doi:10.15376/biores.10.3.4694-4711 fatcat:chmfu5aikfdpbce2fwpav6se5e