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

Xin Xu, Mingqi Liu, Xianjun Dai

Abstract


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 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.

Keywords


Xylanase; Bacillus amyloliquefaciens; Expression; Enzyme properties; Xylooligosaccharides

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