Recombinant Bacillus amyloliquefaciens xylanase A expressed in Pichia pastoris and generation of xylooligosaccharides from xylans and wheat bran

In this study, BaxA (GenBank: KM624029), which encodes the Bacillus amyloliquefaciens xylanase A (BaxA), was highly expressed in Pichia pastoris GS115 under the control of the AOX1 promoter. The recombinant xylanase, namely rePBaxA, was purified to homogeneity by using Ni-affinity resin and its molecular weight was 35.0 kDa. The optimum temperature and pH of rePBaxA were 50 °C and 5.0, respectively. The kinetic parameters Michaelis-Menten constant (Km) and maximum reaction rate (Vmax) of rePBaxA were 5.41 mg/mL and 22.42 μmol/min/mL, respectively. High-performance liquid chromatography results showed that after 6 h of hydrolysis, rePBaxA released xylose-xylohexaose (X1-X6) mixture from beechwood and birchwood xylan, with xylobiose (X2) and xylotriose (X3) as the major products, respectively. The hydrolyates from oat spelt, wheat bran insoluble xylan and pretreated wheat bran by rePBaxA included X2-X6, with X6 having the highest concentration. The mode of action analysis revealed that rePBaxA was an endo-acting xylanase with transglycosylation activity. X2 might be the minimum oligomer hydrolyzed by rePBaxA. The pretreated wheat bran and wheat bran insoluble xylan could be directly hydrolyzed by rePBaxA. This study provided a basis for using agricultural waste by-products as substrates for manufacting value-added probiotics, namely, xylooligosaccharides.