Analysis of the xynB5 gene encoding a multifunctional GH3-BglX β-glucosidase-β-xylosidase-α-arabinosidase member in Caulobacter crescentus.

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-L-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (Km 0.24 ± 0.0005 mM, Vmax 0.041 ± 0.002 µmol min(-1) mg(-1) and Kcat/Km 0.27 mM(-1) s(-1)), followed by β-xylosidase (Km 0.64 ± 0.032 mM, Vmax 0.055 ± 0.002 µmol min(-1) mg(-1) and Kcat/Km 0.14 mM(-1)s(-1)) and finally α-L-arabinosidase (Km 1.45 ± 0.05 mM, Vmax 0.091 ± 0.0004 µmol min(-1) mg(-1) and Kcat/Km 0.1 mM(-1) s(-1)). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.