Analysis of the xynB5 gene encoding a multifunctional GH3-BglX beta-glucosidase-beta-xylosidase-alpha-arabinosidase member in Caulobacter crescentus

作者：Justo Priscila Innocenti; Correa Juliana Moco; Maller Alexandre; Kadowaki Marina Kimiko; da Conceicao Silva Jose Luis; Gandra Rinaldo Ferreira; Garcia Simao Rita de Cassia

来源：Antonie van Leeuwenhoek International Journal of General and Molecular Microbiology, 2015, 108(4): 993-1007.

DOI：10.1007/s10482-015-0552-x

• Cloning and expression
• xynB genes
• Caulobacter crescentus
• Hemicellulose
• Agro-industrial residues

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted beta-glucosidase-beta-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), beta-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 beta-glucosidase (pNPG: p-nitrophenyl-beta-D-glucoside) beta-xylosidase (pNPX: p-nitrophenyl-beta-D-xyloside) and alpha-arabinosidase (pNPA: p-nitrophenyl-alpha-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 A degrees C for beta-glucosidase and alpha-l-arabinosidase and 60 A degrees C for beta-xylosidase. BglX-V-Ara predominantly presented beta-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (K-m 0.24 +/- A 0.0005 mM, V-max 0.041 +/- A 0.002 A mu mol min(-1) mg(-1) and K-cat/K-m 0.27 mM(-1) s(-1)), followed by beta-xylosidase (K-m 0.64 +/- A 0.032 mM, V-max 0.055 +/- A 0.002 A mu mol min(-1) mg(-1) and K-cat/K-m 0.14 mM(-1)s(-1)) and finally alpha-l-arabinosidase (K-m 1.45 +/- A 0.05 mM, V-max 0.091 +/- A 0.0004 A mu mol min(-1) mg(-1) and K-cat/K-m 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.

• 出版日期2015-10