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

• Published in: Antonie van Leeuwenhoek Vol. 108; no. 4; pp. 993 - 1007
• Main Authors: Justo, Priscila Innocenti, Corrêa, Juliana Moço, Maller, Alexandre, Kadowaki, Marina Kimiko, da Conceição-Silva, José Luis, Gandra, Rinaldo Ferreira, Simão, Rita de Cássia Garcia
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01.10.2015
• Subjects: alpha-N-arabinofuranosidase; beta-glucosidase; beta-Glucosidase - genetics; beta-Glucosidase - metabolism; Biomedical and Life Sciences; catalytic activity; Caulobacter crescentus; Caulobacter crescentus - enzymology; Caulobacter crescentus - genetics; Cloning, Molecular; Computational Biology; Enzyme Stability; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; fibronectins; Gene Expression; gene overexpression; genes; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; hemicellulose; Hydrogen-Ion Concentration; Life Sciences; Medical Microbiology; Microbiology; Molecular Weight; Original Paper; Plant Sciences; plasmids; polymerase chain reaction; Protein Structure, Tertiary; recombinant proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; saccharification; sequence analysis; Soil Science & Conservation; Substrate Specificity; Temperature; xylan 1,4-beta-xylosidase; Xylosidases - genetics; Xylosidases - metabolism; Cloning and expression; Hemicellulose; Agro-industrial residues; genes
• ISSN: 0003-6072; 1572-9699
• DOI: 10.1007/s10482-015-0552-x

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⁻¹s⁻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.