β-Xylosidases (EC 3.2.1.37) are among the principal glycosyl hydrolases involved in the breakdown of hemicelluloses, catalyzing the reduction of xylooligosaccharides to free xylose. All GH39 β-xylosidases structurally characterized to date display a modular multi-domain organization that assembles a tetrameric quaternary structure. In this work, the crystal structure and the SAXS molecular envelope of a new GH39 β-xylosidase from Caulobacter crescentus (CcXynB2) have been determined. Interestingly, CcXynB2 is a monomer in solution and comparative structural analyses suggest that the shortened C-terminus prevents the formation of a stable tetramer. Moreover, CcXynB2 has a longer loop from the auxiliary domain (the long α-helix-containing loop) which makes a number of polar and hydrophobic contacts with the parental (α/β)(8)-barrel domain, modifying the accessibility and the molecular topography of the catalytic interface. These interactions also maintain the accessory domain tightly linked to the catalytic core, which may be important for enzyme function and stability.

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.

In the present work, the gene xynB2, encoding a β-xylosidase II of the Glycoside Hydrolase 39 (GH39) family, of Caulobacter crescentus was cloned and successfully overexpressed in Escherichia coli DH10B. The recombinant protein (CcXynB2) was purified using nickel-Sepharose affinity chromatography, with a recovery yield of 75.5 %. CcXynB2 appeared as a single band of 60 kDa on a sodium dodecyl sulfate polyacrylamide gel and was recognized by a specific polyclonal antiserum. The predicted CcXynB2 protein showed a high homology with GH39 β-xylosidases of the genus Xanthomonas. CcXynB2 exhibited an optimal activity at 55 °C and a pH of 6. CcXynB2 displayed stability at pH values of 4.5-7.5 for 24 h and thermotolerance up to 50 °C. The K (M) and V (Max) values were 9.3 ± 0.45 mM and 402 ± 19 μmol min(-1) for ρ-nitrophenyl-β-D-xylopyranoside, respectively. The purified recombinant enzyme efficiently produced reducing sugars from birchwood xylan and sugarcane bagasse fibers pre-treated with a purified xylanase. As few bacterial GH39 family β-xylosidases have been characterized, this work provides a good contribution to this group of enzymes.

The species Eugenia involucrata DC. is a plant native to Brazil and is traditionally used for intestinal problems, however, little research has documented about its biological potential and phytochemical profile. Thus, the objective of this study was to carry out preliminary phytochemical prospecting, antimicrobial and antioxidant potential of E. involucrata extracts. Using the E. involucrata leaves, aqueous and organic extracts were obtained using the following solvents (ethanol, methanol, hexane, acetone, dichloromethane and ethyl acetate). The phytochemical prospecting detected the presence of saponins, steroids, flavonoids and tannins in the extracts. Ethanolic and methanolic extracts presented antimicrobial activity for most of the bacterial strains tested, as well as for yeast Candida albicans, with concentrations between 3.12 and 50 mg/mL. The ethanolic and metanolic extract presented high free radical sequestration potential (>90%). The methanol extract showed an IC50 value statistically equal to that found for the commercial antioxidant BHT (p <0.05). The crude extracts obtained with ethanol and methanol were the most promising. These results suggest that methanolic, ethanolic and aqueous extracts are a promising source of natural bioactive.

No abstract