A novel xylan degrading β-D-xylosidase: purification and biochemical characterization
2012
Michelin, Michele | Peixoto-Nogueira, Simone C. | Silva, Tony M. | Jorge, João A. | Terenzi, Héctor F. | Teixeira, José A. | de Lourdes T. M. Polizeli, Maria
Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular β-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. β-xylosidase is a glycoprotein (39 % carbohydrate content) and has a molecular mass of 137 kDa by SDS-PAGE, with optimal temperature and pH at 70 °C and 3.0–5.5, respectively. β-xylosidase was stable in acidic pH (3.0–6.0) and 70 °C for 1 h. The enzyme was activated by 5 mM MnCl2 (28 %) and MgCl2 (20 %) salts. The β-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-β-D-xylopyranoside, exhibiting apparent Km and Vmax values of 0.66 mM and 39 U (mg protein)−1 respectively, and to a lesser extent p-nitrophenyl-β-D-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel β-D-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by β-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose.
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