Purification and Characterization of Extracellular β-Xylosidase and β-Glucosidase from Aspergillus fumigatus

Abstract

A β-xylosidase (β-D-xyloside xylohydrolase, EC 3.2.1.37) and β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) extracted from a wheat bran culture of Aspergillus fumigatus were purified up to 90-fold and 131-fold, respectively, by ammonium sulfate precipitation, gel filtration, ion exchange chromatography, and hydroxylapatite chromatography. Molecular weights of the β-xylosidase and β-glucosidase were 360, 000 and 380, 000, respectively, each consisting of four identical subunits. The isoelectric points of β-xylosidase and β-glucosidase were at pH 5.4 and 4.5, respectively. The optimum temperature for the β-xylosidase was 75°C, being stable up to 65°C for 20 min and for the β-glucosidase was 65°C, being stable up to 60°C for 20 min. The optimum pH for both enzymes was about 4.5, being stable between 2 and 8 at 50°C for 20min. Both enzymes were inhibited by Fe³⁺, Cu²⁺, Hg²⁺, SDS, and p-chloromercuribenzoate. The apparent Michaelis constants of the β-xylosidase were 2.0 and 23.8 HIM for p-nitrophenyl-β-xyloside and xylobiose, respectively, and those of the β-glucosidase were 1.4, 11.4, and 24.8 HIM for p-nitrophenyl-β-glucoside, gentiobiose, and cellobiose, respectively. To produce xylose from crude xylooligosaccharides prepared by steam-explosion of cotton seed waste (DP≤10, 53%, total sugars =150 g/liter), the crude enzyme from A. fumigatus (β-xylosidase activity = 14.7 units/ml, xylanase activity = 20 units/ml) could hydrolyze the substrate at 55°C and pH 4.5 resulting in almost complete conversion to xylose (160g/liter).