Purification and Characterization of an Extracellular β-Xylosidase of Termitomyces clypeatus

Abstract

Termitomyces clypeatus liberated β-xylosidase (EC 3.2.1.37) optimally in xylan medium but poorly in cellulose medium. The enzyme activity reached 5–6% of that of xylanase liberated in xylan medium. The culture filtrate enzyme, purified 5-fold by ammonium sulfate precipitation, BioGel P-200, and DEAE-Sephadex anion exchange chromatographies at pH 5.0, was homogeneous (190 kDa) in polyacrylamide gel electrophoresis (PAGE) and in high-performance gel permeation liquid chromatography (HPGPLC) but contained high amounts of cellobiase and sucrase and gave multiple protein bands in SDS-PAGE (SDS = sodium dodecyl sulfate) . The aggregate was subsequently resolved by DEAE−anion exchange chromatography at pH 6.0 into a number of β-xylosidase fractions with decreasing sucrase contents. The sucrase free β-xylosidase fraction was subsequently purified to 55.6-fold by hydrophobic interaction chromatography on a phenyl−sepharose column. The enzyme was a homogeneous 94 kDa protein, both in SDS−PAGE and HPGPLC. The physicochemical properties of the enzyme were similar to those of other fungal β-xylosidases, and the enzyme had no unrelated glycosidase activities. The purified (94 kDa) and aggregated forms (190 kDa) of β-xylosidase had the same pH optima (5.0), temperature optima (60 °C), substrate specificities, and sensitivities toward end product inhibition by xylose or to the actions of SDS, urea, and guanidine hydrochloride. But aggregated enzyme was reasonably stable in the pH and temperature ranges where purified enzyme was completely inactive. The protein−protein aggregation appeared to confer additional stability to the β-xylosidase toward extracellular denaturing conditions.