Isolation of Aspergillus flavus MO-5 Producing Two Types of Intracellular α-D-Xylosidases : Purification and Characterization of α-D-Xylosidase I

Abstract

One strain (MO-5) of fungi producing α-D-xylosidase was isolated from sake koji by a method using glucose oxidase. MO-5 was identified as an Aspergillus flavus strain, produced no aflatoxin (B1, B2, G1, and G2). Two different types of α-D-xylosidases were detected in the cell-free extract. One of the enzymes (α-D-xylosidase I) was purified to an electrophoretically pure state by successive chromatography on Q-Sepharose, Phenyl Superose, PL-SAX, and TSK-gel G3000SW_XL. The purified enzyme hydrolyzed p-nitrophenyl α-D-xylopyranoside (α-p-NPX), methyl α-D-xylopyranoside, isoprimeverose [α-D-xylopy-ranosyl-(1→6)-D-glucopyranose], and xyloglucan oligosaccharide. The activity of this enzyme was highly specific for α-D-xylosidic linkages. The apparent K_m and V_max of the enzyme for α-p-NPX and isoprimeverose were 1.32 mM and 4.4 μmol/min/mg protein, and 4.00 mM and 58.8 μmol/min/mg protein, respectively. This enzyme had an apparent molecular weight of 100, 000 by SDS-polyacrylamide gel electrophoresis and 400, 000 by gel filtration chromatography (TSK-gel G3000SW_XL). The enzyme showed the highest activity at pH 4.5 and 45°C, and was stable from pH 4.5 to 6.0 and at temperatures up to 45°C. The activity was inhibited by SDS and Hg²⁺, and slightly by Cu²⁺ and Fe³⁺. This enzyme showed transfer action at high concentrations of isoprimeverose and transfer products were detected, and it had transxylosylation activity on maltose from isoprimeverose as a donor, too. From the results of the enzymatic digestion and the partial acidolysis of these prepared transfer products, their structures may be trisaccharides in which the xylosyl residues were transferred to isoprimeverose and maltose forming α-D-linkages. Accordingly, this enzyme was indicated to be able to transfer xylosyl residues to form α-D-xylosidic linkages.