Isolation of Aspergillus flavus MO-5 producing two types of intracellular alpha-D-xylosidases: purification and characterization of alpha-D-xylosidase I

Abstract

One strain (MO-5) of fungi producing alpha-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 alpha-D-xylosidases were detected in the cell-free extract. One of the enzymes (alpha-D-xylosidase I) was purified to an electrophoretically pure state by successive chromatography on Q-Sepharose, Phenyl Superose, PL-SAX, and TSK-gel G3000SWXL. The purified enzyme hydrolyzed p-nitrophenyl alpha-D-xylopyranoside (alpha-p-NPX), methyl alpha-D-xylopyranoside, isoprimeverose [alpha-D-xylopyranosyl-(1-->6)-D- glucopyranose], and xyloglucan oligosaccharide. The activity of this enzyme was highly specific for alpha-D-xylosidic linkages. The apparent Km and Vmax of the enzyme for alpha-p-NPX and isoprimeverose were 1.32 mM and 4.4 mumol/min/mg protein, and 4.00 mM and 58.8 mumol/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 G3000SWXL). The enzyme showed the highest activity at pH 4.5 and 45 degrees C, and was stable from pH 4.5 to 6.0 and at temperatures up to 45 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)