Purification and Characterization of an Intracellular α-D-Xylosidase II from Aspergillus flavus MO-5

Abstract

α-D-Xylosidase II activity from Aspergillus flavus MO-5 was increased roughly 5- to 10-fold by use of xylose instead of methyl α-D-xylopyranoside (α-MX) as a carbon source. The enzyme 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 isoprimeverose [α-D-xylopyranosyl-(1→6)-D-glucopyranose] and p-nitrophenyl α-D-xylopyranoside (α-p-NPX), but not α-MX or xyloglucan oligosaccharide. The apparent K_m and V_max of the enzyme for α-p-NPX and isoprimeverose were 0.97 mM and 28.0 μmol/min/mg protein, and 47.62 mM and 2.0 μmol/min/mg protein, respectively. This enzyme had an apparent molecular weight of 67, 000 by SDS-polyacrylamide gel electrophoresis and 180, 000 by gel filtration chromatography (TSK-gel G3000SW_XL). The enzyme showed the highest activity at pH 6.0 and 40°C, and was stable in the pH range from 6.0 to 7.0 and at the temperatures up to 40°C. The activity was inhibited by Cu²⁺, Zn²⁺, Hg²⁺, p-CMB, SDS, Fe³⁺, and N-ethylmaleimide. This enzyme had nothing in common with α-D-xylosidase I and four α-D-xylosidases reported already.