PURIFICATION AND CHARACTERIZATION OF AN INTRACELLULAR ALPHA-D-XYLOSIDASE II FROM ASPERGILLUS-FLAVUS MO-5

Alpha-D-Xylosidase II activity from Aspergillus flavus MO-5 was increased roughly 5- to 10-fold by use of xylose instead of methyl alpha-D-xylopyranoside (alpha-MS) 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 G3000SWXL. The purified enzyme hydrolyzed isoprimeverose [alpha-D-xylopyranosyl-(1 --> 6)-D-glucopyranose] and p-nitrophenyl alpha-D-xylopyranoside (alpha-p-NPX), but not alpha-MX or xyloglucan oligosaccharide. The apparent K(m) and V(max) of the enzyme for alpha-p-NPX and isoprimeverose were 0.97 mM and 28.0 mumol/min/mg protein, and 47.62 mM and 2.0 mumol/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 G3000SWXL). The enzyme showed the highest activity at Ph 6.0 and 40-degrees-C, and was stable in the Ph range from 6.0 to 7.0 and at the temperatures up to 40-degrees-C. The activity was inhibited by Cu2+, Zn2+, Hg2+, p-CMB, SDS, Fe3+, and N-ethylmaleimide. This enzyme had nothing in common with alpha-D-xylosidase I and four alpha-D-xylosidases reported already.