PURIFICATION, CHARACTERIZATION, AND SUBSTRATE-SPECIFICITY OF A NOVEL HIGHLY GLUCOSE-TOLERANT BETA-GLUCOSIDASE FROM ASPERGILLUS-ORYZAE

Aspergillus oryzae was found to secrete two distinct beta-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by gluco se. The minor form, which was induced most effectively on quercetin (3 ,3',4',5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total beta-glucosidase activity but exhibited a high tolerance to glucose inhibition, This highly glucose-tolerant beta-glucosidase ( designated HGT-BG) was purified to homogeneity by ammonium sulfate pre cipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pi of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electropho resis, respectively. Using p-nitrophenyl-beta-D-glucoside as the subst rate, we found that the enzyme was optimally active at 50 degrees C an d pH 5.0 and had a specific activity of 1,066 mu mol min(-1) mg of pro tein(-1) and a K-m of 0.55 mM under these conditions. The enzyme is pa rticularly resistant to inhibition by glucose (K-i, 1.36 M) or glucono -delta-lactone (K-i, 12.5 mM), another powerful beta-glucosidase inhib itor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity ty pe of fungal beta-glucosidase. It exhibits exoglucanase activity and h ydrolyzes (1-->3)- and (l-->6)-beta-glucosidic linkages most effective ly. This enzyme was able to release flavor compounds, such as geraniol , nerol, and linalol, from the corresponding monoterpenyl-beta-D-gluco sides in a grape must (pH 2.9, 90 g of glucose liter(-1)). Other flavo r precursors (benzyl- and 2-phenylethyl-beta-D-glucosides) and prunin (4',5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitt erness of citrus juices, are also substrates of the enzyme. Thus, this novel beta-glucosidase is of great potential interest in wine and fru it juice processing because it releases aromatic compounds from flavor less glucosidic precursors.