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 glucose. 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 precipitation, 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 electrophoresis, respectively. Using p-nitrophenyl-beta-D-glucoside as the substrate, we found that the enzyme was optimally active at 50 degreesC and pH 5.0 and had a specific activity of 1,066 micromol min-1 mg of protein-1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki, 1. 36 M) or glucono-delta-lactone (Ki, 12.5 mM), another powerful beta-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal beta-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1-->3)- and (1-->6)-beta-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-beta-D-glucosides in a grape must (pH 2.9, 90 g of glucose liter-1). Other flavor precursors (benzyl- and 2-phenylethyl-beta-D-glucosides) and prunin (4',5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel beta-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.
Riou
C
C
Laboratoire de Microbiologie et Technologie des Fermentations, Institut National de la Recherche Agronomique, Institut des Produits de la Vigne, F-34060 Montpellier Cedex 2, France. riou@ensam.inra.fr
Salmon
J M
JM
Vallier
M J
MJ
Günata
Z
Z
Barre
P
P
D001236:Aspergillus oryzae / Q000201:enzymology* / Q000254:growth & development; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004790:Enzyme Induction; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011794:Quercetin / Q000494:pharmacology; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*
D009844:Oligosaccharides; D011794:Quercetin; D001617:beta-Glucosidase; D005947:Glucose