Purification and characterization of vanilla bean (Vanilla planifolia Andrews) beta-D-glucosidase.
Vanilla bean beta-D-glucosidase was purified to apparent homogeneity by successive anion exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme is a tetramer (201 kDa) made up of four identical subunits (50 kDa). The optimum pH was 6.5, and the optimum temperature was 40 degrees C at pH 7.0. K(m) values for p-nitrophenyl-beta-D-glucopyranoside and glucovanillin were 1.1 and 20.0 mM, respectively; V(max) values were 4.5 and 5.0 microkat.mg(-1). The beta-D-glucosidase was competitively inhibited by glucono-delta-lactone and 1-deoxynojirimycin, with respective K(i) values of 670 and 152 microM, and not inhibited by 2 M glucose. The beta-D-glucosidase was not inhibited by N-ethylmaleimide and DTNB and fully inhibited by 1.5-2 M 2-mercaptoethanol and 1,4-dithiothreitol. The enzyme showed decreasing activity on p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, and p-nitrophenyl-beta-D-xylopyranoside. The enzyme was also active on prunasin, esculin, and salicin and inactive on cellobiose, gentiobiose, amygdalin, phloridzin, indoxyl-beta-D-glucopyranoside, and quercetin-3-beta-D-glucopyranoside.
Odoux
Eric
E
Département FLHOR, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), TA50/16, 34398 Montpellier Cedex 5, France.
Chauwin
Audrey
A
Brillouet
Jean-Marc
JM
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D031669:Vanilla / Q000201:enzymology*; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D001617:beta-Glucosidase