Purification and Characterization of Vanilla Bean (Vanilla planifolia Andrews) β-d-Glucosidase

Abstract

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.

The C-Domain of Oleuropein β-Glucosidase Assists in Protein Folding and Sequesters the Enzyme in Nucleus

“…S8). Although this outcome seems to be contradictory, plant b-glucosidases are known to have a compact quaternary structure that remains active in concentrations of up to 3.2% SDS and 5% b-mercaptoethanol (Esen andGungor, 1991, 1993) or even exhibit enhanced activity in the presence of reducing agents (Esen, 1992;Odoux et al, 2003). Because a single fluorescent band was detected in olive mesocarp protein extracts reacting with oleuropein that had a similar molecular mass compared to the transiently expressed olive b-glucosidase in tobacco cells, OeGLU is the major oleuropein-specific b-glucosidase in olive.…”

Influence of curing procedures on sensory quality of vanilla beans

“…The pH of the aqueous suspensions was measured at 5.1. Purified β-glucosidase has been shown to be inactive and unstable below pH 6.5 [23].…”

Combination of Rigorous and Statistical Modeling for Process Development of Plant‐Based Extractions Based on Mass Balances and Biological Aspects

“…Localization of vanillin and b-D-glucosidase in the green vanilla capsules over the cross section has been already investigated by Odoux et al [5,[9][10][11][12][13]. They could prove that both the vanillin precursor and the enzyme which is needed for conversion can be found in the placenta tissue, the secretory papillae, and the inner mesocarp.…”

“…The characteristic aroma is obtained through a curing process during which vanillin and other aroma constituents are developed as a result of different biochemical transformations. During the process of curing, the typical flavor is obtained by hydrolysis of nonvolatile glycosidic flavor precursors mainly composed of glucovanillin by a b-glucosidase [5].…”