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

Vanilla bean β-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 °C at pH 7.0. K m values for p-nitrophenyl-β-d-glucopyranoside and glucovanillin were 1.1 and 20.0 mM, respectively; V max values were 4.5 and 5.0 μkat·mg - 1. The β-d-glucosidase was competitively inhibited by glucono-δ-lactone and 1-deoxynojirimycin, with respective K i values of 670 and 152 μM, and not inhibited by 2 M glucose. The β-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-β-d-fucopyranoside, p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl-β-d-galactopyranoside, and p-nitrophenyl-β-d-xylopyranoside. The enzyme was also active on prunasin, esculin, and salicin and inactive on cellobiose, gentiobiose, amygdalin, phloridzin, indoxyl-β-d-glucopyranoside, and quercetin-3-β-d-glucopyranoside. Keywords: Vanilla planifolia Andrews; vanilla bean; β-glucosidase; glucovanillin hydrolysis