Handbook of Vanilla Science and Technology

Breakdown of a glucoside requires hydrolysis of the glucosidic bond between sugar and aglycone. In most natural glucosides, the bond is of the β-type. In vitro, this can be achieved non-enzymatically, especially at low pH and at increased temperature. In biological conditions the reaction is catalysed by β-glucosidase, a hydrolytic enzyme splitting β-glucosidic bonds between sugar and aglycone. p-Glucosidase usually occurs in plant tissues that accumulate or store bound secondary metabolites in the form of β-glucosides. Activity of β-glucosidase is the most important factor in formation of vanilla aroma during the curing of vanilla beans (Havkin-Frenkel et al. 2003). Although glucovanillin has long been postulated as the source of vanillin formed during the curing of the beans (Goris 1924), there is not much data concerning vanilla bean β-glucosidase itself (Arana 1943; Ranadive et al. 1983; Wild-Altamirano 1969; Hanum 1997). The enzyme was purified and characterized by Odoux et al. (2003). It consists of a 201 kDa tetramer composed of four identical 50kDa subunits. It exhibits very narrow optimum activity at pH 6.5 and a temperature optimum of 40°C. The enzyme was stable at neutral and alkaline pH, but is very unstable at pH below 6. Surprisingly, this β-glucosidase, despite purification to electrophoretic homogeneity, exhibited broad substrate specificity. It was active with several natural glycosides such as glucovanillin, prunasin, esculin, and salicin, as well as the artificial substrates 4-nitrophenyl- β-D-glucopyranoside, 4-nitrophenyl-β-D-fucopyranoside, 4-nitrophenyl-β-D-galactopyranoside, and 4-nitrophenyl-β-D-xylopyranoside. Looking at its broad specificity, it can be described rather as β-D-glycosidase than a β-D-glucosidase (Odoux et al. 2003). However, since the gene encoding the enzyme was not cloned and the activity of the recombinant enzyme characterized, it is possible that the activity characterized by Odoux et al. 2003 was originating from multiple enzymes. Activity of other glycosyl hydrolases, such as α-galactosidase, β-galactosidase, and β-mannosidase, has been reported in V. planifolia (Havkin-Frenkel and Belanger 2007). It will require future research to determine if the activities described above originate from separate enzymes, or rather are a result of the broad substrate specificity of β-glucosidase.

During the curing process, activity of glycosyl hydrolases liberate vanilla flavor components from their bound glycosidic forms. These hydrolytic enzymes and their substrates show different spatial distribution in the cell and in the tissues. The hydrolytic enzymes are located predominantly in the cytosolic fraction of the cell, whereas the glucoside substrates are stored in vacuolar compartments. In vanilla beans, β-glucosidase is predominantly located in the outer part of the fruit tissue and glucovanillin is in the inner placental part of the fruit (Joel et al. 2003; Havkin-Frenkel et al. 2003). Thus, after partial killing of the beans during the curing process, disorganization of the cellular compartments enables contact of β-glucosidase with the accumulated glucosides and the release of aroma constituents. Curing is a complex process also involving activity of other enzymes such as proteases, peroxidases, and polyphenoloxidases. The oxidative enzymes, peroxidases, and polyphenoloxidases, are responsible for browning of the pod tissue and for the formation of volatile ketones, aldehydes, and hydrocarbon derivatives (Adedji et al. 1993), which perhaps may also add to the final vanilla flavor.

REFERENCES

Achnine, L., Blancaflor, E.B., Rasmussen, S. and Dixon, R.A. (2004) Colocalization of L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis. The Plant Cell, 16, 3098-3109.

Adedji, J., Hartman, T.G. and Ho, C.-T. (1993) Flavor characterization of different varieties of Vanilla beans. Perfumer & Flavorist, 18, 25-33.

Arana, F.E. (1943) Action of a β-glucosidase in the curing of vanilla. Food Research, 8, 343-351.

Bartlett, D.J., Poulton, J.E. and Butt, V.S. (1972) Hydroxylation of p-coumaric acid by illuminated chloroplasts from spinach beet leaves. FEBS Letters, 23, 265-267.

Blount, J.W., Korth, K.L., Masoud, S.A., Rasmussen, S., Lamb, C. and Dixon R.A. (2000) Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry into the phenylpropanoid pathway. Plant Physiology, 122, 107-116.

Bolwell, G.P., Bell, J.N., Cramer, C.L., Schuch, W., Lamb, C.J. and Dixon, R.A. (2005) L-Phenylalanine ammonia-lyase from Phaseolus vulgaris. Characterisation and differential induction of multiple forms from elicitor-treated cell suspension cultures. European Journal of Biochemistry, 149, 411-419.