Handbook of Vanilla Science and Technology

Glycosylation is carried out by glycosyltransferases (GTs), which transfer nucleotide-diphosphate activated sugars (UDP-sugar) to low molecular weight substrates. These enzymes occur in minute quantities in plant tissues as a set of related enzymes and thus are hard to separate and purify. GTs are very labile, making purification quite a difficult task. Those involved in secondary metabolism are usually soluble proteins of molecular weight between 45 and 60 kDa (Vogt and Jones 1997). Because a variety of secondary metabolites occur in the form of glycosides of different sugars, there are also several UDP-activated-sugars donating a sugar moiety to these low molecular substrates. The most frequent is UDP-glucose (in this case the respective glycosides are called glucosides), but UDP-galactose, UDP-rhamnose, and others are also found (Vogt and Jones 1997). Avery important feature of glycosides is that they are not volatile. Glycosylation of a volatile aglycone makes it nonvolatile and allows for its accumulation at high concentrations in specialized tissues and cell compartments. Release of toxic aglycones by glycosylhydrolases is often induced as part of the defense response to microbial, fungal, or animal attack.

Although glucosides of some of the proposed vanillin biosynthesis intermediates have been found in green vanilla beans, it is still unknown if the glucosylated compounds actually serve as intermediates in the pathway. Addition of the sugar moiety to the intermediates may be a side reaction of the glucosyltransferase, “trapping” them from further modification. The added sugar moiety can be expected to change the shape of the substrate molecule and its interaction with the biosynthetic enzyme. For example, Van OMT-3, discussed above, was highly active with myricetin but not with a glycosylated derivative, myricitrin (Li et al. 2006). The answer to this question will require the isolation and characterization of the enzymes involved.

The concentration of glucovanillin (vanillin- β-D-glucoside) found in green vanilla pods is around 14% on a dry weight basis. Vanillin, the aglucone moiety in glucovanillin, comprises 45.8% by weight of the compound, which is also the percentage yield of vanillin when glucovanillin is hydrolyzed to completion. For example, 14 g of glucovanillin when hydrolyzed to completion, yields 6.4 g of vanillin. An actual recovery of 6 g vanillin therefore represents around 94% of the theoretically possible recovery. This level of vanillin recovery has been achieved after 7 days of curing under controlled laboratory conditions (Havkin-Frenkel et al. 2003). In traditional curing methods, however, the vanillin losses are considerably larger. A typical vanillin content of 2% on a dry weight basis of cured beans, represents a recovery of about 30%.

17.7 β-GLYCOSYL HYDROLASES AND CURING