The evaluation of β-glucosidase activity produced by wine-isolated yeasts

[摘要] ENGLISH ABSTRACT:~-Glucosidases constitute a major group of biologically important enzymes thatcatalyze the hydrolysis of glycosidic linkages in ~-glucosides, as well as in glycosidesthat contain only carbohydrate residues, e.g. cellobiose. These enzymes occur in allliving kingdoms and perform a variety of functions in organisms ranging from bacteriato highly evolved mammals. Three different types of ~-glucosidases are found inhumans, each with its own function: glucocerebrosidase (a deficiency causesGaucher disease), lactase-phlorizin hydrolase (a deficiency results in lactoseintolerance) and cytosolic ~-glucosidase (responsible for the hydrolysis of ~-glucosides ingested with foods of plant and animal origin). In plants, the functions of~-glucosidases include pigment metabolism, biomass conversion and cyanogenesis,a function it shares with insect ~-glucosidases. Microbial ~-glucosidases, as part ofthe cellulase enzyme system that is responsible for the hydrolysis of cellobiose andshort-chain oligosaccharides into glucose, playa role in the conversion of cellulosicbiomass to liquid fuel. These microbial ~-glucosidases also playa very important rolein the enhancement of fruit and wine aromas through the liberation of monoterpenols.Monoterpenols play an invaluable role in the flavor and aroma of grapes andwine, and are present as free, volatile and odorous molecules, as well as flavorless,non-volatile glycosidic complexes. These complexes most often occur as 6-0-~-Dxvlopyranosyl-B-Dcqlucopyranosides, 6-0-~-D-glucopyranosyl-~-D-glucopyranosides,6-0-a-L-arabinofuranosyl-~-D-glucopyranosides, 6-0-a-L-rhamnopyranosyl-~-Dglucopyranosides,or 6-0-~-D-apiofuranosyl-~-D-glucopyranosides of mainly linalool,geraniol, nerol, a-terpineol and hotrienol. Two mechanisms exist for the release ofmonoterpenes from glycosidically bound non-volatile precursors: acid hydrolysis andenzymatic hydrolysis. As high temperature acid hydrolysis causes a rearrangementof the monoterpene aglycones, the focus has shifted towards the more efficientenzymatic hydrolysis that does not result in modifications of the intrinsic aromaticcharacter of the wine.The endogenous ~-glucosidases of grapes (Vitis vinifera), as well as of the wineyeast Saccharomyces cerevisiae, exhibit very low activity towards the glycosideprecursors, and thus the focus has increasingly fallen on the addition of exogenous~-glucosidases to enhance wine flavor. Fungal, bacterial and some yeast ~-glucosidases have been indicated as effective aroma liberators, but these enzymesare not always suitable for use under the harsh conditions that prevail duringwinemaking (i.e. low pH, low temperatures, and high ethanol and glucoseconcentrations). The limited enzyme activities of the abovementionedmicroorganisms have resulted in a search among non-Saccharomyces yeasts for ~-glucosidases that can withstand these conditions.The ~-glucosidase activities of 20 wine-associated non-Saccharomyces yeastswere quantified, characterized and assessed to determine the efficiency with whichthey could liberate monoterpenols from their terpenyl-glycosides. The Debaryomycespseudopolymorphus l3-glucosidase from intracellular crude cell extracts exhibited themost suitable combination of properties in terms of functionality at wine pH,resistance to wine-associated inhibitory compounds (glucose, ethanol and sulfurdioxide), high substrate affinity and large aglycone-substrate recognition. This yeaststrain was also used, in conjunction with S. cerevisiae VIN13, for the small-scalefermentation of Chardonnay juice. The results indicated that the l3-glucosidase of D.pseudopolymorphus had definite potential as a wine aroma-enhancing enzyme, asthe concentrations of free terpenols (nerol, geraniol and citronellol) were significantlyincreased during fermentation.Future experimental work would include an in-depth study of the kineticcharacteristics of the l3-glucosidases (both cytosolic and cell-associated) exhibitingthe highest terpenol-liberating activity under winemaking conditions. The next stepwould then be the cloning and expression of the most efficient l3-glucosidase gene ina commercial wine yeast. Such a recombinant wine yeast would release grapederivedaroma compounds from their non-volatile precursors during single culturefermentations, thereby increasing the sensorial quality of wine.

[发布机构] Stellenbosch University