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

Potgieter, Nydia, 1977- (2004-03)

Thesis (MSc)--University of Stellenbosch, 2004.

Thesis

ENGLISH ABSTRACT: ~-Glucosidases constitute a major group of biologically important enzymes that catalyze the hydrolysis of glycosidic linkages in ~-glucosides, as well as in glycosides that contain only carbohydrate residues, e.g. cellobiose. These enzymes occur in all living kingdoms and perform a variety of functions in organisms ranging from bacteria to highly evolved mammals. Three different types of ~-glucosidases are found in humans, each with its own function: glucocerebrosidase (a deficiency causes Gaucher disease), lactase-phlorizin hydrolase (a deficiency results in lactose intolerance) 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 of the cellulase enzyme system that is responsible for the hydrolysis of cellobiose and short-chain oligosaccharides into glucose, playa role in the conversion of cellulosic biomass to liquid fuel. These microbial ~-glucosidases also playa very important role in the enhancement of fruit and wine aromas through the liberation of monoterpenols. Monoterpenols play an invaluable role in the flavor and aroma of grapes and wine, 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 of monoterpenes from glycosidically bound non-volatile precursors: acid hydrolysis and enzymatic hydrolysis. As high temperature acid hydrolysis causes a rearrangement of the monoterpene aglycones, the focus has shifted towards the more efficient enzymatic hydrolysis that does not result in modifications of the intrinsic aromatic character of the wine. The endogenous ~-glucosidases of grapes (Vitis vinifera), as well as of the wine yeast Saccharomyces cerevisiae, exhibit very low activity towards the glycoside precursors, 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 enzymes are not always suitable for use under the harsh conditions that prevail during winemaking (i.e. low pH, low temperatures, and high ethanol and glucose concentrations). The limited enzyme activities of the abovementioned microorganisms 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 yeasts were quantified, characterized and assessed to determine the efficiency with which they could liberate monoterpenols from their terpenyl-glycosides. The Debaryomyces pseudopolymorphus l3-glucosidase from intracellular crude cell extracts exhibited the most suitable combination of properties in terms of functionality at wine pH, resistance to wine-associated inhibitory compounds (glucose, ethanol and sulfur dioxide), high substrate affinity and large aglycone-substrate recognition. This yeast strain was also used, in conjunction with S. cerevisiae VIN13, for the small-scale fermentation of Chardonnay juice. The results indicated that the l3-glucosidase of D. pseudopolymorphus had definite potential as a wine aroma-enhancing enzyme, as the concentrations of free terpenols (nerol, geraniol and citronellol) were significantly increased during fermentation. Future experimental work would include an in-depth study of the kinetic characteristics of the l3-glucosidases (both cytosolic and cell-associated) exhibiting the highest terpenol-liberating activity under winemaking conditions. The next step would then be the cloning and expression of the most efficient l3-glucosidase gene in a commercial wine yeast. Such a recombinant wine yeast would release grapederived aroma compounds from their non-volatile precursors during single culture fermentations, thereby increasing the sensorial quality of wine.

AFRIKAANSE OPSOMMING: I3-Glukosidases vorm deel van 'n groot groep biologies belangrike ensieme wat die hidrolise van glikosidiese bindings binne l3-glukosiede,sowel as binne glikosiede wat slegs uit koolhidraatresidue bestaan, soos bv. sellobiose, kataliseer. Hierdie ensieme kom in alle koningkryke van lewende organismes voor en verrig 'n wye verskeidenheid funksies binne organismes wat wissel van bakterieë tot hoogs ontwikkelde soogdiere. Drie verskillende tipes l3-glukosidases,elk met sy eie funksie, kom in mense voor: glukoserebrosidase ('n gebrek hieraan lei tot Gaucher-siekte), laktaseflorizinhidrolase ('n gebrek hieraan gee aanleiding tot laktose-intoleransie) en sitosol l3-glukosidase (verantwoordelik vir die hidrolise van l3-glukosiede wat saam met voedsel van plant en dier oorsprong ingeneem word). Die funksies van 13- glukosidase binne plante sluit in pigmentmetabolisme, biomassa-omsetting en sianogenese, wat ook 'n funksie van insek l3-glukosidases is. Mikrobiese 13- glukosidases, as deel van die sellulase-ensiemsisteem wat verantwoordelik vir die hidrolise van sellobiose en kortketting-oligosakkariede na glukose is, speel 'n rol in die omsetting van sellulosebiomassa na brandstof. Hierdie mikrobiese 13- glukosidases speelook 'n baie belangrike rol in die verbetering van vrugte- en wynaroma deur die vrystelling van monoterpenole. Monoterpenole speel 'n belangrike rol in die geur en aroma van druiwe en wyn, en kom voor as vry, vlugtige en aromatiese molekules, asook geurlose, nie-vlugtige glikosidies-gebonde komplekse. Hierdie komplekse is meestal in die vorm van 6-0- I3-D-xilopiranosiel-I3-D-glukopiranosiede, 6-0-a-L-arabinofuranosiel-I3-D-glukopiranosiede, 6-0-I3-D-glukopiranosiel-I3-D-glukopiranosiede, 6-0-a-L-ramnopiranosiel- I3-D-glukopiranosiede,of 6-0-I3-D-apiofuranosiel-I3-D-glukopiranosiedevan hoofsaaklik linalool, geraniol, nerol, a-terpineol en hotrienol. Monoterpenole kan op een van twee maniere van hul suikermolekules vrygestel word: suurhidrolise of ensimatiese hidrolise. Die hoë temperature waarby suurhidrolise plaasvind veroorsaak 'n herrangskikking van die monoterpeen aglikone, en die fokus het gevolglik verskuif na die meer effektiewe ensimatiese hidrolise wat nie verandering van die intrinsieke aromatiese karakter van die wyn tot gevolg het nie. Die endogene l3-glukosidases van druiwe (Vitis vinifera) en die wyngis Saccharomyces cere visiae , toon baie lae aktiwiteit ten opsigte van die aromatiese voorlopers, en dus word daar toenemend op die toevoeging van eksogene 13- glukosidases tot die wyn gefokus om meer geur vry te stel. Daar is bevind dat 13- glukosidases van fungiese, bakteriële en gis oorsprong effektiewe aromavrystelIers is, maar hierdie ensieme is nie altyd gepas vir gebruik in wyn nie, aangesien dit 'n omgewing is met 'n lae pH, lae temperatuur, en hoë etanol- en glukosekonsentrasies. Die beperkte ensiemaktiwitiet van bogenoemde mikroorganismes het gelei tot 'n soeke onder nie-Saccharomyces giste na l3-glukosidases wat in die wynomgewing kan funksioneer. Die ~-glukosidase-aktiwiteit van twintig wyn geassosieerde nie-Saccharomyces giste is gekwantifiseer en gekarakteriseer om te bepaal tot watter mate dit monoterpenole van hul terpeniel glikosiede kan vrystel. Die intrasellulêre ~- glukosidase teenwoordig in the selekstrak van Debaryomyces pseudopolymorphus, het die belowendste resultate getoon ten opsigte van funksionaliteit by wyn se pH, weerstand teen wyn geassosieerde inhibeerders (glukose, etanol en swaweidioksied), hoë substraataffiniteit en breë aglikoon-substraat herkenning. Hierdie gisras is ook in kombinasie met S. cerevisiae VIN13 gebruik vir die kleinskaalse fermentasie van Chardonnay sap. Die resultate het getoon dat die ~- glukosidase van D. pseudopolymorphus wel potensiaal het om wynaroma te verhoog, aangesien die konsentrasie van ongebonde terpenole (nerol, geraniol en citronellol) aansienlik tydens fermentasie toegeneem het. Toekomstige eksperimentele werk sluit in, onder meer, In in-diepte studie van die kinetiese eienskappe van die ~-glukosidases (beide sitesolies en sel-geassosieerd) wat die meeste terpenole onder wynrnaakkondisies vry stel, asook die klonering en uitdrukking van die enkele ~-glukosidasegeen met die hoogste aktiwiteit, in In kommersiële wyngis. Só In rekombinante wyngis sal die vrystelling van druifgebaseerde aromakomponente van hul nie-vlugtige, geurlose voorlopers tydens enkel-kultuur fermentasies teweeg bring.

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