| Summary: | Thesis (MSc)--University of Stellenbosch, 2004. === 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.
|
|---|
