Optimization of β-glucosidase activy in recombinant Saccharomyces cerevisiae strains

[摘要] ENGLISH ABSTRACT: Wine is a complex medium. Wine aroma, flavour and colour are important qualityfactors, but these can be influenced by many factors, such as grape-derivedcompounds that exist as free volatiles and also as glycosidically bound. Thechemical composition of wine is determined by factors such as grape variety,geographic position, viticulture condition, microbial ecology of the grape and thewinemaking process. The varietals aroma is determined by both the volatile andthe non-volatile compounds, such as monoterpenes, norisoprenoids and benzenederivatives, which are naturally present in the wine. Monoterpenes are veryimportant in the flavour and aroma of grapes and wine. They can be found ingrapes and wine either in the free, volatile and odorous form, or in theglycosidically-bound, non-volatile and non-odorous form. The ratio ofglycosidically-bound compounds to free aroma compounds is very high in theGewürztraminer, Muscat and Riesling cultivars in particular.The glycosidic bonds can be hydrolysed either by the acid method or by usingenzymes. The acid method is disadvantageous because it can modify themonoterpenes, whereas enzymatic hydrolysis has the advantage of not modifyingthe aroma character. The enzyme method of breaking the glycosidic bonds occursin two successive steps: initial separation of glucose from the terminal sugar by ahydrolase (a-L-arabinofuranosidase, a-L-rhamnosidase or β-apiosidase,depending on the aglycone moiety), followed by the breaking of the bond betweenthe aglycone and glucose by β-glucosidase.The enzyme β-glucosidase can be obtained from many plant (Vitis vinifera),bacterial, yeast or fungal sources. Most of the enzymes produced by these sourcesare not functional under the winemaking conditions of low pH, low temperature,high glucose and high ethanol content. However, β-glucosidases from fungalorigins, particularly from Aspergillus spp., are tolerant of winemaking conditions.The idea of using the β-glucosidase gene from the fungus Aspergillus kawachii(BGLA), which is linked to the cell wall and the free β-glucosidase, was todetermine if anchoring the enzyme to the cell wall will increase the activity of theenzyme compared to the free enzyme. Four plasmids, pCEL 16, pCEL 24, pDLG97 and pDLG 98, were used in this study. BGLA that was cloned into the plasmidspCEL 24 and pDLG 97 was linked to CWP2, and in pDLG 98 it was linked to AGa1anchor domains. All the plasmids were genome-integrated and expressed in thereference strain Saccharomyces cerevisiae 303-1A. All the transformants weregrown in 2% cellobiose and showed higher biomass production compared to thereference strain. β-Glucosidase activity was also assayed and transformed strainW16 showed a fourfold increase in activity compared to the reference strain. Therewas no significant increase in the activity of the other transformed strains, W24,W97 and W98. Enzymatic characterisation for optimum pH and temperature was done – for all strains the optimum pH was 4 and the optimum temperature was40ºC.The recombinant strains together with the reference strain were used to makewine from Gewürztraminer grapes. The levels of numerous monoterpenes wereenhanced in the resultant wines. The concentration of nerol was increasedfourfold, that of citronellol twofold, and geraniol was 20% higher than in the wildtype. There was also an increase in the levels of linalool and a-terpinol, but thiswas not significant. In wines produced with W97, W98 and W24, monoterpenelevels did not show a significant difference.In future, the expression of the W16 expression cassette in an industrial wineyeast strain could be performed. In combination with the production of enzymessuch as a-arabinofuranosidase, a-rhamnosidase and β-apiosidase, which areinvolved in the first step of enzymatic hydrolysis, this wine strain could release thebound monoterpenes and enhance the aroma of the wine.

[发布机构] Stellenbosch University