| β-Glucosidases (EC3.2.1.21) is the key enzyme which releases alkyl and arylβ-glycosides, and disaccharide glucosides and short oligosaccharides of non-volatileglycosidic precursors present in fruit juices, musts and wines. It plays an important role in thehydrolysis of wine aroma precursors. Due to the absence of special β-glucosidase inwinemaking, it is difficult to enhance or modify wine flavor in industry. The application ofenzyme preparation rich in β-glucosidase was restricted by many factors in grape wine. So,more and more research have focused on the improvement of wine quality by the applied ofβ-glucosidases. It has been a difficulty and a focus of the enhancement of wine quality tostudy β-glucosidase by the orientation of wine flavor. Although wine industry is booming inChina and the production and sale of wines are increasing, there is still a long way to go toreach the quality and characteristics of foreign wine. The application of proper β-glucosidasewith excellent performance in Chinese wines to improve the quality and embody the spcialstyle will contribute the theory and practical application to the winemaking and wine flavor inChina.Based on the little research and applications of β-glucosidase in Chinese wine, theproducer of β-glucosidase was isolated and screened from the enology environment of wineryof Jiaodong peninsula, belonged to Changyu Group Company Ltd.(1) Screening of wine yeast for β-glucosidase activity with high sugar and low pHtolerances. Among the10species,4exhibited higher β-glucosidase activity. Trichosporonasahii (F6) exhibited the highest β-glucosidase activity. This is the first description ofβ-glucosidase activity involved in Trichosporon yeast. The resistances of F6β-glucosidase tomain enological factors (high sugar, low pH, and alcohol) and the capacity to hydrolyzearoma precursors from glycoside extract was investigated compared with the β-glucosidasesfrom other species yeasts. The results showed that β-glucosidase of F6had stronger resistanceto low pH and better ability to hydrolyze aroma precursors than other β-glucosidases.(2) The production enhancement and characteristics of β-glucosidase from F6strain. Themaximum β-glucosidase production (0.216U/mL) of F6strain was achieved after theoptimization of medium, which led to an increase in β-glucosidase production of F6by1.74times than the basal medium YPD. The β-glucosidase of F6was purifed successively throughammonium sulphate precipitation, hydrophobic interaction chromatography, anion exchangechromatography, and gel fltration. Two β-glucosidases were obtained, namely BG1and BG2.The molecular weights of BG1and BG2were160kDa and30kDa, respectively. Due to thelower Km, BG1had more affinity for pNPG and higher catalytic efficiency than BG2. BG1also exhibited better resistance to SDS and β-mercaptoethanol, and the latter suggested theprobable absence of a disulfde bond in the active catalytic site of BG1. The better resistanceof BG1to many factors derived from the predominance of β-sheet structure. TheSDS-resistant and high β-sheet secondary structure of BG1suggests higher kinetic stabilitythan BG2. Additionally, BG1had the ability to synthesize oligosaccharides bytransglycosylation. Due to its unique characteristics, BG1from T. asahii appears to represent a novel β-glucosidase. These properties mark BG1as a potential enzyme for a wide range ofindustrial application.(3) β-Glucosidase BG1of F6was added to the glycosides extract and grape must toinvestigate the hydrolysis ability to aroma precursors under the two conditions, comparedwith commercial β-glucosidases from Aspergillus niger (AS) and almond (Al). The resultsrevealed that the three β-glucosidases exhibited different hydrolysis abilities under the grapeextract conditions. BG1had stronger ability to hydrolyze the precursors of terpenes, somealdehydes, ketones, and alcohols compounds; Al had more affinity for some glycosides ofaldehydes and ketones, AS exhibited stronger ability to release β-damascenone, C6compounds and some terpenes from their precursors. However, under the winemakingconditions, the three β-glucosidases did not show the similar performances. In comparisonwith AS and Al, BG1exhibited good performance under winemaking conditions. Due to thedifferent affinity for anthocyanins, the three β-glucosidases exhibited different hydrolysisdegrees to the main anthocyanin glycosides from Cabernet Gernischt wine, such asmalvidol-3-glucoside, malvidol-3-acetylglucoside, and malvidol-3-coumarylglucosid. BG1exerted the lowest hydrolysis degree, followed by Al. However, AS showed the highesthydrolysis ability. These results revealed that BG1has better property on protectinganthocyanin glycosides than commercial β-glucosidases. All of these will provide BG1wideapplication domain in industry.(4) Investigation of hydrolysis mechanism on aroma glycosides. The differentperformances of the three β-glucosidases under the two conditions suggested that theactivities of β-glucosidases were inhibited or inactivated by some factors during winemakingprocess. The extents of inhibition were different according to different β-glucosidase. Toinvestigate the possible reason, the effects of different pH and glucose concentrations on thesecondary structures and activity of β-glucosidases were studied. The assay of secondarystructures of different β-glucosidases revealed that glucose exerted reversible effects on thestructures of β-glucosidases. The activities of β-glucosidases recovered after removing theglucose. Among the three β-glucosidases, AS showed the highest sensitivity to glucose.Glucose inhibited the activity of AS resulting in a decrease in α-helix and β-sheet structuresand an increase in random coil structure. Low pH showed stronger inhabitation on theactivities of β-glucosidases and exhibited irreversible influence on the structures and activitiesof β-glucosidases. When the pH value was further decreased, the contents of α-helix andβ-sheet decreased and β-turn and random coil increased with decreasing pH. The lowercontent of α-helix and higher content of β-sheet gave BG1stronger resistance against glucoseand low pH and provides this β-glucosidase with a great impetus for application duringwinemaking process.All of these results showed that the β-glucosidase of F6strain exhibited more superiorproperties than commercial β-glucosidases from Aspergillus niger and almond, especially, thelow hydrolysis ratio of pigments in the wine treated by the β-glucosidase BG1of F6. Themechanism was explored for the different performances of the β-glucosidases between thehydrolysis of glycoside extract and the winemaking conditions. The results of this workprovided both the proper β-glucosidase and the valuable guidance of theory and practice for the usage of this enzyme. |
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