Studies On Optimum Fermentation Conditions And Some Characterization Of β-glucosidase In Vintage Red Yeast

Wine arome is one of the key factors that influence the wine quality. β-glucosidase hassignificant effect on improving wine aroma that hydrolyzes the bound flavor precusor to freearoma substances.The β-glucosidase from the saccharomyces cerevisiae which can adapt towinemaking conditions has a proper enzyme activity. So it is very important for increasingwine aroma to optimize the fermentation conditions and research on the characterization ofβ-glucosidase.In this study, p-NPG as substrate, determine wavelength400nm, absorbance value wasdetermined by colorimetric method and β-glucosidase activity was calculated according to thestandard curve of p-nitrophenol. On basis of single factor experiment containing theconcentration of substrate, reaction temperature, time and pH, optimum parameters of yeastβ-glucosidase activity determination were optimized by using orthogonal experiment: theconcentration of substrate5mmol/L, reaction temperature45℃, reaction time13min and pH6.2.This paper selected Vintage red from several kinds of saccharomyces cerevisiae, whichenzyme activity were the highest. The fermentation conditions were optimized by using singlefactor and orthogonal experiment, and the optimum group were obtained: fermentationtemperature30℃，fermentation time46h and initial pH of medium5.2.The crude enzyme was obtained by acetone precipitation method. Through ion exchangecolumn chromatography, purification fold was19.41and the yield was38.67%.Electrophoretic purity of β-glucosidase was determined by SDS-PAGE electrophoresis. Oneelectrophoresis band was attained and molecular mass was about45kD.Research on the characterization of the purified β-glucosidase proved that thethermostability of β-glucosidase was stable in the temperature range from20℃to40℃andpH range from5.0to10.0. The enzyme activity was inhibited by Al3+、Cu2+strongly, but K+、Mg2+、Ca2+、Zn2+、Na+have no effect on the activities of β-glucosidase.Glucose and alcohol had no significantly inhibitory effect on β-glucosidase activity.When glucose concentration was20%, the enzyme activity only decreased17.61%. Ethanolconcentration in the range of0to20%even had active effect on β-glucosidase enzymeactivity. While glucose and alcohol showed inhibitory effect on enzyme production of VintageRed yeast, The relative activity was reduced to12.98%when alcohol concentration was12%.