Studies On The Properties And Hign-Density Liquid Fermentation Model Of β-Glucosidase From Brettanomyces Sp. Obtained By Genome Shuffling

P-glucosidase(EC3.2.1.21), aslo known as p-D-glucosidase glucohydrolase, which is an essential ingredient of the composition of cellulase. In this study, the two parental strains U1and V1, which were carried out three times genome shuffling, were obtained using P-glucosidase enzyme activity as indicator from B.anomalus PSY-001with UV and ultrasonic as mutagent. By using the screening medium, and P-glucosidase enzyme activity as indicator, a fusion strain F3-25high-yield beta-glucosidase was attained. Optimize the fermentation conditions of the F3-25, and develop the strains of high-density liquid fermentation model. The purification of P-glucosidase produced by F3-25were investigated with AKTA purifier-900protein separation and purification workstation. The properties of β-glucosidase was aslo discussed. This paper provides a reliable basis for the industrial production.Main contents and results of the study are as follows:1, In this study, the strain producing β-glucosidase B.anomalus PSY-001was UV mutagenesis and ultrasonic mutagenesis, screened from the mutant strains yielding beta-glucosidase from the two parental strains U1and V1, which was carried out three times genome shuffling. By the screening of the screening medium, and then to the P-glucosidase enzyme activity as an indicator, filter out the high-yield P-glucosidase fusion strain F3-25. The strain F3-25produced P-glucosidase activity is nearly eight times larger than the original strain.2, The optimum culture medium of fusion strain F3-25was optimized by Design Expert software, which were composed of:the bran concentration of53.15g/L, the concentration of yeast extract of3.03g/L, KC1of0.204g/L, CaCl2of0.611g/L.Fusion strains of the F3-25flask fermentation conditions were determined by single factor and orthogonal experiments:the fermentation temperature of30℃,250mL conical bottled liquid volume of20mL, the shaker is150r/min inoculation3%, the initial pH of5.3, The fed-batch fermentation conditions of F3-25were optimized. The growth kinetics model of fusion strain F3-25was obtained by Orign8.0software. The model is4, The purification of P-glucosidase produced by F3-25were investigated with AKTA purifier-900protein separation and purification workstation using Sephadex G-100 gel filtration chromatography. β-glucosidase purification rate was reached96.2%. Electrophoretic purity of β-glucosidase was detected by SDS-PAGE electrophoresis. Two electrophoresis band were attained, which molecular mass were estimated to be50.3KDa and67.8KD respectively. The content of two subunits were40.5%and57.3%respectively anlalyzed by Bandscan.The optimum temperature of F3-25was60℃. The optimal pH was5.0. Theβ-glucosidase has good stability within the range of40℃-50℃and the pH5.0-6.0. Fe3+,Mn2+and Ca2+can promote the activities of β-galactosidase while Mg2+,Al3+,Cu2+, Zn2+can inhibit the activities of β-galactosidase.