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β-glucosidase are hydrolases, which catalyze hydrolysis of many withβ-D-glucoside bond glycosidic substances, such as amygdalin, soy isoflavone glycosides, etc.β-glucosidase enzy-me is widely used.It can be used to enhance the natural flavor of fruit and vegetable products, but also can improve the biological activity of soy isoflavones. At home and abroad have been produced using microbial fermentation ofβ-glucosidase enzyme studies, but the mold produc-ing bacteria is mainly due to mold growth cycle is long, large energy consumption, is not con-ducive to the realization of industrial production. In this study, conventional methods of micr-obial studies in order ofβ-glucosidase-producing strain selection, optimization of enzyme pr-oduction, strain identific- ation, enzyme kinetics, enzyme extraction and crude soy isoflavone glycosides and other enz- ymatic processes have been studied.Objectives:To screen out 1-2 strains producingβ-glucosidase of bacterial strains, and the use of their production ofβ-glucosidase enzymes for conversion of soy isoflavone glycosides, the soy isoflavones daidzein Enzymatic production of a new enzyme source.Methods:In this study, selective medium filter selected to separate the different materials, the highest activity by DNS method selected bacterial strains; The use of single-factor test and response surface analysis tests to optimize conditions for enzyme production, and by colony morphology, physiological and biochemical tests and 16SrRNA sequence analysis of strain id-entification;The use of ammonium sulfate precipitation, dialysis, ultrafiltration and DEAE-52 ion-exchange chromatography ; The use ofβ-glucosidase produced by crude extract of soybean isoflavone glycosides to trastrains producingβ-glucosidase were isolated and purified and its enzymatic properties of the studynslational research, the single factor experiments and ort-hogonal test to determine the amount of enzyme, pH, temperature, hydrolysis time, the best equipped.Results:1. From natto screened a highβ-glucosidase of bacterial strains, the test number NX-3. NX-3 strains producingβ-glucosidase of the best conditions for medium 1.5% glucose, 2% soybean peptone, soybean flour 1%, KH2PO40.1%, NaCl 0.5%,CaCl20.05%, MgSO4.7H2O 0.02%, fermentation temperature 36℃, pH 7.0, fermentation time 42h, medium volume 50ml/300ml flask, speed 150r/min, activity increased from 110.1IU/mL to 155.2IU/mL, increased by 40%. NX-3 strain identified as Bacillus subtilis(Bacillus subtilis).2.The purified enzyme specific activity from the initial 2.5 U/mg increased to 25.3U/mg, specific activity increased 9.1-fold, by SDS-PAGE to determine the NX-3 strains producingβ-glucosidase molecular weight of 40KDa. NX-3 strains producing (3-glucosidase optimum temperature is 50℃, the temperature exceeds 60℃large changes in enzyme stability; at pH 5.0, pH 3-6 range in the more stable enzyme; metal ions Mg2+ activity of the enzyme can pro-mote, K+, Ca2+ activity of the enzyme had no effect, Ag+, Hg2+inhibit the enzyme activity.3. When the substrate concentration of soy isoflavones 10g/L, the best ratio of conversio-n enzyme:substrate 2.5:1, pH value 5.0, temperature 40℃, hydrolysis time 2.5h.Under these conditions, the measured conversion rate of isoflavone glucoside 67.4%.Conclusion:This study selected a highβ-glucosidase of bacterial strains, shortening the production cycle of the enzyme fermentation, reducing the waste of resources, is conducive to industrial production, while production of soy isoflavone glycosides into the foundation.
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