Study On The Extraction And Separation Of β-glucosidase From The Buckwheat And Its Kinetic Of Enzymic Reaction

β-glucosidase is an important component of the cellulose complex.It widely exists in the animal,plant and Microorganisms.lt can take part in bio transformation of the cellulose material,and hydrolyzes the disaccharide of cellobiose to glucose. It is an enzymic which can hydrolyze the flavour material.It can take part in the glucoside transforms to polyphenol.The research studied on the extraction and separation ofβ-glucosidase from the buckwheat,then P-glucosidase from the buckwheat was studied from the kinetic of enzymic reaction.The reaseach was the foundation for the applicatiion ofβ-glucosidase.Main results were as follow:(1)The extraction conditions ofβ-glucosidase from the buckwheat:C6H8O7-C6H5Na3O7 buffer,pH 4.6,centrifugal speed 5000 r/min. Under the optimum extraction conditions, the extracted P-glucosidase activity concentration was 597.96×106 U/L, the enzyme specific activity was 0.397×106 U/mg.(2)Seperation and purificationβ-glucosidase from the buckwheat:the range of ammonium sulfate precipitation of P-glucosidase was 30～45%,in the rang the enzyme specific activity was 0.66x 106 U/mg.(3)β-glucosidase purified by dialysis:the sample of the last step was purified by dialysis,then the enzyme specific activity was 0.91 x 106 U/mg.(4)β-glucosidase purified by SephadexG-150:the result of this step was expressed two ranges which were higer than others.(5) The molecular weights of the two ranges by SDS-page were estimated to be 1.87×106U/mg,1.78×106 U/mg.(6)The results of the purification multiple:in the 30～45% saturated solubility,the purification multiple was 1.66.After the dialysis step,the purification multiple was 2.29.After SephadexG-150 step,the purification multiple was 4.71.(7)The results of the kinetic of enzymic reaction were as follow:①The parameter of the kinetic of enzymic reaction:the result showed the Michaelis equation was y= 0.6183x+0.4948,R2=0.9938,the Km and Vmax were 1.25 mmol/L and 2.02 mmol/min.②The optimum temperature of the kinetic of enzymic reaction:the Optimum temperature was 50℃,the stability of P-glucosidase was below 40℃.③The optimum pH of kinetic of enzymic reaction was 5.pH 5,the relative enzyme activity was highest by the stability of P-glucosidase.④The result of pNPG concentration effect on kinetic of enzymic reaction:when the range of pNPG concentration was below 1mmol/L,the response rate was fast by the increasing concentration.When the range of pNPG concentration was 1～5mmol/L,the response rate was slow by the increasing concentration.When the range of pNPG concentration was up 5mmol/L,the response rate was not increasing.⑤When the concentration of Vc was 4 mmol/L,the relative enzyme activity was the highest.The Mg+ iron can promote the relative enzyme activity.The Na+ and Ca2+ irons can not promot the relative enzyme activity.⑥The parameter of the kinetic of enzymic reaction after attending Ag+ iron:the Michaelis equation was y=2.1827x+0.4948,R2=0.9972,the Km and Vmax were 4.41 mmol/L,2.02 mmol/min.Through comprasion parameters of enzymic reaction,Ag+ was of inhibition for the enzymic reaction,but of the competitive inhibition.