?-glucosidase In Rhizome Of Dioscorea Zingiberensis C.H.Wright

P-D-Glucosidase(EC3.2.1.21),also known as P-D-glucoside glucohydrolase,alias gentian disaccharidase,cellobiose(cellobias,CB or ?-G)And bitter amygdylase.It belongs to the cellulase,which is an important constituent of the cellulolytic enzyme system,capable of hydrolyzing the non-reductive beta-D-glucose bond at the end,while releasing ?-D-glucose and the corresponding ligand.In this study,we mainly studied the purification and basic enzymological properties of ?-glucosidase from the rhizome of Dioscorea zingiberensis C.H.Wright.In order to further study the effects of P-glucosidase in the rhizome of Dioscorea zingiberensis C.H.Wright,Which provide the foundation for the further application of the specific ?-glucosidase 26-O-?-glucosidase(Furostanol glycoside 26-0-?-glucosidase,F26G),which is highly pharmacologically active.The results indicate as follows:(1)The optimum conditions for the extraction of ?-glucosidase from the rhizome of Dioscorea zingiberensis C.H.Wright were studied.According to the single factor experiment and the three factors experiment,the P-glucosidase crude enzyme solution The best solution for the extraction of 0.01mol/L,pH7.2 potassium phosphate buffer at 0? under the conditions of extraction 3.5h.(2)The crude enzyme solution was first separated by salting-ammonium sulfate precipitation in the first stage of purification.After the two stages of ammonium sulfate precipitation,the final determination of ammonium sulfate precipitation area of 35%to 55%when the enzyme concentration was the largest.(3)After salting out,the collected enzyme solution was dialyzed and desalted and concentrated.The concentrated sample was collected as a sample for the next stage of separation and purification.The enzyme activity was 152.2U/g.(4)Purification of the second stage of the use of ion separation chromatography,using DEAE-cellulose DE52 anion exchange column,the enzyme activity to obtain a higher tube enzyme,protein eluent KCl concentration of 0.12mol/L and 0.16mol/L.measured enzyme activity about 145U/g and152U/g.(5)The third stage of the purification was carried out using a Butyl-650M TOYO PEARL hydrophobic column to give the starting protein when the eluate was an ammonium sulfate solution of 0.4mol/L,and when the ammonium sulfate solution was 0.1M Most of the target protein will flow out.(6)The time cost and economic cost were the best at the condition of the reaction time was 45 minutes and the substrate concentration was 10 mmol/L.(7)The optimum temperature of ?-glucosidase is 50? and it is stable at 40?.The optimum pH is 5,which is stable in weak alkaline environment.(8)The experimental results showed that Fe2+ had inhibited the activity of?-glucosidase in the rhizome of Dioscorea zingiberensis C.H.Wright.Furthermore the inhibitory effect of Cu2+ and Zn2+ on ?-glucosidase was significantly higher than that of the other metal ions in the experimental group.The two organic solvents,acetic acid and formaldehyde,had the greatest inhibitory effect on ?-glucosidase activity.However the inhibitory effect is not so obvious relative to metal ions.The largest inhibition of ?-glucosidase activity is the organic matter called SDS,but other organic matter on the enzyme activity have a certain degree of inhibition which had not much difference.(9)Four kinds of glycoside substrates were used as the substrate specificity of ?-glucosidase in the rhizome of Dioscorea zingiberensis C.H.Wright.It was found that ?-glucoside was highly effective in the hydrolysis of p-nitrophenyl?-D-glucopyranoside,but was not obvious in the role of p-nitrophenyl?-D-galactopyranoside,p-nitrophenyl ?-D-xyloside and p-nitrophenyl glucuronide.?-Glucosidase is specific for the hydrolysis of p-nitrophenyl beta-D-glucopyranoside.(10)The Km of ?-glucosidase in rhizome of Dioscorea zingiberensis was measured as 1.31mmol/L,Vmax is 2.06mmol/L/min.