Purification And Characterizationof Rutin Degrading Isoenzymes In Tartary Buckwheat Seed

Tartary buckwheat is abundant in flavonoid compounds.Being its main component,rutin can improve human beings’ immunity and has accessory treatment effect for diabetes,hypertension and cataract.However,tartary buckwheat powder contains highly active rutin-degrading enzyme,so in tatary buckwheat food processing,this enzyme can cause rutin degradation,thus reduce its nutritional value significantly.In this paper,we conducted a systematic method study on the purification methods and characterization of rutin-degrading enzymes based on the gel detection method of rutin-degrading enzymes.We obtained the following results：Rutin-degrading enzyme have strong methanol and acetone resistance.Based on this result,crude enzyme extraction mixed with the same volumn of methanol to precipitate the protein impurities.Then the obtained enzyme solution can be concentrated by acetone precipitation.Based on the previous PAGE active staining,by electrophoresed in a slab gel with a 20%running gel and a 4%stacking gel and boost the concentration of methanol in substrate solution,we can find there are at least 5 kinds of RDEs in tartary buckwheat seeds.The lower detection limit of RDE in the silver staining method was 1.92μg for displaying the isozymes,which was much less sensitive than activity-staining method.With the method,the molecular mechanism of the formation of RDEs through Mass Spectrometry can be explored.Based on solubility difference between quercetin and rutin,two high-abundant isozymes are purified.Analyzing the two isozymes’s tolerance of organic solvent,we can find that rutin-degrading enzyme has excellent tolerance of the nine organic solvents.The activity of enzymes 1 and 2 was little change with 0%-70%of methanol.Ethanol,propanol and ethylene glycol can promote the activity of both enzymes.The activity of enzymes 1 and 2 will be inhibited with 70%chloroform,while the activity of enzymes 1 was inhibited by 15%with 80%toluene.The both enzymes can be inhibited by xylene,while carbon tetrachloride and acetone have no such effect.This study will definitely have some instructional significance to the producing of quercetin through enzymic method and other industrial applications of rutin-degrading enzyme.Substrate specificity analysis discover that the two purified isozymes can both hydrolyze rutin and quercetin-3-o-glucoside,which are both belong to the derivatives of quercetin.This resμlt shows that the specificity presented in prodcing quercetin throμgh the rutin-degrading enzyme may have some relations with the sugar moiety of quercetin.Analyzing the two isozymes by catalytic kinetic analysis,we find that the km of the two isozymes were 0.07mol/L and 0.075mol/L respectively.And they both have strong pH and thermal stabilities.These basic data can lay the basis for a deeper study of the rutin-degrading enzyme.