The Optimized Purification Method's Of Rutin-Degrading Enzyme In Tartary Buckwheat And Its Metabolic Profile During Seed Germination Process

Tartary buckwheat is rich in bio-flavonoids,which give the plant high medicinal value.Being the main component of tartary buckwheat flavonoids, rutin has accessory treatment effect for hypertension,hyperlipidemia,heart disease, stroke and gastricism. However, tartary buckwheat powder contains highly active rutin-degrading Enzyme, which have strong pH and thermal stabilities. Rutin-degrading enzyme catalyzes specificly the hydrolysis of rutin.So in tatary buckwheat food processing, this enzyme can cause rutin degradation, thus reduce its medicinal value significantly. In this paper, we conducted a systematic study about the testing and purification methods of rutin-degrading enzyme and its metabolic profiles during tartary buckwheat seed germination process. We obtained the following results:1 Based on solubility difference between quercetin and rutin, We establish the method using PAGE active staining to detect rutin-degrading enzyme activity.The rssults showed the method can detect the activities of rutin-degrading enzyme and its isozymes efficiently, there are at least four rutin-degrading enzyme isozymes present in tartary buckwheat seeds. Then we use different sample loadings of purified Rutin-degrading Enzyme to test this method, the resluts proved that isozyme staining intensity is directly proportional with the content to rutin-degrading enzyme content. and the intensity of hybridization signal was consistent with the activity changing of Rutin-degrading Enzyme. The content of total flavonoids increased during the whole process.2 By studying the metabolic profiles of Rutin-degrading Enzyme during Seed Germination Process of Tartary Buckwheat,wo found the Rutin-ding Enzyme distributes both in embyro and endotesta.During the sprouting process, the activity of Rutin-degrading Enzyme did not change significantly during the first three days. there was a sharp decrease in the forth day,,and Rutin-degrading Enzyme activity was disappear in the fifth day when the embryo was completely absorbed by the young leaf. But activity of Rutin-degrading Enzyme kept highly active in the endotesta during all sampling period.PAGE analysis demonstrated that Rutin-degrading Enzyme has three isozymes in embyro,and the isozymes disappeared as the activity of Rutin-degrading Enzyme decreased in the forth day, while Rutin-degrading Enzyme has three isozymes during the whole germination process in endotesta. Rutin-degrading Enzyme protein was detected by western blotting using polyclonal antibody of rutin-degrading enzyme prepared by our laboratory,3 Tolerability experiment of rutin-degrading enzyme was conducted using different concentrations of methanol. The result showed that, when methanol concentration is less than 50%, activity of rutin-degrading enzyme was not influenced. The activity of rutin-degrading enzyme begins to decrease shrply when methanol concentration increased to 60% and vanishes at 70% methanol concentration. Based on this result, we mix crude enzyme extraction, which was extracted by 10 times volume of acetic acid buffer solution (pH 5.0,0.2 mol/L), with the same volumn of methanol to precipitate the protein impurities, cation exchange chromatography and the gelatin filtration chromatography were then utilized to purified the 58kD rutin-degrading enzyme. SDS-PAGE and Western blotting analysis confirmed the reliability of this purification method. This purification system simplified our labrotary's former rutin-degrading enzyme purification route.Through this study,we established the method use PAGE to detect Rutin-degrading enzyme, analyzed the metabolic profiles of Rutin-degrading Enzyme during seed germination process of tartary buckwheat,and reported isozymes of Rutin-degrading Enzyme for the first time.A new operative purification route was established by the tolerability experiment of rutin-degrading enzyme with methanol.These results provided background information and technical support for revealing the metabolism and biological function of Rutin-degrading Enzyme in the further research.