Purification And Characteration Of Rutin-Degarding Enzyme In Tartary Buckwheat Seeds

Along with improvement of people's lives, hypertesion and hyperglycemia, hyperlipoidemia have become unavoidable problems of people health, because of their dangerous incidence of diseases, disability and death. During prevention and treatment of those diseases, bioflavonoid has been widely accepted, basing on its potential medicinal function. Tartary buckwheat is famous as"the most promising greenfood in 21st century"for its rich nutrition and special medicinal roles. Tartary buckwheat seeds contain rich bioflavonoid, 80% of which is rutin. With regards to ruitn's medical function, rutin has antioxidative, antihypertension, cardiovascular diseases activities. However, rutin-degrading enzyme(RDE)with high activity and stronge duration in tartary buckwheat seeds, to most extent, limits extraction, utilization and processing of rutin.Ammonium sulfate precipitation, phenyl sepharose CL-4B, CM-cellulose, gel filtration and electrophoresis were employed to purify RDE in tartary buckwheat seeds, with yield of 7.39% and purification fold of 13.15. Both PAGE and SDS-PAGE displayed singal band, with molecular weight of 66kD on SDS-PAGE. Efficient poly-cloned antibody (1:10000) of RDE was preparaed by using purified RDE to immunize healthy rabbits, which laid foundations of further research on physiological roles of RDE. N-terminal peptide sequence was sequenced: TVSRSSFPDGFLFGL. Compared with the known proteins database by the BLAST program, the sequence did not show significant similarity to any other protein seqeunce. Besides, MALDI-TOF-MS also demonstrated no similar proteins reported previously.To study thermal and pH change of RDE, the purified RDE had activity from pH 3.0 to pH 8.0, along with optimal pH of 5.0. In the range of 20℃to 70℃, the purified RDE also presented its enzymatic activity with optimal temperature of 50℃. With respect to kinatics of RDE, its Km and Vmax were 38.4U mg-1 and of 0.16M, respectively. Cu²⁺, Al²⁺, Zn²⁺, Mn²⁺ and EDTA inhibited the RDE to different degrees. Moreover, in this paper, modified SDS-PAGE was introduced into detect RDE activity, resulting in improving resolution.This paper established a method to purify RDE in tartary buckwheat seeds and further studied on its kinetics and stability. All results provide a preliminary research and discussion on cloning RDE gene, studying rutin metabolism and physiological roles of RDE in tartary buckwheat.