PURIFICATION AND CHARACTERIZATION OF THE RUTIN-DEGRADING ENZYMES IN TARTARY BUCKWHEAT SEEDS

Two distinct rutin-degrading enzymes (RDEs) have been purified from tartary buckwheat (Fagopyrum tataricum) seeds. Both enzymes were purified by chromatography on DEAE-Sepharose, gel filtration on Sephacryl S-200 and chromatography on Mono-S columns. The highly purified RDEs I (early fraction in Mono-S chromatography) and II (late fraction) contained ca 14 and 11% carbohydrate, respectively. Both enzymes have the same optimal pH of 5, the same K-m value for rutin and are completely inactivated by incubation at 80 degrees for 30 min. The activities of both enzymes were strongly inhibited by CuSO4 (K-i=1.0 mM). RDEs I and II did not act on other flavonoids such as naringin and hesperidin. The purified RDEs yielded a single protein band upon SDS-PAGE. Both gel filtration and PAGE indicated M(r)s of 70 000 for both the native RDE I and II. SDS-PAGE of the enzyme proteins yielded a subunit band with a M(r) of 68 000. It is concluded that RDE I and II were composed of monomers.