PURIFICATION AND CHARACTERIZATION OF THE QUINATE - OXIDOREDUCTASE FROM PHASEOLUS-MUNGO SPROUTS

Abstract

A quinate:oxidoreductase which catalyses the interconversion of (-)-quinic acid and 3-dehydroquinic acid, was extracted from the homogenate of Phaseolus mungo sprouts bought from a supermarket and purified by ammonium sulphate fractionation, anion exchange (DEAE-cellulose), hydrophobic interaction (Phenyl-Sepharose), affinity (Blue Sepharose) chromatography and gel filtration (FPLC Superose 12). Unlike most quinate:oxidoreductases of higher plants reported, which are absolutely NAD(H) specific, the enzyme from mungbean sprouts can use both NAD(H) and NADP(H) as a cofactor. Although the K(m)-values for NAD(H) and NADP(H) differ by one order of magnitude, no difference between the apparent maximum activities with NAD(H) and NADP(H) as cofactors is observed. The purified enzyme has a specific activity of about 0.5 mukat mg-1 protein (quinic to 3-dehydroquinic acid, pH 9.8, NAD) and 2.83 mukat mg -1 protein (3-dehydroquinic to quinic acid, pH 8.2, NADH). Upon gel filtration the enzyme has a M(r) of 160 000. SDS- PAGE of the peak fraction results in a single protein band of about 55 000. This result suggests that the enzyme might be a trimer.