CHARACTERIZATION AND PURIFICATION OF O-DIPHENOLASE FROM BULRUSH MILLET (PENNISETUM-TYPHOIDES STAPF AND HUBBARD)

The O-diphenolase in germinating millet (Pennisetum typhoides) was ext racted in cold buffer at pH 7.0 and subjected to some biochemical anal ysis. The best substrate for the enzyme was found to be catechol With an apparent K-m of 7.14 mM and a V-max Of 689.7 unit ml(-1), but it ha d moderate activity towards other diphenols and polyphenols such as pr otocatechuic acid, pyrogallol and 2,6 dihydroxy benzoic acid. No activ ity was detected towards monophenols (e.g tyrosine) while meta-substit uted phenols (e.g. resorcinol and phloroglucinol) were poorly utilised . L-cysteine inhibited the enzyme strongly; ascorbic acid and sodium m etabisulphite were moderate inhibitors; while EDTA did not inhibit the enzyme at 10 mM. Two pH optima at 5.0 and 7.5 and a sharp temperature profile With an optimum temperature of 50 degrees C were exhibited. W hen subjected to 65 degrees C treatment for 5-10 minutes, the enzyme l ost 10-20% of its initial activity while a residual 20% enzyme activit y remained after exposure to 80 degrees C for one hour. Through a comb ination of gel filtration and ion exchange chromatography, a purificat ion of 34 fold was achieved and the molecular weight of the enzyme was found to be 109.9 KD.