PARTIAL PURIFICATION OF POLYPHENOL OXIDASE FROM PLUMS (Prunus domestica L., cv. STANLEY)

Polyphenol oxidase (PPO) was extracted and purified from Stanley plums (Prunus domestica L.) Crude PPO showed pH optima of 5.8 to 6.4 with different substrates. Heating for 5 min at 75C completely inactivated this enzyme. Plum PPO was stable at -20C for 16 weeks. K_mof this enzyme ranged from 17.5 mM with 4-methylcatechol to 31.2 mM with chlorogenic acid. The enzyme was purified 36-fold through (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose and Sephadex G-100. PAGE of crude and purified plum PPO showed 7 and 3 bands, respectively, when stained for activity with catechol. The molecular weight of 3 subunits of purified PPO was estimated in the range of 45–66 kD.     

CHARACTERISTICS OF γ-GLUTAMYL TRANSPEPTIDASE AND ALLIINASE OF ONION AND THEIR EFFECTS ON THE ENHANCEMENT OF PYRUVATE FORMATION IN ONION MACERATES

The activity of alliinase monitored in onions stored for 32 weeks at ambient temperature (mean value of 20C) only slightly increased whereas the activity of γ-glutamyl transpeptidase increased gradually. Purified alliinase and transpeptidase showed single protein bands on SDS-PAGE, with molecular weights between protein markers of 40–69 kDa and 116–205 kDa, respectively. Molecular weights of native transpeptidase and alliinase determined by Sephadex G-200 column chromatography were estimated to be 120 and 200 kDa, respectively. Transpeptidase and alliinase had isoelectric points of 5.2 and 4.8, pH optima of 9.0 and 7.5, temperature optimum of 40C and activation energies (E_a) of 15.8 and 16.6 kJ/mol, respectively. Inhibitor studies suggested that the conditions for optimal alliinase activity did not depress the activity of transpeptidase. Transpeptidase in conjunction with alliinase enhanced pyruvate production in onion macerates over and above that catalyzed by either enzyme alone.