APPLICATION OF A NEW CONTINUOUS FLOW SPECTROPHOTOMETRIC METHOD FOR THE CHARACTERIZATION OF POLYPHENOL OXIDASE NATURALLY IMMOBILIZED ON COCONUT FIBER

Abstract

The association of continuous flow injection and spectrophotometry affords a simple, novel and rapid way of monitoring continuously the activity of naturally immobilized enzymes in their natural environment, thus eliminating cumbersome purification. The method was applied to determine the activity of polyphenol oxidase (PPO) enzymes naturally immobilized on coconut (Cocus nucifera, L.) fiber tissues. Maximum enzyme activity occurred at a temperature of 25C and at pH 6.0 using catechol as substrate. Thermal stability was assayed in a temperature range of 20 to 75C. The PPO exhibited excellent thermal stability, with only 50% loss in its activity at 75C after 4.3 min exposure. For catechol apparent Michaelis-Menten constant (apparent Km), apparent Vmax and the apparent activation energy were 9.1 × 10−3 mol L−1, 0.20 abs min−1 and 10.5 kcal mol−1, respectively. The immobilized PPO showed high activity for o-diphenols. The reactivity order was caffeic acid > pyrogallol > catechol. Complete inhibition of the enzyme was observed with 1 × 10−3 mol L−1 concentration of cyanide, thiourea, L-cysteine, ascorbic acid, sodium sulfite, nitrates of cadmium, zinc and mercury, individually. Benzoic acid, 3-hydroxy-benzoic acid, 4-acetamidephenol, sodium azide, resorcinol, L-cystine and EDTA at equal concentrations inhibited PPO partially.