STUDIES ON KINETIC PARAMETERS AND BIOCHEMICAL CHARACTERISTICS OF POLYPHENOL OXIDASE PURIFIED FROM JACKFRUIT (ARTOCARPUS HETEROPHYLLUS) WASTE

摘要

Objectives: Polyphenol oxidase activity was extensively studied in jackfruit for its role in enzymatic browning. PPO and the phenolic compound play a vital role in defensive mechanism against pest and diseases. Thus, to facilitate further studies in jack fruit waste, Polyphenol oxidase [PPO] was purified and characterized. Methods: Partial Purification of PPO from waste done through a sequential process of ammonium sulfate precipitation, dialysis and ion-exchange chromatography [DEAE- Cellulose]. Then the partially purified PPO was subjected to check various parameters like molecular weight and kinetic activity, the following characteristics of enzyme are checked: SDS-PAGE, pH, temperature, thermal stability, heat inactivation, metal ions, surfactants and inhibitor. Results: Purified PPO resulted in ~23 folds enriched in the specific activity of 1360 [μkat/mg] and it was found to be the monomer with a molecular weight of 63 kDa revealed by Coomasie Brilliant Blue staining. PPO exhibited optimum activity at pH 7.0 and temperature 20 o C. PPO showed the maximum stability between pH 6.4- 7.6 at 10 o C - 40 o C. PPO showed the enzyme activity towards Diphenol> Triphenol> Monophenol, the substrate specificity was especially high towards the catechol at 0.1 M. The PPO activity was activated by Mn 2+ , Triton X- 100, EDTA, Sorbic acid and Citric acid, but inhibited by L- cysteine, Ascorbic acid, SDS, Cetyl trimethyl ammonium bromide [CTAB], K + , Zn 2+ , Ca 2+ and Mg 2+ . Kinetic constant for PPO was found to be k m = 15.82 mM and V max = 2182 U/ml min using catechol as substrate. Conclusion: Partial Purification of PPO from waste done through a sequential process of ammonium sulfate precipitation, dialysis and ion-exchange chromatography [DEAE- Cellulose]. The best substrate for PPO was identified as catechol [diphenol] and best inhibitor was L-cysteine and ascorbic acid. Keywords : Polyphenol oxidase, PPO purification, Catechol, Ascorbic acid and L- Cysteine.