Characterization of poly phenol oxidase in two in vitro regenerated cultivars of Mucuna: Mucuna pruriens L. and Mucuna prurita H

摘要

Polyphenol oxidases (PPOs EC 1.14.18.1) were isolated from Mucuna pruriens and Mucuna prurita and confi rmed as tyrosinases involved in L-DOPA production. PPOs were extracted by using a 0.05 M phosphate buff er, pH 7.0. The purifi ed enzyme was resolved into a single band by PAGE, the enzyme was confi rmed as PPO by activity staining, and SDS-PAGE analysis revealed that the purifi ed PPO of both the species is a tetramer. Substrate specifi city experiments were carried out with catechol, L-DOPA, L-tyrosine, and p-cresol. Of these, catechol was evaluated as the most suitable substrate based on the determined K_m and V_(max) values. The optimum pH and temperature were determined to be 6.5 to 7.0 and 30 °C, respectively, with catechol as substrate. Inhibitor studies were carried out and, of the 6 inhibitors tested, L-ascorbic acid, citric acid, L-cysteine, and potassium cyanide were the most eff ective against the PPOs of both the cultivars. Th e PPOs have both monophenol and polyphenol oxidase activities, with low K_m and high V_(max) values for catechol, p-cresol, and L-tyrosine, and high K_m and low V_(max) values for L-DOPA. Th e results suggest that the purifi ed PPO forms isolated from 2 Mucuna species in the present study showed an affinity towards not only both catechol and p-cresol, but also L-tyrosine, confirming that the isolated PPO is tyrosinase and it might be responsible for the L-DOPA production in the Mucuna species. The comparative studies reveal that enzyme activity was slightly greater in crude extracts of M. pruriens compared to crude extracts of M. prurita, while the fold purity was greater in a partially purifi ed fraction of M. prurita than it was in M. pruriens. Th e isolated enzyme can be further exploited for the overproduction of L-DOPA from in vitro cultures of the Mucuna species by biotechnology approaches.