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

Authors: RAGHAVENDRA SATHYANARAYANA, VADLAPUDI KUMAR, CHAPEYIL KUMARAN RAMESH, MAHADEVAPPA PARMESHA, MAHABOOB HABEEBULLA MOINUDDIN KHAN

Abstract: Polyphenol oxidases (PPOs EC 1.14.18.1) were isolated from Mucuna pruriens and Mucuna prurita and confirmed as tyrosinases involved in L-DOPA production. PPOs were extracted by using a 0.05 M phosphate buffer, pH 7.0. The purified enzyme was resolved into a single band by PAGE, the enzyme was confirmed as PPO by activity staining, and SDS-PAGE analysis revealed that the purified PPO of both the species is a tetramer. Substrate specificity 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 effective against the PPOs of both the cultivars. The 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. The results suggest that the purified 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 purified fraction of M. prurita than it was in M. pruriens. The isolated enzyme can be further exploited for the overproduction of L-DOPA from in vitro cultures of the Mucuna species by biotechnology approaches.

Keywords: Mucuna pruriens, Mucuna prurita, polyphenol oxidase, L-tyrosine, L-DOPA

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