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

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. | R. SATHYANARAYANA, V. KUMAR, C. K. RAMESH, M. PARMESHA, M. H. M. KHAN Turk J Biol 35 (2011) 575-583 © TÜBİTAK doi:10.3906/biy-0912-26 Characterization of poly phenol oxidase in two in vitro regenerated cultivars of Mucuna: Mucuna pruriens L. and Mucuna prurita H. Raghavendra SATHYANARAYANA1,*, Vadlapudi KUMAR1, Chapeyil Kumaran RAMESH2, Mahadevappa PARMESHA2, Mahaboob Habeebulla Moinuddin KHAN3 1 Department of Biochemistry, (Kuvempu University-P.G. Centre) Davangere University, Shivagangothri, Davangere - 577 002, Karnataka - INDIA 2 Department of Biotechnology, Sahyadri Science College, Shivamogga, 577203, Karnataka - INDIA 3 Department of Chemistry, Jawaharlal Nehru National College of Engineering, 577201 - SHIVAMOGGA Received: 14.12.2009 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 Km and Vmax 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 Km and high Vmax values for catechol, p-cresol, and L-tyrosine, and high Km and low Vmax values for L-DOPA. The results suggest that the purified PPO forms isolated from 2 Mucuna species in the present study showed