Polyphenol oxidase Activity during Development of Saffron (Crocus sativus L.) Corm

Polyphenol oxidase (EC 1.14.18.1) (PPO) is a copper-containing enzyme which can be found throughout the phylogenetic tree. PPO is the rate-limiting enzyme in the melanin biosynthesis pathway. It hydroxylates monophenols and oxidizes diphenols to o-quinone. PPO activity was determined, using L-DOPA as substrate, in extracts prepared from saffron corms. Apparent Km and Vmax/mg prot. at pH 6.7 were, respectively, 4.3 mM and 6.6 x 10-5 mM/min, 4.1 mM and 2.9 x 10-5 mM/min, 4 mM and 0.45 x 10-5 mM/min, 4 mM and 6.9 x 10-5 mM/min, 3.7 mM and 6.5 x 10-5 mM/min, 5.1 mM and 17 x 10-5 mM/min and the catalytic efficiency, calculated per mg extract protein, was respectively 1.5 x 10-5, 0.7 x 10-5, 0.11 x 10-5, 1.7 x 10-5, 1.7 x 10-5 and 3.3 x 10-5 min-1 for extracts prepared from saffron corms cultivated in liquid medium for 0, 3, 6, 10, 20 and 30 days. The maximum substrate oxidation rate was found in extracts from corms grown for 30 days. PPO activity dropped by a factor of 2 after 3 days cultivation and then gradually increased to reach the initial value after 10 days cultivation and to almost triple after 30 days cultivation. Phenolic compounds are thought to be sequestered in cell vacuoles and simple phenols as well as most of the phenolic compounds are intermediates and derivates of the shikimate and phenyl propanoid pathways. The decrease in PPO activity at the beginning of development may be involved in these processes. Non-denaturing gel electrophoresis followed by activity staining showed at least 3 isoenzymes. Overall data suggest that various isoenzymes were differentially expressed during development in Crocus sativus corm.

Polyphenol oxidase Activity during Development of Saffron (Crocus sativus L.) Corm Keywords:

Kinetics , Kojic acid , L-DOPA , pH profile