Phenoloxidase From Larva Anomala Corpulenta Motsch And Its Inhibitors

Phenoloxidase (PO, EC.1.14.18.1), tyrosinase or catechol oxidase, is a structure-complex and multifunctional copper-containing enzyme. PO is distributed in animals, plants, microorgnisms, insect and man, which localized in melanocytes. It is one of the key enzymes in the development process of insects, the enzyme possesses an important function in metamorphism developing and immunity system. Currently, many studies focused on this field.In the present paper, the kinetic properties of phenoloxidase from larva Anomala corpulenta Motsch one kind of Coleoptera insect, were determined after the enzyme was partially purified by35%(NH4)2SO4. The inhibitory effects on the PO from A. corpulenta Motsch activity by kojic acid, arbutin, morin, quercitin,4-hexylresorcino and4-n-dodecylresorcino were determined, and the mechanism of the four inhibitors were also investigated. The results could be summarized as follows:1. Properties of the PO from larvae of A. corpulenta Motsch purified by (NH4)2SO4were determined. The optimal temperature and pH of the enzyme for the oxidase of L-DOPA were determined to be at50℃and at pH7.5, respectively. The kinetic parameters for the oxidation of L-DOPA and catechol by the PO were11.11mmol·L-1and9.52mmol·L-1, respectively.2. Studies the inhibitory effects by kojic acid, arbutin, morin, quercitin,4-hexylresorcino and4-n-dodecylresorcino on the activity of PO. The results showed that these inhibitors had inhibitory effects on the enzyme activity. Kojic acid, morin, quercitin,4-hexylresorcino and4-n-dodecylresorcino exhibited sensitive. The IC50were0.65mmol·L-1,1.05mmol·L-1,0.15mmol·L-1,1.7μmol·L-1,1.2μmol·L-1, respectively.3. Studies the inhibitory effects by kojic acid, arbutin, morin, quercitin,4-hexylresorcino and4-n-dodecylresorcino on the activity of PO. The results showed that these inhibitors had reversible inhibitory effects on the enzyme activity.4. Studies the inhibitory effects mechanism by kojic acid, arbutin, morin, quercitin,4-hexylresorcino and4-n-dodecylresorcino on the activity of PO. The results showed that kojic acid, morin, quercitin,4-hexylresorcino and4-n-dodecylresorcino exhibited competitive inhibition and the inhibitory constants (KI) were determined to be1.59mmol·L-1,0.54mmol·L-1,0.112mmol·L-1,1.52μmol·L-1and1.0μmol·L-1, respectively. Arbutin exhibited noncompetitive inhibition and the inhibitory constants (KI) was2.98mmol·L-1.