Properties of Phenoloxidase (PO) from Different Insects in Garden and Inhibitory Effect on PO Activity by Some Inhibitors

The phenoloxidase (Phenoloxidase, EC.1.14.18.1, referred PO) widely exist in different animals, plants and micro-organisms in vivo. The enzyme has activity of the monophenolase (Monophenoloxidase, MPO) activity and the diphenol enzyme (Diphenoloxidase, DPO), catalytic monophenol (Monphenols), hydroxylation diphenols (such as L-DOPA), and the diphenol re-oxidized into quinones. It is an important enzymes of insects, insect metamorphosis and immune system plays an important role. Phenoloxidase has important theoretical significance and prospects for the development, a lot of research work is concentrated in this area, screening, design, synthetic phenol oxidase inhibitors become a hot topic. In this paper, the the 2 kinds Lepidoptera Lepidoptera insects gypsy moth Lymantria dispar, Sidemia armyworm Sidemia depravata and the two kinds of Coleoptera Coleoptera insects walnut flat leaf beetle Gastrolina depressa, the the North China black Melolonthidae of Holotrichia oblita four garden insects phenol oxidase test object, its partially purified and were compared to the same mesh of different types and different insects phenol oxidase enzymatic properties of several metal ions with several organic solvents explore these four insects PO Reference predecessors on the basis of agricultural pests the PO inhibitor as well as fruits, vegetables, color fixative, select kojic acid, quercetin, 4 - dodecyl resorcinol and 4 - hexyl hydroquinone as effectors of lepidopteran pests - gypsy moth larvae phenoloxidase activity suppression parameter; choose ascorbic acid and L-cysteine ??lepidopteran - Sidemia the armyworm larvae and Coleoptera Head - walnut of flat leaf beetle adults as the enzyme source phenol oxidase inhibition were measured, which were efficient inhibitors inhibition kinetics studies. The results showed that: 1. Different saturation ammonium sulfate precipitation, the gypsy moth (Lymantria dispar), walnut flat the Chrysomelidae (Gastrolina depressa) Sidemia armyworm (Sidemia depravata) and North China big black gill beetles (Holotrichia oblita ) phenol oxidase partially purified. Lepidopteran insects gypsy moth larvae and Sidemia the armyworm larvae phenol oxidase enzyme present in the 35% saturation ammonium sulfate solution precipitated, the two insects optimum pH values ??were 6.5, 7.0, the optimum The temperature is not the same, 35 ℃ and 25 ℃, respectively. Catechol, naphthalene, no gallic acid and L-dopa as a substrate, the reaction product of the absorption peak (characteristic absorption peaks at 410,334 and 465 nm), hydroquinone, resorcinol, no obvious absorption peak as substrate. L-dopa, catechol and coke did Km values ??of gallic acid and enzyme order 3.19,10.74 and 19.26 mmol · L-1. Catechol and L-dopa as a substrate, the reaction product absorption peak (characteristic absorption peaks at 410 and 475nm), hydroquinone, resorcinol as a substrate, no significant absorption peak. The short sword catechol and L-dopa as substrate passage of armyworm phenol oxidase enzymatic reaction Michaelis constant Km were 1.44 and 37.49 mmol · L-1. Coleoptera insects walnut the flat leaf beetle and North China big black gill the beetle adults phenol oxidase enzyme present in the 40% saturation ammonium sulfate solution precipitated its optimum pH 7.5,7.0, two insects optimal the same temperature are 40 ℃, with L-dopa and catechol as substrate the North China a big black Melolonthidae and walnut of flat leaf cresol oxidase Km values ??were 3.02 and 61.25mmol L-1, 15.01 and 9.17 mmol · L-1. 2. study of four insect phenoloxidase activity of metal ions, the results showed that: K and Na walnut flat leaf beetle, Sidemia armyworm larvae, North China big black gill beetle adults PO activity had no effect; However, the K mild inhibition of gypsy moth larvae. Mg2 gypsy moth larvae phenol oxidase inhibition; walnut flat leaf beetle, Sidemia armyworm larvae PO activation; to China the Beida black Melolonthidae adult Mg2 at low concentrations PO activation, high concentration when suppressed. Ba2 walnut flat leaf beetle can activate Sidemia PO Budworm, but China the Beida black Melolonthidae PO no significant impact. Cu2 on gypsy moth larvae, armyworm larvae of Sidemia and North China the black Melolonthidae adult PO impact are in the low concentrations activate the role of the high concentration inhibition role. Cu2 walnut flat leaf beetle phenol oxidase significantly inhibited. Activate the role of the Zn2 pair Sidemia armyworm larvae PO; However, significant inhibition of walnut the flat leaf beetle adults PO; ions at low concentrations of gypsy moth larvae and the North China black Melolonthidae adult PO activation, high concentration When inhibition role. The study also found that Mn2 on short sword the passage of armyworm larvae PO no significant activation or inhibition; However, at low concentrations can activate the North China big black Melolonthidae PO high concentration was underway inhibition. 3. Study of organic solvents four insect phenoloxidase activity results: the ether can significantly suppression Sidemia of armyworm larvae and North China large black gills beetle adults of PO activity. The methanol walnut flat leaf beetle, Sidemia armyworm larvae of gypsy moth larvae, and North China big black Melolonthidae PO has a significant inhibitory effect. Acetone ethanol and glycerol can inhibit walnut flat leaf beetle and North China black Melolonthidae adult PO activity, but has a minor role in the activation of ethanol on Sidemia armyworm larvae of the gypsy moth larvae and North a big black Melolonthidae adult The phenoloxidase there a significant inhibitory effect, but no significant activation or inhibition of Sidemia armyworm larvae phenoloxidase. 4. Research effector four insect phenoloxidase enzyme inhibition, the results showed that: catechol as substrate, quercetin and 4 - dodecyl resorcinol can significantly inhibit the dance moth larvae phenoloxidase activity, suppression of gypsy moth larvae phenoloxidase activity concentration (IC50) were 0.076 mmol · L-1 and 0.372 mmol · L-1, and these two inhibitors are gypsy moth larvae phenol the competitive inhibitor oxidase inhibition constant Ki 31.71 mmol · L-1 and 0.192 mmol · L-1, respectively. Research 4HR and kojic The results show that: of the 4HR of the pest monophenolase diphenol activity of inhibition kinetics of of four insects phenol oxidase showed strong inhibition IC50 of 0.00041 mmol / L and 0.00035 mmol / L. Wherein 4HR monophenol expression of enzyme activity, the lag time significantly prolonged effect, a concentration of 0.0002 mmol / L monophenol enzyme activity can be expression of the lag time from the extended to 253 s 181 s; while a concentration of 0.0005 mmol / L when the lag time was extended to 372 s. Catechol as substrate, 4HR diphenols enzyme inhibition performance of typical competitive type of inhibition, inhibition constant KI 0.00015 mmol / L Kojic acid showed strong inhibition of the gypsy moth monophenolase diphenols activity of IC50 of 0.06 mmol / L and 0.92 mmol / L. Kojic acid significantly extend the effect of the the monophenol enzyme expression hysteresis time, the concentration of 0.1 mmol / L the monophenol enzyme can express the hysteresis time from 306 s extended to 732 s; When the concentration of 0.15mmol / L , the lag time was extended to 900 s. Catechol as substrate, kojic acid and of diphenols enzyme inhibition performance for typical competitive type of inhibition, inhibition constant KI of 0.51 mmol / L Catechol as substrate, walnut flat leaf beetle phenol oxidase is not very sensitive for ascorbic acid, ascorbic acid, only about 27% of the enzyme inhibition relative activity and ascorbic acid of the enzyme inhibition with reversible effects; but Sidemia the armyworm larvae phenol oxidase of ascorbic acid are more sensitive ascorbic acid on Sidemia armyworm larvae phenol oxidase inhibition concentration (IC50) of 0.36 mmol · L-1, and the inhibitor is Sidemia armyworm larvae phenoloxidase a competitive inhibitor of irreversible inhibition constant (Ki) of 0.0768 mmol · L-1. Catechol as substrate, walnut flat leaf beetle phenol oxidizing enzyme is very sensitive to L-cysteine, L-cysteine ??can be almost completely inhibit the enzyme activity, and the inhibitor is walnut flat The leaf beetle phenol oxidase an irreversible competitive inhibitor, and the inhibition constant (Ki) of 0.40 mmol · L-1. Although of Sidemia armyworm larvae phenoloxidase is also more sensitive to L-cysteine, L-cysteine ??inhibition concentration (IC50) phenoloxidase activity on Sidemia armyworm larvae were 0.0185 mmol · L -1, but of Sidemia armyworm larvae phenoloxidase a reversible competitive inhibitor, and the inhibition constant (Ki) of 0.0742 mmol · L-1.