Properties Of Phenoloxidase(PO) From Pieris Rapae(L.) (Lipidoptera: Pieridae) And The Inhibitory Effect On Po Activity By Some Inhibitors

Phenoloxidase (EC.1.14.18.1, PO) is the main enzyme that is widelydistributed in animals, plants and microorganism, which is one of key enzymesin the development process of insects. The enzyme possesses an importantfunction in metamorphism developing and immunity system. In the presentpaper we have done some researches including comparison on properties ofPO from Pieris rapae (L.) in different stages and instars, and purification ofPO from the insect. Properties of the enzyme and effects of some effectors onthe activity of PO were also done. The results could be summarized asfollows: 1. The activity of PO from Pieris rapae (L.) in different stages and instarswas different. PO activity was gradualy improved with the instars increased,however, PO activity was gradually decreased with the pupae days increases.The enzyme activity of the 5th instar larva was the hightest and that of the 5thday pupae was the lowest. 2. The properties of PO from Pieris rapae (L.) in different stages andinstars were different and compared each other. The optimum temperature ofthe 3rd instar, the 4th instar, the 5th instar and the pupae were 36.0℃, 38.5℃,43.0℃ and 45.5℃, respectively. Meanwhile, the optimum pH was the samevalue in all of the stages and instars, and the optimum pH was determined tobe at 7.0, what's more, the energy of activation (Ea) for the oxidation ofcatechol were 43.10, 36.50, 25.79 and 30.10 kJ·mol-1, repectively. Theoptimum temperature, pH and the Ea for the oxidation of catechol all were thesame value in different days pupae, the optimum temperature was 50.0℃, theoptimum pH was 7.0, and the Ea was 58.8 kJ·mol-1. 3. The PO was purified by (NH4)2SO4. Much of the activity was in the 3薛超彬:菜青虫酚氧化酶的性质及抑制剂对其活性的抑制作用研究deposition of 35% saturated (NH4)2SO4, PO was purified 3.08-fold with arecovery of 69.52%. After the enzyme was chromatographed on SephadexG-100 gel filtration, PO was purified 6.22-fold with a recovery of 42.50%. 4. The properties of PO showed that the optimum pH was 7.0 and theenzyme had a stable activity if the pH reaction system between 6.5~7.4. Theoptimum temperature was 42℃, and the enzyme had a stable activity if thetemperature reaction system less than 32℃. The kinetic parameter for theoxidation of L-DOPA by PO was determined, the Km was 1.35 mmol·L-1, theEa was 42.77 kJ·mol-1. The Km was 19.20 mmol·L-1 for the oxidation ofcatechol by PO in this paper. 5. Studies the effects of some metal ions on the PO activity. The resultsshowed that K+,Li+ and Na+ had no any influence on the enzyme activity.Mg2+, Ba2+ and Ca2+ activated the PO and the activated order were Mg2+,Ca2+and Ba2+ from strong to weak. The transitional metal ions had differenteffect on the activity of the PO, Mn2+, Co2+ and Zn2+ were activators to theenzyme and Cu2+ enhanced the enzyme activity when it was at theconcentration of 0~0.10mmol·L-1, but the activity was inhibited by Cu2+ whenthe concectration went over to 0.13 mmol·L-1. 6. Studies the effects of some organic solvents on the activity of PO. Theresults showed that these solvents had inhibitory effects on the enzyme activity.The IC50 were 2.13, 2.04, 2.25, 1.06, 2.00 and 0.47mmol·L-1 by ethanol,propylene glycol, glycerol, propyl alcohol, acetone and dioxane, and the IC50were 3.39 and 1.00 μmol·L-1 by formaldehyde and glutaraldehyde,respecitively. 7. The inhibitory effects on the activity of PO by quercetin,4-hexylresorcino, 4-n-dodecylresorcino, benzoic acid and its familycompounds, benzaldehyde and cupferron were deterimined. (1) Quercetin was a reversible inhibitor of PO. The IC50 was0.13mmol·L-1. The inhibitory mechanism was competitive, and the inhibitoryconstant (KI) was determined to be 0.143mmol·L-1. (2) 4-hexylresorcino and 4-n-dodecylresorcino were reversible inhibitors 4山东农业大学硕士学位论文of P...