南美白对虾酚氧化酶催化不同底物的生化特性研究

为揭示南美白对虾酚氧化酶（phenoloxidase,PO）催化不同底物时的生化特性,本文以L-多巴、甲基多巴、邻苯二酚为底物,分别测定南美白对虾酚氧化酶催化这三种底物时的最适温度、最适p H,酶促动力学常数K_m、V_max值、活化能。结果表明,南美白对虾PO酶催化三种底物的最适温度分别为45、40、30℃,最适p H分别为6.8、7.2、8.0,酶促动力学常数K_m分别为2.5003、5.8661、1.8429 mmol/L,V_max分别为0.0624、0.1008、0.1692ΔA/min,活化能Ea分别为15.2158、18.1981、10.4696 k J/mol;邻苯二酚与PO酶的结合能力较强,但其结合受外界温度的影响较大;而PO酶与L-多巴和甲基多巴的结合受外界温度影响较小。由此可得,底物结构越简单,越容易与PO酶活性中心结合;底物与酶的结合能力越强,酶活化底物时所需的活化能越小;在PO酶研究中,如果影响因素有温度这一条件时,不应该选择邻苯二酚作为底物。 

Biochemical characteristics of phenol oxidase in Penaeus vannamei on different substrates

To reveal the changes of biochemical characteristics( phenoloxidase,PO) of Penaeus vannamei in catalyze with different substrates. In this paper,L-dopa,methyldopa,catechol were taken as substrates,the optimum temperature of PO were measured by enzymatic oxidation with the three substrates,the p H optimum,enzyme kinetics constants K_m,V_max the value of activation energy were determined. The results showed that when PO enzyme catalyzed L-dopa,methyldopa and catechol,the optimum temperature were 45,40,30 ℃,the optimum p H was 6.8,7.2,8.0,the enzymatic kinetic constants were 2.5003,5.8661,1.8429 K_m mmol / L and V_max were 0.0624,0.1008,0.1692 ΔA / min,activation energy Ea were 15.2158,18.1981,10.4696 kJ / mol,respectively. Catechol binding with PO was stronger,but the combination was greatly influenced by the outside temperature,while the effects of temperature on PO combination with L-dopa and methyldopa were less.So it can be speculated that the simpler the substrate structure was the easier the combination with the active center of the PO; the stronger the enzyme substrate binding ability was the less activation energy was desired.In PO enzyme studies,if the temperature was included in factors,catechol should not be chosen as a substrate.