【作者】 周成刚；

【导师】 罗万春；

【作者基本信息】 山东农业大学 ， 农药学， 2008， 博士

【摘要】 酚氧化酶(Phenoloxidase,EC.1.14.18.1,简称PO)广泛存在于动物、植物和微生物等不同生物体内。该酶具有单酚酶(Monophenoloxidase,MPO)活性和二酚酶(Diphenoloxidase, DPO)活性,可催化单酚(Monphenols)羟化成二酚(如L-DOPA),并把二酚再度氧化成醌。它是昆虫体内的一种重要酶类,在昆虫的变态发育和免疫系统中起着重要作用。由于酚氧化酶具有重要的理论意义和发展前景,大量的研究工作集中在此领域,筛选、设计、合成酚氧化酶抑制剂成为研究热点。本文以2种鳞翅目Lepidoptera昆虫舞毒蛾Lymantria dispar、淡剑袭夜蛾Sidemia depravata和2种鞘翅目Coleoptera昆虫核桃扁叶甲Gastrolina depressa、华北大黑鳃金龟Holotrichia oblita四种园林昆虫酚氧化酶为试验对象,对其进行了部分纯化,并分别比较了同一目不同种类及异目昆虫酚氧化酶的基本酶学特性,初步探讨了几种金属离子与几种有机溶剂对这四种昆虫PO的影响,并在参考前人对农业害虫PO抑制剂以及对水果、蔬菜护色剂研究的基础上,选择了曲酸、槲皮素、4-十二烷基间苯二酚和4-己基间苯二酚作为效应物研究其对鳞翅目害虫——舞毒蛾幼虫酚氧化酶活性的抑制参数;并选择抗坏血酸和L-半胱氨酸对鳞翅目——淡剑袭夜蛾幼虫和鞘翅目——核桃扁叶甲成虫为酶源的酚氧化酶的抑制作用进行了测定,其中对高效抑制剂的抑制动力学进行了研究。结果表明:1.经不同饱和度硫酸铵分级沉淀,将舞毒蛾(Lymantria dispar)、核桃扁叶甲(Gastrolina depressa)、淡剑袭夜蛾(Sidemia depravata)和华北大黑鳃金龟( Holotrichia oblita)酚氧化酶部分纯化。鳞翅目昆虫舞毒蛾幼虫和淡剑袭夜蛾幼虫酚氧化酶的最大酶活力均存在于35%饱和度硫酸铵溶液沉淀中,两虫的最适pH值分别为6.5,7.0,其最适温度也不相同,分别为35℃和25℃。以邻苯二酚、焦性没食子酸和L-多巴作底物时,反应产物有吸收峰出现(特征吸收峰分别为410、334和465nm),而以对苯二酚、间苯二酚作底物时未见明显吸收峰。L-多巴、邻苯二酚和焦性没食子酸与酶的Km值依次为3.19、10.74和19.26 mmol·L-1。以邻苯二酚和L-多巴作底物时,反应产物有吸收峰出现(特征吸收峰分别为410和475nm),以对苯二酚、间苯二酚作底物时也未见明显吸收峰。以邻苯二酚和L-多巴为底物的淡剑袭夜蛾酚氧化酶酶促反应的米氏常数Km分别为1.44和37.49 mmol·L-1。鞘翅目昆虫核桃扁叶甲成虫和华北大黑鳃金龟成虫酚氧化酶的最大酶活力均存在于40%饱和度硫酸铵溶液沉淀中,其最适pH值分别为7.5、7.0,两虫最适温度相同均为40℃,以L-多巴和邻苯二酚为底物时,华北大黑鳃金龟和核桃扁叶甲酚氧化酶的Km值分别为3.02和61.25mmol·L-1,15.01和9.17 mmol·L-1。2.研究金属离子对四种昆虫酚氧化酶活力的影响,结果表明:K+和Na+对核桃扁叶甲成虫、淡剑袭夜蛾幼虫、华北大黑鳃金龟成虫PO的活性基本没有影响;但K+对舞毒蛾幼虫有轻微的抑制作用。Mg2+对舞毒蛾幼虫酚氧化酶有抑制作用;但对核桃扁叶甲成虫、淡剑袭夜蛾幼虫PO有激活作用;Mg2+在低浓度时对华北大黑鳃金龟成虫PO具有激活作用,高浓度时抑制。Ba2+能激活核桃扁叶甲成虫、淡剑袭夜蛾幼虫中的PO,但对华北大黑鳃金龟PO无显著的影响。Cu2+对舞毒蛾幼虫、淡剑袭夜蛾幼虫和华北大黑鳃金龟成虫PO的影响都是在低浓度时对其有激活作用,高浓度时表现为抑制作用作用。而Cu2+对核桃扁叶甲成虫酚氧化酶具有显著抑制作用。Zn2+对淡剑袭夜蛾幼虫PO有激活作用;但对核桃扁叶甲成虫PO有显著抑制作用;该离子在低浓度时对舞毒蛾幼虫和华北大黑鳃金龟成虫PO有激活作用,高浓度时表现为抑制作用作用。研究还发现Mn2+对淡剑袭夜蛾幼虫PO无显著的激活或抑制作用;但在低浓度时能激活华北大黑鳃金龟PO,高浓度又起抑制作用。3.研究有机溶剂对四种昆虫酚氧化酶活力的影响,结果表明:乙醚能显著的抑制淡剑袭夜蛾幼虫和华北大黑鳃金龟成虫PO的活性。甲醇对舞毒蛾幼虫、核桃扁叶甲成虫、淡剑袭夜蛾幼虫和华北大黑鳃金龟PO具有显著的抑制作用。乙醇和丙三醇能抑制核桃扁叶甲成虫和华北大黑鳃金龟成虫PO的活性,但是乙醇对淡剑袭夜蛾幼虫具有轻微的激活作用,丙酮对舞毒蛾幼虫和华北大黑鳃金龟成虫酚氧化酶有显著的抑制作用,但对淡剑袭夜蛾幼虫酚氧化酶无显著的激活或抑制作用。4.研究效应物对四种昆虫酚氧化酶活力的抑制作用,结果表明:以邻苯二酚为底物时,槲皮素和4-十二烷基间苯二酚都能显著的抑制舞毒蛾幼虫酚氧化酶活力,对舞毒蛾幼虫酚氧化酶活力的抑制中浓度(IC50)分别为0.076 mmol·L-1和0.372 mmol·L-1,并且这两种抑制剂都是舞毒蛾幼虫酚氧化酶的竞争性抑制剂,其抑制常数Ki分别为31.71 mmol·L-1和0.192 mmol·L-1。研究4HR和曲酸对四种昆虫酚氧化酶的抑制动力学,结果表明:4HR对该虫的单酚酶和二酚酶活性均表现很强的抑制作用,其IC50分别为0.00041 mmol/L和0.00035 mmol/L。其中,4HR对单酚酶活力表达的迟滞时间有明显的延长效应,浓度为0.0002 mmol/L时可使单酚酶活力表达迟滞时间从181 s延长到253 s;而当浓度为0.0005 mmol/L时,其迟滞时间则延长至372 s。以邻苯二酚为底物时,4HR对二酚酶的抑制作用表现为典型的竞争型抑制类型,抑制常数KI为0.00015 mmol/L。曲酸对舞毒蛾的单酚酶和二酚酶活性均表现较强强的抑制作用,其IC50分别为0.06 mmol/L和0.92 mmol/L。曲酸对单酚酶活力表达的迟滞时间也有明显的延长效应,浓度为0.1 mmol/L时可使单酚酶活力表达迟滞时间从306 s延长到732 s ;而当浓度为0.15mmol/L时,其迟滞时间则延长至900 s。以邻苯二酚为底物时,曲酸对二酚酶的抑制作用表现为典型的竞争型抑制类型,抑制常数KI为0.51 mmol/L。以邻苯二酚为底物时,核桃扁叶甲成虫酚氧化酶对抗坏血酸不甚敏感,抗坏血酸只能抑制该酶大约27%的相对活性,并且抗坏血酸对该酶的抑制作用具有可逆效应;但淡剑袭夜蛾幼虫酚氧化酶对抗坏血酸较为敏感,抗坏血酸对淡剑袭夜蛾幼虫酚氧化酶活力抑制中浓度(IC50)为0.36 mmol·L-1,且该抑制剂是淡剑袭夜蛾幼虫酚氧化酶的一种不可逆竞争性抑制剂,其抑制常数(Ki)为0.0768 mmol·L-1。以邻苯二酚为底物时,核桃扁叶甲成虫酚氧化酶对L-半胱氨酸非常敏感,L-半胱氨酸几乎可以完全抑制该酶的活性,并且该抑制剂是核桃扁叶甲成虫酚氧化酶的一种不可逆竞争性抑制剂,其抑制常数(Ki)为0.40 mmol·L-1。虽然淡剑袭夜蛾幼虫酚氧化酶对L-半胱氨酸也较为敏感, L-半胱氨酸对淡剑袭夜蛾幼虫酚氧化酶活力抑制中浓度(IC50)分别为0.0185 mmol·L-1,但淡剑袭夜蛾幼虫酚氧化酶的一种可逆竞争性抑制剂,其抑制常数(Ki)为0.0742 mmol·L-1。更多还原

【Abstract】 Phenoloxidase (PO, EC.1.14.18.1), is a copper-containing enzyme widely distributed in plants, microorganisms and animals. This multifunctional enzyme catalyzes two distinct reactions, the hydroxylation of monophenol to o-diphenol (monophenolase activity) and the conversion of o-diphenol to the corresponding o-quinone (diphenolase activity). 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 order to screen, design and synthesis PO inhibitors for the importance theory of PO inhibitors and its bright future.In the present paper, the kinetic properties of phenoloxidase from Lymantria dispar , Sidemia depravata , two kinds of Lepidoptera insect, Gastrolina depressa and Holotrichia oblita, two kinds of Coleoptera insects, were determined after the enzyme was partially purified by different staturated (NH4)2SO4 and Sephadex G-100 gel. The effects of some metal ions and several organic solvents on the activity of the PO were also studied. The inhibitory effects on the PO from Lymantria dispar activity by quercetin and 4-dodecylresorcinol were determined, and the mechanism of the two inhibitors were discussed also. The inhibitory effects of tetra-hexylresorcinol and kojic acids on the monophenolase and diphenolase activities of PO were also studied in the present paper. The inhibitory effects on the PO from Gastrolina depressa and Sidemia depravata activity by corbic acid and cysteine were determined, and the mechanism of the two inhibitors were discussed also. The results could be summarized as follows:1. The kinetic properties of phenoloxidase from Lymantria dispar, Sidemia depravata, two kinds of Lepidoptera insects, and Gastrolina depressa , Holotrichia oblita, two kinds of Coleoptera insects, were determined after the enzyme was partially purified by different staturated (NH4)2SO4. The results showed that the optimum pH of phenoloxidase from Lymantria dispar, Gastrolina depressa was 6.5, 7.0 and the optimum temperature was 35℃and 25℃, respectively. The kinetic parameter for the oxidation of L-DOPA, catechol and pyrogallol by PO from Lymantria dispar were determined, the Km were 3.19, 10.74 and 19.26 mmol·L-1, respectively. The kinetic parameter for the oxidation of L-DOPA and catechol by PO from Sidemia depravata were determined too, the Km were 37.49 and 1.44 mmol·L-1, respectively. The results also showed that the optimum pH of phenoloxidase from Gastrolina depressa and Holotrichia oblita were 7.5, 7.0, respectively and the optimum temperature was consistent 40℃. The kinetic parameter for the oxidation of L-DOPA and catechol by PO from Gastrolina depressa and Holotrichia oblita were determined too, the Km were 15.01 and 9.17 mmol·L-1, 3.02 and 61.25 mmol·L-1, respectively.2. The effect of some metal ions on the PO activity was studied. The results showed that alkali metal ions K+ and Na+ had no significantly influences on PO from Gastrolina depressa, Sidemia depravata and Holotrichia oblita activity, however K+ showed a slight inhibition role to PO from Lymantria dispar. Alkali-earth metal ion Mg2+ showed inhibition role to PO from Lymantria dispar and promotion role to PO from Gastrolina depressa and Sidemia depravata. Mg2+enhanced the enzyme activity when it was at the low concentration, but the activity was inhibited by Mg2+ when the concentration went over to 0.40 mmol·L-1. PO from Gastrolina depressa and Sidemia depravata activity were inhibited by Ba2+, but this ion had no significantly influences on PO from Holotrichia oblita activity. The activity of PO from Lymantria dispar, Sidemia depravata and Holotrichia oblita activation and inhibtion were observed at low and high concentration of Cu2+, but Cu2+ showed inhibition role to the PO from Gastrolina depressa activity. Zn2+ showed activation and inhibition role to the activity of PO from Sidemia depravata, Gastrolina depressa, respectively. Zn2+ played the same role as Cu2+ to the activity of PO from Lymantria dispar and Holotrichia oblita. Mn2+ showed no significantly influences on PO from Sidemia depravata. The activity of PO from Holotrichia oblita activation and inhibtion were observed at low and high concentration of Mn2+.3. The effect of some organic solvents on the PO activity was studied. The results showed that ethyl ether showed remarkable inhibition role to the activity of PO from Sidemia depravata and Holotrichia oblita. Ethanol and glycerol also showed remarkable inhibition role to the activity of PO from Gastrolina depressa and Holotrichia oblita, respectively. Ethanol can actived the activity of PO from Sidemia depravata slightly. The activity of PO from Lymantria dispar and Holotrichia oblita can be inhibited by acetone, however, acetone had neither inhibition nor activation to the activity of PO from Sidemia depravata.4. Quercetin and 4-dodecylresorcino showed remarkable inhibition role to the activity of PO from Lymantria dispar. The two inhibitors concentrations leading to 50% (IC50) activity lost were estimated to be 0.076 mmol·L-1 and 0.372 mmol·L-1 , respectively, using catechol as substrate. Both quercetin and 4-dodecylresorcino were reversible competitive inhibitors and the inhibitory constants (Ki) were determined to be 31.71 mmol·L-1 and 0.192 mmol·L-1 repectively. The inhibitory effects of tetra-hexylresorcinol and kojic acids on the phenoloxidase from Lymantria dispar were also stuided, using L-tyrosine or catechol as substrate. The results showed that the IC50 were estimated to be 0.00041 mmol/L for monophenolase activity and 0.00035 mmol/L for diphenolase, respectively. Tetra-hexylresorcinol extended the lagtime of the enzyme for oxidation of L-tyrosine. The lagtime extended from 181 s to 253s by 4-hexylresorcinol with 0.0002 mmol/L, and the lagtime extended from 181 s to 372s by the natural source compound with 0.0005 mmol/L. The results of inhibition kinetics analyzed by Lineweaver-Burk plots indicated that 4-hexylresorcinol was a competitive inhibitor for the oxidation of catechol and the inhibition constant was determined to be 0.00015 mmol/L.The results showed that the IC50 were estimated to be 0.06 mmol/L for monophenolase activity and 0.92 mmol/L for diphenolase, respectively. Kojic acids extended the lagtime of the enzyme for oxidation of L-tyrosine. The lagtime extended from 306 s to 702s by the kojic acids with 0.1 mmol/L, and the lagtime extended from 306 s to 900s by the compound with 0.15 mmol/L. The results of inhibition kinetics analyzed by Lineweaver-Burk plots indicated that kojic acids was a competitive inhibitor for the oxidation of catechol, and the inhibition constant was determined to be 0.51 mmol/L.PO from Gastrolina depressa was insensitive to ascorbic acid. The PO activity decreased with ascorbic acid showing a 27% most reduction as compared with no inhibitor in and scorbic acid was a reversible competitive inhibitors to this PO . PO from Sidemia depravata was sensitive to ascorbic acid which was a reversible competitive inhibitors for this PO and the Ki were estimated to be 0.0768 mmol·L-1. PO from Gastrolina depressa was sensitive to cystein and this inhibitor which was an irreversible competitive inhibitors and the inhibitory constants (Ki) were determined to be 0.40 mmol·L-1 can almost inhibited the activity completely. PO from Sidemia depravata was sensitive to cystein which was a reversible competitive inhibitors for this PO and the Ki were estimated to be 0.0742 mmol·L-1.更多还原