【作者】 王树栋；

【导师】 罗万春；

【作者基本信息】 山东农业大学 ， 农药学， 2005， 硕士

【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。该酶是昆虫体内的一种重要酶类,在昆虫的正常发育过程中具有重要的生理功能:（1）参与表皮的硬化和黑化;（2）对卵壳的鞣化作用;（3）参与伤害防御;及（4）加速伤口的愈合。研究昆虫酚氧化酶及其抑制剂对发展专化性的环境友好害虫控制剂具有重要意义。本论文就小菜蛾酚氧化酶初纯化方法、酶学特性及效应物对酶活力的影响等进行了研究。结果表明:1. 小菜蛾酚氧化酶酶活力存在于40%饱和度硫酸铵的沉淀中,回收率达68.09 %,该初纯化酶的比活力比粗酶液提高4.525 倍。经Sephadex G –100 凝胶过滤层析进一步纯化,测得酶制剂比活力为粗酶的6.526 倍,回收率为31.21%。2. 小菜蛾不同虫态及相同虫态不同龄期的酚氧化酶活力存在差异。幼虫期随着幼虫龄期增大酚氧化酶活力逐渐提高,化蛹后酶活力降低,以4龄幼虫酶活力最高。3. 研究小菜蛾酚氧化酶的酶学特性得出,酚氧化酶最适pH 值为6.5,pH 在6.0～7.0 范围内酶稳定性较好。最适温度为35℃,35℃以上可使该酶迅速失活,25℃～40℃范围内酶稳定性较好。4. 研究金属离子对酶活力的影响,结果表明:金属离子Mg²⁺、Mn²⁺和Zn²⁺对该酶二酚酶有不同程度的激活作用;K⁺、Ag⁺、Fe²⁺和Fe³⁺对二酚酶有不同程度的抑制作用,且Fe²⁺和Fe3+可以明显缩短该酶催化L-酪氨酸的迟滞时间,但对单酚酶稳态酶活力影响不明显;Cu²⁺ 和Cu⁺在低浓度范围内对酶都有不同程度的激活作用,但在较高浓度范围内对该酶却有一定的抑制作用;5. 研究有机溶剂对酶活力的影响,结果表明:甲醇、乙醇、丙酮和二甲苯对酶的催化作用表现不同的抑制作用,苯和二甲基亚砜在较低浓度范围内对酶均有一定的抑制作用,但在一确定浓度范围内有激活作用。6. 曲酸对小菜蛾酚氧化酶表现可逆抑制效应,为竞争型抑制类型。曲更多还原

【Abstract】 Phenoloxidase (PO, EC.1.14.18.1), is a copper-containing enzymewidely distributed in plants , microorganisms and animals. Thismultifunctional enzyme catalyzes two distinct reactions, the hydroxylation ofmonophenol to o-diphenol (monophenolase activity) and the conversion ofo-diphenol to the corresponding o-quinone (diphenolase activity). In insects,phenoloxidase is also a widely distributed enzyme playing important roles innormal developmental processes, such as cuticular tanning, scleration,wound healing, production of opsonins, and encapsulation and noduleformation for defense against foreign pathogens. In the present paper we havedone research on the purification of PO from the diamondback moth P.Xylostella (L.) (Lepidoptera Plutellidae), the properties of the enzyme and theeffects of some effectors on its activity. The results could be summarized asfollows:1. The PO was purified by (NH4)2SO4. Much of the activity was in thedeposition of 40% saturated (NH4)2SO4, PO was purified 4.525-fold with arecovery of 68.09%. After the enzyme was chromatographed on SephadexG-100 gel filtration, PO was purified 6.526-fold with a recovery of 31.21%.2. The activity of PO from P. Xylostella (L.) in different stages and instarswas different. The activity was gradualy improved with the instars increased,however, it was decreased in the pupal period. The enzyme activity of the 4thinstar larva was the highest.3. The properties of PO showed that the optimum pH was 6.5 and theenzyme would have a stable activity if the pH reaction system is between 6.5 ~7.0; The optimum temperature was 35℃, and the enzyme had a stableactivity if the temperature reaction system is between 25 ~ 40℃.4. The effect of some metal ions on the PO activity was studied. The更多还原