凡纳滨对虾N-乙酰-β-D-氨基葡萄糖苷酶活力调控的研究
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摘要
凡纳滨对虾(Litopenaeus vannamei)是全世界三大养殖对虾中单位产量最高的虾种。N-乙酰-β-D-氨基葡萄糖苷酶(N-Acetyl-β-D-glucosaminidase,简称NAGase,EC.3.2.1.52)在对虾的蜕壳发育和营养代谢中发挥着重要作用。因此,研究凡纳滨对虾在患病过程的酶学变化,探讨环境污染物、饲料添加剂、水产养殖常用药物等对NAGase活力的影响与调控,以及对虾活体急毒性试验的开展,探究重金属离子对凡纳滨对虾NAGase活力的影响,将有利于对虾养殖业发展,为对虾养殖水环境污染监测提供更加充分、直观的科学依据,并填补国内外在这方面的研究空白。本文以凡纳滨对虾壳膜与内脏所提NAGase为对象,展开以下几方面的研究。
(1)跟踪考察分析健康和患红体病的凡纳滨对虾壳膜与内脏NAGase的活力和性质的差异。测得健康对虾壳膜NAGase的活力为34.80 U/mg,内脏NAGase的活力为35.34 U/mg;而患红体病对虾壳膜NAGase的活力为38.32 U/mg,内脏NAGase的活力为20.02 U/mg。结果表明:两种来源对虾的NAGase活力、基本酶学性质等均存在差异。对虾患红体病后,壳膜与内脏NAGase的催化反应动力学常数K_m和V_m值、活化能均较高,但其最适温度较低,pH稳定性及热稳定性较差。同时,对虾患病前后,壳膜与内脏来源的NAGase对外界各影响因子(Hg~(2+)、Zn~(2+)、Cu~(2+)、Cd~(2+)、pb~(2+)、Al~(3+)等重金属离子污染物:乙醇、甲醛、苯酚、甲醇、二氧六环、DMF、DMSO等有机污染;脲、NBS、SDS等常用化学修饰剂及变性剂)的敏感性也发生了变化。
(2)养殖水体常用消毒药物中,次氯酸钠、三氯异氰尿酸、季氨盐对凡纳滨对虾壳膜与内脏来源的NAGase的活力有不同程度的影响。同时,次氯酸钠、三氯异氰尿酸对两种来源的酶的抑制为不可逆过程。青霉素钾、链霉素、庆大霉素、卡那霉素等对壳膜与内脏来源的NAGas活力基本上没有影响;养殖常用的内服、外用抗菌药物中,盐酸环丙沙星对两种来源的酶有激活作用,而诺氟沙星和磺胺甲嗯唑对酶活力则有不同程度的抑制作用。磺胺甲嗯唑对壳膜NAGase表现为混合型可逆抑制,对内脏来源的NAGase表现为非竞争性抑制;诺氟沙星对内脏来源的NAGase为混合型可逆抑制。考察几种养殖常用维生素对内脏NAGase活力的影响,其中VB_(12)、烟酸、核黄素等对酶活力基本上没有影响;而VB_1、VB_6、抗坏血酸等对该酶活力均有不同程度的抑制作用,进一步研究了VB_6的抑制作用动力学,其对酶的抑制作用为非竞争性可逆抑制。
(3)对凡纳滨对虾进行养殖试验,研究重金属离子对对虾NAGase活力影响,在不同时间取样并考察Cu~(2+)、Zn~(2+)、Cd~(2+)、Pb~(2+)、Hg~(2+)等重金属离子对凡纳滨对虾壳膜及内脏NAGase活力的影响。结果显示5种重金属离子低浓度组在短时间内对凡纳滨对虾壳膜与内脏的NAGase活力没有显著影响,48h后随作用时间的延长,重金属离子使两种来源的NAGase活力均逐渐降低,表现为显著性抑制的剂量效应关系。
The production per unit of Litopenaeus vannamei tops among three major prawns in the world.And it is known that N-Acetyl-β-D-glucosaminidase(NAGase EC.3.2.1.52) plays a pivotal role in molting and nutritious metabolism of prawns. Hence,our study of enzymatic changes of prawns when sick,the effects of environmental pollutants,forage addition and common drugs used for aquatic breeding on the activities of NAGase,along with the study of acute toxic assays on prawn living bodies and the effects of heavy metal ions on activity of NAGase of prawns will benefit the prawn breeding industry and provide a more sufficient and direct scientific basis for monitoring the pollution in aquatic environment for culturing the prawn.Meanwhile,our study fulfills the blank in this field both domestically and internationally.Our study focuses on the NAGase extracted from Litopenaeus vannamei and falls into three categories:
(1) Study of differences of activities and properties of NAGase in prawns between health ones and the ones with "red body disease(RBD)".Our results show that the activities of NAGase in shell membrane and viscera of healthy prawns are 34.80 U/mg and 35.34 U/mg,respectively;while the activities of NAGase in shell membrane and viscera of prawns with RBD are 38.32 U /mg and 20.02 U/mg, respectively.This illustrate that there are differences between healthy prawns and the ones with RBD in activity and basic enzymatic properties of NAGase.Although catalytic reaction kinetic constant K_m and V_m value and activity energy of NAGase from the shell membrane and viscera of prawns with RBD are higher,the optimal temperature of NAGase is lower;also pH and thermal stabilities of NAGase are worse. At the same time,the sensitivity of NAGase changes between healthy prawns and prawns with RBD to outside factors,including heavy metal ions like Hg~(2+),Zn~(2+),Cu~(2+), Cd~(2+),Pb~(2+),Al~(3+);organic pollutants like ethanol,formaldehyde,phenol,methanol, dioxane,DMF and DMSO;common chemical dressing agent and denaturant like urea, NBS and SDS.
(2) Study of different effects of common antidote for aquatic bodies,such as sodium hypochlorite,symclosene and quaternary ammonium on the activity of NAGase from shell membrane and viscera of Litopenaeus vannamei.Meanwhile,the inhibitions of sodium hypochlorite and symclosene to these two different enzymes are irreversible.Moreover,our results show that benzylpenicillin sotassium,streptomycin, gentamicin and kanamycin have no effect on the activities of these two kinds of NAGase.Our results also show that among common antibiotics,ciprofloxacin activates these two kinds of NAGase while norfloxacin and sulfalene inhibit them. Sulfalene shows mixed reversible inhibition on NAGase from shell membrane while noncompetitive inhibition on NAGase from viscera;norfloxacin shows mixed reversible inhibition on NAGase from viscera.We also study the effects of several common vitamins on the activity of NAGase extracted from viscera.Our data illustrate that VB_(12),niacin and lactoflavin have no significant effect on the enzyme activity while VB_1,VB_6 and ascorbic acid inhibit it in distinct degrees.Further kinetic study shows that the inhibition of VB_6 on the enzyme is noncompetitive.
(3) Study of culture conditions for Litopenaeus vannamei,and different effects of heavy metal ions on NAGase activities.We study how different metal ions such as Cu~(2+),Zn~(2+),Cd~(2+),Pb~(2+),Hg~(2+) affect the activity of NAGase extracted from shell membrane and viscera of Litopenaeus vannamei.Our results show that no significant change in the activity of NAGase extracted from shell membrane and viscera of Litopenaeus vannamei treated with these five metal ions in short time.Nevertheless, the activities of these two NAGase decrease after 48 hours treatment with metal ions, which indicates a significant dose-dependent inhibition.
(1)跟踪考察分析健康和患红体病的凡纳滨对虾壳膜与内脏NAGase的活力和性质的差异。测得健康对虾壳膜NAGase的活力为34.80 U/mg,内脏NAGase的活力为35.34 U/mg;而患红体病对虾壳膜NAGase的活力为38.32 U/mg,内脏NAGase的活力为20.02 U/mg。结果表明:两种来源对虾的NAGase活力、基本酶学性质等均存在差异。对虾患红体病后,壳膜与内脏NAGase的催化反应动力学常数K_m和V_m值、活化能均较高,但其最适温度较低,pH稳定性及热稳定性较差。同时,对虾患病前后,壳膜与内脏来源的NAGase对外界各影响因子(Hg~(2+)、Zn~(2+)、Cu~(2+)、Cd~(2+)、pb~(2+)、Al~(3+)等重金属离子污染物:乙醇、甲醛、苯酚、甲醇、二氧六环、DMF、DMSO等有机污染;脲、NBS、SDS等常用化学修饰剂及变性剂)的敏感性也发生了变化。
(2)养殖水体常用消毒药物中,次氯酸钠、三氯异氰尿酸、季氨盐对凡纳滨对虾壳膜与内脏来源的NAGase的活力有不同程度的影响。同时,次氯酸钠、三氯异氰尿酸对两种来源的酶的抑制为不可逆过程。青霉素钾、链霉素、庆大霉素、卡那霉素等对壳膜与内脏来源的NAGas活力基本上没有影响;养殖常用的内服、外用抗菌药物中,盐酸环丙沙星对两种来源的酶有激活作用,而诺氟沙星和磺胺甲嗯唑对酶活力则有不同程度的抑制作用。磺胺甲嗯唑对壳膜NAGase表现为混合型可逆抑制,对内脏来源的NAGase表现为非竞争性抑制;诺氟沙星对内脏来源的NAGase为混合型可逆抑制。考察几种养殖常用维生素对内脏NAGase活力的影响,其中VB_(12)、烟酸、核黄素等对酶活力基本上没有影响;而VB_1、VB_6、抗坏血酸等对该酶活力均有不同程度的抑制作用,进一步研究了VB_6的抑制作用动力学,其对酶的抑制作用为非竞争性可逆抑制。
(3)对凡纳滨对虾进行养殖试验,研究重金属离子对对虾NAGase活力影响,在不同时间取样并考察Cu~(2+)、Zn~(2+)、Cd~(2+)、Pb~(2+)、Hg~(2+)等重金属离子对凡纳滨对虾壳膜及内脏NAGase活力的影响。结果显示5种重金属离子低浓度组在短时间内对凡纳滨对虾壳膜与内脏的NAGase活力没有显著影响,48h后随作用时间的延长,重金属离子使两种来源的NAGase活力均逐渐降低,表现为显著性抑制的剂量效应关系。
The production per unit of Litopenaeus vannamei tops among three major prawns in the world.And it is known that N-Acetyl-β-D-glucosaminidase(NAGase EC.3.2.1.52) plays a pivotal role in molting and nutritious metabolism of prawns. Hence,our study of enzymatic changes of prawns when sick,the effects of environmental pollutants,forage addition and common drugs used for aquatic breeding on the activities of NAGase,along with the study of acute toxic assays on prawn living bodies and the effects of heavy metal ions on activity of NAGase of prawns will benefit the prawn breeding industry and provide a more sufficient and direct scientific basis for monitoring the pollution in aquatic environment for culturing the prawn.Meanwhile,our study fulfills the blank in this field both domestically and internationally.Our study focuses on the NAGase extracted from Litopenaeus vannamei and falls into three categories:
(1) Study of differences of activities and properties of NAGase in prawns between health ones and the ones with "red body disease(RBD)".Our results show that the activities of NAGase in shell membrane and viscera of healthy prawns are 34.80 U/mg and 35.34 U/mg,respectively;while the activities of NAGase in shell membrane and viscera of prawns with RBD are 38.32 U /mg and 20.02 U/mg, respectively.This illustrate that there are differences between healthy prawns and the ones with RBD in activity and basic enzymatic properties of NAGase.Although catalytic reaction kinetic constant K_m and V_m value and activity energy of NAGase from the shell membrane and viscera of prawns with RBD are higher,the optimal temperature of NAGase is lower;also pH and thermal stabilities of NAGase are worse. At the same time,the sensitivity of NAGase changes between healthy prawns and prawns with RBD to outside factors,including heavy metal ions like Hg~(2+),Zn~(2+),Cu~(2+), Cd~(2+),Pb~(2+),Al~(3+);organic pollutants like ethanol,formaldehyde,phenol,methanol, dioxane,DMF and DMSO;common chemical dressing agent and denaturant like urea, NBS and SDS.
(2) Study of different effects of common antidote for aquatic bodies,such as sodium hypochlorite,symclosene and quaternary ammonium on the activity of NAGase from shell membrane and viscera of Litopenaeus vannamei.Meanwhile,the inhibitions of sodium hypochlorite and symclosene to these two different enzymes are irreversible.Moreover,our results show that benzylpenicillin sotassium,streptomycin, gentamicin and kanamycin have no effect on the activities of these two kinds of NAGase.Our results also show that among common antibiotics,ciprofloxacin activates these two kinds of NAGase while norfloxacin and sulfalene inhibit them. Sulfalene shows mixed reversible inhibition on NAGase from shell membrane while noncompetitive inhibition on NAGase from viscera;norfloxacin shows mixed reversible inhibition on NAGase from viscera.We also study the effects of several common vitamins on the activity of NAGase extracted from viscera.Our data illustrate that VB_(12),niacin and lactoflavin have no significant effect on the enzyme activity while VB_1,VB_6 and ascorbic acid inhibit it in distinct degrees.Further kinetic study shows that the inhibition of VB_6 on the enzyme is noncompetitive.
(3) Study of culture conditions for Litopenaeus vannamei,and different effects of heavy metal ions on NAGase activities.We study how different metal ions such as Cu~(2+),Zn~(2+),Cd~(2+),Pb~(2+),Hg~(2+) affect the activity of NAGase extracted from shell membrane and viscera of Litopenaeus vannamei.Our results show that no significant change in the activity of NAGase extracted from shell membrane and viscera of Litopenaeus vannamei treated with these five metal ions in short time.Nevertheless, the activities of these two NAGase decrease after 48 hours treatment with metal ions, which indicates a significant dose-dependent inhibition.
引文
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