【作者】 张伟妮；

【导师】 黄小红；

【作者基本信息】 福建农林大学 ， 动物学， 2014， 博士

【摘要】 尼罗罗非鱼是我国最重要的养殖鱼类之一。“新吉富”尼罗罗非鱼是在吉富罗非鱼的基础上研发的具有自主知识产权的新品种,因具有卓越的生长优势被农业部在全国范围内推广。本论文对该鱼N-乙酰-β-D-氨基葡萄糖苷酶(NAGase)的性质进行了描述,并研究了外界环境条件对该酶性质的影响。经40%～55%的硫酸铵分级沉淀、DEAE纤维素(DE-32)离子交换层析、Sephadex G-200凝胶过滤层析、DEAE-Sephadex(A-50)层析柱从“新吉富”尼罗罗非鱼精巢组织分离纯化得到比活力为4100U·mg-1的NAGase,经电泳检测为单一纯。该酶的分子量为118.0kD,水解底物对-硝基苯-N-乙酰-β-D-氨基葡萄糖苷(pNP-NAG)的最适pH和最适温度分别为pH5.7和55℃。37℃条件下该酶的pH稳定范围为3.3-8.1,当温度高于45℃时该酶开始失活。该酶水解底物pNP-NAG遵循米氏方程,米氏常数(Km)和最大反应速度(Vm)分别为0.67mm01·L-1和23.26μm01-L-1·min-1。金属离子对“新吉富”尼罗罗非鱼NAGase的活力影响实验表明：Li+,Na+,K+,Mg2+和Ca2+等金属离子对该酶活力基本没有影响,重金属离子对该酶的抑制强弱顺序为Hg2+>Zn2+>Cu2+>Pb2+>Mn2+.Cu2+和Zn2+对该酶抑制的IC50分别为1.23和0.28mm01·L-1,其抑制机理均为可逆抑制,其中Cu2+的抑制类型为非竞争型,Zn2+的抑制类型为竞争型。这两种金属离子均能明显影响该酶的pH稳定性和热稳定性,也都能改变酶的构象从而影响到其内源荧光的发射。Hg2+对该酶呈强烈的不可逆抑制作用,IC50值仅为2.70μm01·L-1,而且Hg2+也能明显缩短该酶的热稳定和pH稳定范围。进一步通过底物反应的动力学方法研究了Hg2+对该酶的抑制动力学,建立了相应的动力学模型,经测定Hg2+对自由酶和酶与底物复合物的微观抑制速度常数k1和k’2分别为4.42×10-4mmol·L-1.s.-1and7.06×10-5mm01·L-1·s-1.说明在Hg2+的抑制作用下,底物的存在对于该酶有明显的保护作用。本文还测定了罗非鱼养殖过程中的一些常用药物对该酶活力的影响,在测定浓度范围内内服药物对罗非鱼NAGase的活力影响不大,盐酸强力霉素却对该酶有强烈的激活作用。而常用消毒剂强氯精和甲醛则对酶活力有明显的抑制作用,其IC50值分别为0.25mg.mL-1和3.2%。强氯精对该酶呈反竞争性可逆抑制作用,而甲醛呈竞争性可逆抑制。两种消毒剂均能明显缩短该酶的热稳定和pH稳定性。进一步建立了该酶在这两种消毒剂中的失活动力学模型,测定了其抑制速度常数。用嗜水气单胞菌322A腹腔感染健康“新吉富”尼罗罗非鱼,测定其内脏和血清NAGase比活力的变化规律,发现感染后第1,3,5,9,13,17,21d注射组的肝脏NAGase比活力明显高于对照组,呈极显著性差异(P<0.01)；肾脏NAGase比活力只在第1d和第3d极显著性高于对照组(P<0.01)；脾脏NAGase比活力与对照组没有显著性差异；血清中没有检测到NAGase活力。更多还原

【Abstract】 Nile tilapia(Oreochromis niloticus) is one of the most important farmed fish in China. New GIFT Nile tilapia is a new strain selected from GIFT (Genetically Improved Farmed Tilapia) by Shanghai Fishery University (China). It has been certified as a desirable strain by the National Certification Committee of Wild and Bred Varieties in January2006, and announced by the Ministry of Agriculture to expand in China because of its good growth performance. In this dissertation, we describe the enzymatic characterization and effects of environmental condition on its activity of NAGase from New GIFT Nile tilapia.N-acety1-/β-D-glucosaminidase (NAGase) from spermary of New GIFT Nile tilapia was purified to be PAGE homogeneous by the following techniques:(NH4)2SO4fractionation (40%-55%), DEAE-cellulose (DE-32) ion exchange chromatography, Sephadex G-200gel filtration and DEAE-Sephadex (A-50). The specific activity of the purified enzyme was4100U·mg-1. The enzyme molecular weight was estimated as118.0kD. The optimum pH and optimum temperature of the enzyme for hydrolysis ofpNP-NAG was to be at pH5.7and55℃, respectively. The enzyme was stable in a pH range from3.3to8.1at37℃, and inactive at temperature above45℃. Kinetic studies showed that the hydrolysis of p-Nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) by the enzyme followed Michaelis-Menten kinetics. The Michaelis-Menten constant (Km) and maximum velocity (Vm) were determined to be0.67mmol-L"1and23.26μmol·L-1min-1, respectively. The enzymatic characterizations of NAGase from tilapia were special to the other animals, which were correlated with its living habit.Effects of metal ions on tilapia NAGase had been testied. The enzyme activity was inhibited by the following heavy metal ions in decreasing order: Hg2+>Zn2+>Cu2+>Pb2+>Mn2+. However, the ions Li+, Na+5K+, Mg2+and Ca2+had almost no influence on enzyme activity. TheIC50of Cu2+and Zn2+were1.23and0.28mmol-L"1, respectively. They both inhibited the enzyme reversibly, while the inhibition type of Cu2+was noncompetitive and Zn2+was competitive. Both of the two metal ions had significant influence on the thermal and pH stability of the enzyme. Besides, Cu+and Zn+could both change the conformation of tilapia NAGase. Based on the results we got, CuSO4and ZnSO4should be used very carefully as insecticides in tilapia cultivation since they both had significant effects on the enzyme.In this paper, we determined the kinetics of HgCl2-mediated inhibition of NAGase, and our results showed that it was irreversible inhibition with an IC50value at2.70μM. Moreover, Hg2+reduced the thermal and pH stability of the enzyme. We determined the inhibition kinetics of Hg2+by using the kinetic method of substrate reaction. With this inhibition model, the microscopic rate constants for the reaction of Hg2+with free enzyme (k1) and the enzyme-substrate complex (K1) were determined to be4.42×10-4mM-1·s-1and7.06×10-5mM-1·s-1, respectively, indicating that the presence of substrate can protect NAGase from Hg2+inhibition.Effects of some medicines commonly used in tilapia cultivation were also studied. Some antibiotics tested had almost no influence on tilapia NAGase, except Doxycycline hydrochloride had activation. However, disinfectant such as Trichloroisocyanuric acid (TCCA) and formaldehyde could inhibit the enzyme. The IC50value of TCCA and formaldehyde were estimated to be0.25mg·mL-1and3.2%. Appropriate concentration of TCCA leaded to uncompetitive reversible inhibition on tilapia NAGase, while formaldehyde leaded to competitive reversible inhibition. Moreover, TCCA and formaldehyde could both reduce the thermal and pH stability of the enzyme. The inactivation models were both set up, and the rate constants were determined. The results might give some basis to TCCA and formaldehyde used in tilapia culture.Aeromonas hydrophila322A were intraperitoneal injected into healthy New GIFT Nile tilapia, specific activities of NAGase in entrails and serum were then tested. We found that specific activities of NAGase in livers of injected group were apparently higher than control group at1,3,5,9,13,17an21d post-injection (P<0.01); specific activites in kidneys were highly significant than control group at1and3d post-injection (P<0.01); however, there were no significant differences in specific activities of NAGase in spleens. NAGase activities in serum could not be detected.更多还原