University Key Lab for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
School of Life Sciences, Xiamen University, Xiamen 361005, China.
J Biosci Bioeng. 2014 Feb;117(2):153-157. doi: 10.1016/j.jbiosc.2013.07.014. Epub 2013 Sep 5.
N-Acetyl-β-D-glucosaminidase (NAGase) is proved to be correlated with reproduction of male animals. In this study, enzymatic characterizations of NAGase from spermary of Nile tilapia (Oreochromis niloticus) were investigated in order to further study its reproductive function in fish. Tilapia NAGase was purified to be PAGE homogeneous by the following techniques: (NH4)2SO4 fractionation (40-55%), DEAE-cellulose (DE-32) ion exchange chromatography, Sephadex G-200 gel filtration and DEAE-Sephadex (A-50). The specific activity of the purified enzyme was 4100 U/mg. The enzyme molecular weight was estimated as 118.0 kD. 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 be 0.67 mM and 23.26 μM/min, respectively. The optimum pH and optimum temperature of the enzyme for hydrolysis of pNP-NAG was to be at pH 5.7 and 55°C, respectively. The enzyme was stable in a pH range from 3.3 to 8.1 at 37°C, and inactive at temperature above 45°C. The enzyme activity was regulated by the following ions in decreasing order: Hg(2+) > Zn(2+) > Cu(2+) > Pb(2+) > Mn(2+). The IC50 of Cu(2+), Zn(2+) and Hg(2+) was 1.23, 0.28, and 0.0027 mM, respectively. However, the ions Li(+), Na(+), K(+), Mg(2+) and Ca(2+) had almost no influence on enzyme activity. In conclusion, the enzymatic characterizations of NAGase from tilapia were special to the other animals, which were correlated with its living habit; besides, CuSO4 and ZnSO4 should used very carefully as insecticides in tilapia cultivation since they both had strong regulations on the enzyme.
N-乙酰-β-D-氨基葡萄糖苷酶（NAGase）已被证明与雄性动物的生殖有关。在这项研究中，我们对来自尼罗罗非鱼（Oreochromis niloticus）精巢的 NAGase 的酶学特性进行了研究，以便进一步研究其在鱼类中的生殖功能。通过以下技术将尼罗罗非鱼 NAGase 纯化至 PAGE 均一：（NH4）2SO4 分级（40-55%）、DEAE-纤维素（DE-32）离子交换层析、Sephadex G-200 凝胶过滤和 DEAE-Sephadex（A-50）。纯化酶的比活为 4100 U/mg。酶分子量估计为 118.0 kD。动力学研究表明，该酶对 p-硝基苯基-N-乙酰-β-D-氨基葡萄糖苷（pNP-NAG）的水解遵循米氏动力学。米氏常数（Km）和最大速度（Vm）分别确定为 0.67 mM 和 23.26 μM/min。该酶水解 pNP-NAG 的最适 pH 和最适温度分别为 pH 5.7 和 55°C。该酶在 37°C 时在 pH 3.3 至 8.1 的范围内稳定，在 45°C 以上时无活性。Hg(2+)>Zn(2+)>Cu(2+)>Pb(2+)>Mn(2+) 离子对酶活性的调节作用依次降低。IC50 值分别为 1.23、0.28 和 0.0027 mM。然而，Li(+)、Na(+)、K(+)、Mg(2+)和 Ca(2+) 离子对酶活性几乎没有影响。总之，尼罗罗非鱼 NAGase 的酶学特性与其他动物的特性不同，这与其生活习性有关；此外，在罗非鱼养殖中，硫酸铜和硫酸锌应谨慎用作杀虫剂，因为它们对酶都有很强的调节作用。
