Enzymatic characterizations and activity regulations of N-acetyl-β-d-glucosaminidase from the spermary of Nile tilapia (Oreochromis niloticus)

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: Hg2+ > Zn2+ > Cu2+ > Pb2+ > Mn2+. The IC50 of Cu2+, Zn2+ and Hg2+ was 1.23, 0.28, and 0.0027 mM, respectively. However, the ions Li+, Na+, K+, Mg2+ and Ca2+ 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.