Studies On Characterizations Of N-acetyl-β-D-glucosaminidase From New GIFT Nile Tilapia

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.