Inactivation kinetics of formaldehyde on N-acetyl-ß-D-glucosaminidase from Nile tilapia (Oreochromis niloticus).

Formaldehyde is a widely used sanitizer in aquaculture in China, while the appropriate concentration is not available to be used effectively and without damage to tilapia much less to its reproductive function. N-acetyl-ß-D-glucosaminidase (EC 3.2.1.52, NAGase), hydrolyzing the oligomers of N-acetyl-ß-D-glucosamine into monomer, is proved to be correlated with reproduction of male animals. In this paper, NAGase from spermary of tilapia was chosen as the material to study the effects of formaldehyde on its activity in order to further investigate the effects of formaldehyde use on tilapia reproduction. The results showed the relationship between the residual enzyme activity and the concentration of formaldehyde was concentration dependent, and the IC50 value was estimated to be 3.2 ± 0.1 %. Appropriate concentration of formaldehyde leaded to competitive reversible inhibition on tilapia NAGase. Moreover, formaldehyde could reduce the thermal and pH stability of the enzyme. The inactivation kinetics of formaldehyde on the enzyme was studied using the kinetic method of substrate reaction. The inactivation model was setup, and the rate constants were determined. The results showed that the inactivation of formaldehyde on tilapia NAGase was a slow, reversible reaction with partially residual activity. The results will give some basis to determine the concentration of formaldehyde used in tilapia culture.

Irreversible inhibitory kinetics of mercuric ion on N-acetyl-ß-D-glucosaminidase from Nile tilapia (Oreochromis niloticus).

N-acetyl-ß-D-glucosaminidase (EC 3.2.1.52, NAGase), hydrolyzes dimers or trimers of N-acetyl-ß-D-glucosamine (NAG) into monomers and is shown to be important for the reproduction of male animals. NAGase is purified from the spermary of Nile tilapia, and its enzyme activity can be strongly inhibited by mercuric chloride (HgCl2). In this paper, we determined the kinetics of HgCl2-mediated inhibition of NAGase, and our results showed that it was irreversible inhibition with an IC50 value at 2.70±0.02 µM. Moreover, Hg(2+) reduced the thermal and pH stability of the enzyme. We determined the inhibition kinetics of Hg(2+) by using the kinetic method of substrate reaction. With this inhibition model, the microscopic rate constants for the reaction of Hg(2+) with free enzyme (k1) and the enzyme-substrate complex ( [Formula: see text] ) were determined to be 4.42×10(-4) mM(-1) s(-1) and 7.06×10(-5) mM(-1) s(-1), respectively, indicating that the presence of substrate can protect NAGase from Hg(2+) inhibition.

Enzymatic characterizations and activity regulations of N-acetyl-ß-D-glucosaminidase from the spermary of Nile tilapia (Oreochromis niloticus).

N-Acetyl-ß-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-ß-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.