Abstract: This paper studied the character of N-acetyl-β-D-glucosaminidase (NAGase) from the sperm of Putian Black pig. The purification steps involved the following procedures: Ammonium sulfate precipitation, anion-exchange chromatography on DEAE Sepharose Fast Flow, gel filtration chromatography on Sephadex G100. The purified enzyme preparation was homogeneous judged by polyacrylamide gel electrophoresis. It was found that the specific activity of the enzyme was 1561.42 U/mg. The enzyme molecular weight was estimated as 58 ku. The optimal pH value was 5.6 and the optimal temperature was 45 ℃. The enzyme was stable in the pH ranges of 3.6 to 7.8. The enzyme followed typical Michaelis-Menten kinetics for the hydrolysis of pNP-GlcNAc and the K_m and V_m values were determined to be 0.82 mmol/L and 39.23 μmol/(L·min), respectively. The activation energy of the enzyme for hydrolysis of pNP-GlcNAc was 27.30 kJ/mol. The effects of some metal ions on the enzyme activity were determined, Na⁺, K⁺, Mg²⁺, Ca²⁺ had no effects on the enzyme activity, while Zn²⁺, Cu²⁺, Pb²⁺ had inhibitory effect on the enzyme activity.

Key words: Putian Black pig sperm; N-acetyl-β-D-glucosaminidase; isolation and purification; kinetics; metalion