2Department of Animal Science, School of Life Sciences, Foshan Science and Technology College, Foshan 528231, China
* To whom correspondence should be addressed.
Received May 3, 2005; Revision received July 13, 2005
beta-N-Acetyl-D-glucosaminidase was purified from viscera of green crab (Scylla serrata) by extraction with 0.01 M Tris-HCl buffer (pH 7.5) containing 0.2 M NaCl, ammonium sulfate fractionation, and then chromatography on Sephadex G-100 and DEAE-cellulose (DE-32). The purified enzyme showed a single band on polyacrylamide gel electrophoresis, and the specific activity was determined to be 7990 U/mg. The molecular weight of the whole enzyme was determined to be 132.0 kD, and the enzyme is composed of two identical subunits with molecular mass of 65.8 kD. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-NAG) were found to be at pH 5.6 and at 50°C, respectively. The study of its stability showed that the enzyme is stable in the pH range from 4.6 to 8.6 and at temperatures below 45°C. The kinetic behavior of the enzyme in the hydrolysis of pNP-NAG followed Michaelis-Menten kinetics with Km of 0.424 ± 0.012 mM and Vmax of 17.65 ± 0.32 µmol/min at pH 5.8 and 37°C, and the activation energy was determined to be 61.32 kJ/mol. The effects of some metal ions on the enzyme were surveyed, and the results show that Na+ and K+ have no effects on the enzyme activity; Mg2+ and Ca2+ slightly activate the enzyme, while Ba2+, Zn2+, Mn2+, Hg2+, Pb2+, Cu2+, and Al3+ inhibit the enzyme to different extents.
KEY WORDS: enzymatic properties, beta-N-acetyl-D-glucosaminidase, purification, Scylla serrata