Studies On The Properties Of N-Acetyl-β-D-glucosaminidase From Cabbage Butterfly (Pieris Rapae)

A N-Acetyl-β-D-glucosaminidase (NAGase) was purified from the integument of cabbage butterfly (Pieris rapae) by extraction with 50 mmol/L of sodium phosphate buffer (PBS, pH 6.8) containing 0.1 M NaCl and ammonium sulfate fractionation, then chromatography on Sephadex G-200 and Econo-Pac CHT-Пcolumn. The purified enzyme was a single band on polyacrylamide gel electrophoresis and the specific activity was determined to be 8715 U/mg. The molecular weight of whole enzyme was measured to be 106.0 kD by gel filtration on Sephacry-S 200. The enzyme was determined to be a heterodimer with molecular mass of 59.5 and 57.2 kD. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-Nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) were investigated to be at pH 6.2 and at 42 oC, respectively and the Michaelis-Menten constant (Km) was determined to be 0.326 mmol/L at pH 6.2 and 37 oC. The stability of the enzyme was investigated and the results showed that the enzyme was stable at the pH range from 4.0 to 9.0 and at the temperature below 45 oC. The activation energy was 83.86 kJ/mol.The ionization constant, pKe, of ionizing group at the active site of the enzyme was found to be 5.20 at 39.0 oC, and the standard dissociation enthalpy (?Ho) was determined to be 2.18 Kcal/mol. These results showed that the ionizing group of the enzyme active center is the carboxyl group. The enzyme was modified respectively by several chemical modification reagents, such as carbidiimide (EDC), pCMB (p-chloromercuribenzoate), N-Bromosuccinimide (NBS), bromoacetic acid (BrAc), DTT (dithiothreitol), acetic anhydride and acetyl acetone at certain condition, and the residue activity was assayed in normal reaction media. The results showed that the enzyme activity was decreased when the carboxyl group, sulfhydryl group, indolyl group, imidazolyl group and disulfide bond were modified, respectively, which means that these groups were essential for the enzyme catalytic activity. The results also showed that the residues of lysine, arginine were irrespective with the enzyme activity. The reaction mechanism of this enzyme hydrolysis of pNP-NAG was judged to be Ordered Bi-Bi mechanism according to the inhibitory behaviors of the products.Inactivation and unfolding of the NAGase during denaturation in guanidine hydrochloride and urea at different concentrations had been compared, the results...