Studies On The Properties And The Activity Regulation Of N-Acetyl-β-D-glucosaminidase From Green Crab (Scylla Serrata)

N-Acetyl-β--D-glucosaminidase (NAGase, EC 3.2.1.52) was purified from viscera of green crab (Scylla serrata), by extraction with 0.01 mol/L Tris-HCl buffer (pH 7.5) containing 0.2 mol/L NaCl and ammonium sulfate fractionation, then chromatography on Sephadex G-100 and DEAE-cellulose (DE-32). The purified enzyme was a single band on polyacrylamide gel electrophoresis with specific activity to be 7,990 U/mg. The pI value was determined to be 4.68 by isoelectric focusing. The molecular weight of whole enzyme was determined to be 132.0 kD consisted of 1097 amino acid residues and the enzyme is composed of two same subunits with molecular mass of 65.8 kD and there has no s-s bond between each subunit while has s-s bond inside each subunit. The NAGase was a glycoprotein with 12.85%(w/w) glucose. 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 5.6 and at 50 oC, respectively. The results of the stability showed that the enzyme was stable at the pH range from 4.6 to 8.6 and at the temperature below 45 oC. The kinetic behavior of the enzyme in the hydrolysis of pNP-NAG followed Michaelis-Menten kinetics with Km of 0.424±0.012 mmol/L and Vmax of 17.65±0.32μmol/(L·min) at pH 5.8 and 37 oC, and the activation energy was determined to be 61.32 KJ/mol.The residues of cysteine, tryptophan, histidine, lysine andβ-corboxyl of acidic amino acids were necessary for the enzyme activity while The residues of arginine and serine were not necessary for the enzyme activity. Furthermore the s-s bond were not necessary for the enzyme activity too. The pKe value of the ionization of the enzyme active site was determined to be 5.4 at 30℃, and the ?H0 value to be 7.812 kcal/mol. The result suggested that the ionization may be imidazole-group of histidine. The catalytic mechanism of the enzyme for the hydrolysis of pNP-NAG was estimated to be ordered Bi Bi .The results of inactivation of the enzyme in guanidine hydrochloride indicated that the active site of the enzyme is situated in a limited region of the enzyme molecule that is more fragile to denaturants than the enzyme as a whole.The NH4+ and SO3-which were brought because of the eutrophication could inhibited the enzyme activity obviously. The phenol which is the one of main organic contaminations of water had obvious inhibition on the enzyme. Compare with phenol,...