PRELIMINARY STUDIES ON ISOLATION,PURIFICATION AND SOME PROPERTIES OF THE β-N-ACETYL-D-GLUCOSAMINIDASE FROM ERIOCHEIR SINENSIS

A β-N-Acetyl-D-glucosaminidase(EC3.2.1.30) was purified from the viscera of Eriocheir sinensis by ammonium sulfate fractionation,chromatography on DEAE-32,Sephadex G-100 and DEAE-32.The purified enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis.The specific activity of the enzyme was 4490.79U/mg.The molecular weight of the subunits was determined to be 121.21,98.63 and 73.48kD,respectively.The pI value was calculated to be 4.5 by isoelectric focusing.The optimum temperature and pH of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide(pNP-NAG) were determined to be at 45℃ and at pH5.5,respectively.The enzyme was stable in the pH ranges of 4.9 to 9.3 under 37℃and at temperatures below 40℃.The enzyme follows typical Michaelis-Menten kinetics for the hydrolysis of pNP-β-D-GlcNAc.The Km and Vm values were determined to be 0.357mmo/L and 10.41μmol/L·min at pH5.6 and 37℃,respectively.The activation energy of the enzyme for the hydrolysis of pNP-β-D-GlcNAc was to be 76.50kJ/mol.The effects of metal ions on the enzyme were studied.Mg2+,Ca2+ and Ba2+had not influenced the enzyme activity.Na+ activated the enzyme,while,Li+,K+,Zn2+,Hg2+,Pb2+,Cu2+,and Al3+ showed various degrees of inhibitory effects on the enzyme.