Studies On Activity Regulation And Kinetics Of The N-Acetyl-β-D-glucosaminidase From Prawn (Penaeus Vannamei )

N-Acetyl-β-D-glucosaminidase (NAGase, EC3.2.1.52) plays an important role in molting and nutrition assimilation of Penaeus vannamei, and uses in other applications.The NAGase was purified from viscera of Prawn (Penaeus vannamei) and determined to be homogeneous by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE. This purfied enzyme will be used in the following studies.The effects of metal ions on the enzyme were studied. Li+, Na+ and K+ had no influence on enzyme activity. Co2+, Cd2+, Ba2+, Zn2+, Al3+, Pb2+, Cu2+, Fe3+, Ag+, Hg2+ showed various degrees of inhibitory effects on the enzyme. Some inhibition types and inhibition constants had been determined. The results showed that the Ag+ was an uncompetitive inhibitor with KIS of 2.39 mmol/L, and the Zn2+ was a noncompetitive inhibitor with KI of 11.95 mmol/L. The kinetics of inhibition of the enzyme at different concentration of Zn2+ was also study.The effect of Hg2+ on the enzyme activity is irreversible. The inhibitor concentration leading to 50% (IC50) activity lost was estimated to be 0.16 mmol/L. The microscopic rate constants for the reaction of Hg2+ with free enzyme and the enzyme-substrate complex are determined. Comparison of the microscopic rate constants shows that the presence of substrate has a certain protective effect against inactivation by Hg2+. The fluorescence intensity of the enzyme gradually decreased with increasing Hg2+ concentrations, and the ultraviolet spectrum decreased to almost disappear.The acetone inhibited the enzyme at the pH ranged form 4.8 to 5.0. When the pH ranging form 5.2 to 8.0, the acetone activated the enzyme at low concentration and inhibited the enzyme at high concentration. The fluorescence emission intensity of the enzyme gradually decreased with increasing acetone concentrations. The fluorescence excitation intensity of the enzyme also gradually decreased with increasing acetone concentrations, and at the same time the fluorescence excitation peak (at 280 nm) of the enzyme was red-shifted to 290 nm.N,N-Dimethylformamide (DMF) and dimethylsulfoxide (DMSO) both can obviously inactive the enzyme activity. The inactivation mechanisms of DMF and DMSO were reversible .The inactivator concentrations leading to 50% (IC50) activity...