Study On The Properties And Inhibitory Kinetics Of Liver NAGase From Tilapia

N-acetyl-β-D-glucosaminidase(NAGase)is a chitinase that widely found in nature and plays an important role in many biological processes.In this experiment,the tilapia liver was used as a material,and NAGase was isolated and purified by fractional precipitation using ammonium sulfate(30～70%),DEAE-Sepharose Fast Flow ion exchange column chromatography,and Sephadex G-200 molecular sieve column chromatography.The specific activity of the enzyme preparation was 2988 U/mg.The purified enzyme preparation was detected by SDS-PAGE.As a result,there was only one band,and it was considered that the final preparation was determined to be homogeneous by PAGE.The total molecular weight of NAGase in tilapia liver was 61.8 kD as measured by gel chromatography.Combined with SDS-PAGE results,the enzyme had only one subunit.Further study of its enzymatic properties showed that the enzyme had the highest enzyme activity at 50℃,the enzyme activity was stable at 0～55℃,and completely lost its activity at 80℃;the optimum pH of the enzyme was 5.8 and the enzyme activity was relatively stable between 4.0～9.0,when the pH was over 10,the enzyme was almost completely inactivated;the kinetic parameter Km of the enzyme was 0.229 mmol/L,and the Vm was 9.314 μmol·L-1.min-1.The chemical modification method was used to study the functional groups of the liver NAGase active center.It was found that the essential groups of the NAGase active center were disulfide bonds,histidine imidazolyl residues,tryptophan residue thiol,and lysine.The acid oxime-amino group,while modification of the side chain arginine of NAGase did not change the enzyme activity,indicating that it was not a functional group.In this paper,the effect of organic solution on the catalytic activity of NAGase-catalyzed hydrolysis of tilapia liver was studied.The results showed that perfluorohexanoic acid,carbon tetrachloride and acetamide had a greater impact on NAGase activity.The three kinds of organic matters could reversibly inhibit NAGase at a suitable concentration and IC50 was 0.29 mg/mL;0.26 mg/mL and 25 mmol/L,respectively.The lineweaver-Burk method showed that the inhibition type of perfluorohexanoic acid and carbon tetrachloride was competitive inhibition,and the inhibition equilibrium constant KI with free enzyme was 0.18 mg/mL and 0.032 mg/mL,respectively.Whereas,the inhibition of NAGase by acetamide was mixed inhibition with an equilibrium constant KI of 8.1 mmol/L for the free enzyme and 150.72 mmol/L for the enzyme-substrate complex.The substrate reaction method was used to establish the kinetic model of inhibition.The microscopic velocity constant could be obtained by the substrate reaction of NAGase in organic solution.