Some Properties And Activity Regulation Of β-N-Acetyl-D-glucosaminidase From Procam Barus Clarkii

β-N-Acetyl-D-glucosaminidase play an important role in the physiological process the conversion and transformation of the cuticle of insects ,crustaceans and nutrition digestion.Theβ-N-Acetyl-D-glucosaminidase was purified from Procambarus clarkii by ammonium sulfate fractionation, twice tchromatography on DEAE-cellulose (DEAE-32) and Sephadex G-100.The purified enzyme showed a single band on polyacrylamide gel electrophoresis and isoelectric focusing electrophoresis,and the specific activity was 6540.24 U·mg -1.The molecular weight of the whole enzyme was 118.66kD, consisted of 364 amino acid residues.Its isoelectric point is 5.15. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N acetyl-β-D-glucosaminide (pNP-β-D-GlcNAc)were found at pH 5.5 and at 40°C, respectively. The study of its stability showed that the enzyme was stable in the pH range from 3.6 to 5.9 and at temperatures below 50°C. The activation energy was 114.89kJ·mol-1.The kinetic behavior of the enzyme in the hydrolysis of pNP-β-D-GlcNAc followed Michaelis–Menten kinetics with Km of 0.46 mmol·L-1 and Vm of 10.87μmol·L-1·min -1 ,and the kinetic behavior of the enzyme in the hydrolysis of pNP-β-D-GaNAc followed Michaelis–Menten kinetics with Km of 1.04 mmol·L-1 and Vm of 39.40μmol·L-1·min -1 .The effects of metal ions on the enzyme were studied. The results showed that Li2+,K+ had not any effect on enzyme activity in the tested range of concentration. Na+,Ba2+,Ca2+,Co2+ could increase the activity of enzyme at different concentration. Mn2+,Mg2+,Pb2+,Ag+ increase enzyme activity at first then inhibit it. Zn2+,Hg2+,Cu2+,Al3+ were significantly reduce enzyme activity to different degrees. The intensity of inhibition effects of metal ions on the enzyme from big to small in turn: Hg+ > Cu2+ > Al3+ > Zn2+ > Ag+ > Pb2+ > Mn2+ > Mg2+. Mn2+,Ag+,Zn2+,Al3+,Hg+ were reversible anticompetitive inhibitor . The KI were 0.41 mmol·L-1,1.08 mmol·L-1,1.22 mmol·L-1,0.87 mmol·L-1,0.07 mmol·L-1 respectively. Mg2+ was revealed to be a reversible non-competitive inhibitor with a KI of 1.59 mmol?L-1.Pb2+ was revealed to be a reversible competitive inhibitor with a KI of 26.17 mmol?L-1. The effects of amino acids on the enzyme were studied. The results showed that L-Ala,L-Phe,L-Met,L-Pro,L-Gly,L-Thr,L-Ser,L-Gln had not any effect on enzyme activity in the tested range of concentration. L-Arg,L-His,L-Lys reduced the activity of enzyme at different concentration.The IC50 of L-Arg,L-His,L-Lys on the enzyme in turn: 45mmol·L-1,130mmol·L-1,110mmol·L-1. L-Arg,L-His,L-Lys were revealed to be a reversible competitive inhibitor with KI of 10.76mmol?L-1,226.12 mmol?L-1,.157.70 mmol?L-1.We studied the variations of activity ofβ-N-Acetyl-D-glucosaminidase from different organs of Procambarus clarkii in different growth stages. The result s showed that the enzyme activities of the hepatopancreas of Procambarus clarkii in different growth stages are variational.The activity of enzyme from the hepatopancreas of Procambarus clarkii was decreased one by one stage,there was a significant decreasing in stage I(2~3moon's age) and stage II(5~6 moon's age),stage III(9~10 moon's age).But had no significantly decreased in stageII and stage III . The activity of enzyme of the gonad were consistent with variation of shell membrane: decreasing one by one stage ,but there were no significantly decreased between in stageII and stageIII,significantly decreased in stageIII . In addition, the results showed that the enzyme activities of the variations organs of Procambarus clarkii from big to small in turn:The hepatopancreas> The gonad >The shell membrane.Studied some basic properties ofβ-N-Acetyl-D-glucosaminidase from different organs of Procambarus clarkii in different growth stages. The result s showed that the optimum temperature of the enzyme from the hepatopancreas of Procambarus clarkii is 35℃in all stage; the optimum temperature of the activity of enzyme of the gonad were 35℃in stageI,stageII.but 40℃in stageIII.The optimum temperature of the activity of enzyme of of shell membrane were 35℃in stageII,stageIII.but 40℃in stageI. The optimum pH of the enzyme from the hepatopancreas were 5.5 in stageI,stageIII. but 5.2 in stageII;The optimum pH of the activity of enzyme of the gonad were 5.2 in stageII,stageIII,but 5.5 in stageI; The optimum pH of the activity of enzyme of of shell membrane were 4.5 in stageI,stageII,but5.5 in stageIII.