Investigation On The Properties Of N-Acetyl-β-D-glucosaminidase From Epidermis Of Mud Crab (Scylla Serrata)

N-Acetyl-β-D-glucosaminidase(NAGase,EC 3.2.1.52) was purified from epidermis of mud 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 fraction,the chromatography on Sephadex G-100, DEAE-Cellulose,Phenyl Sepharose and ConA Sepharose.The purified enzyme was a single band on polyacrylamide gel electrophoresis and the specific activity was determined to be 668.90 U/mg.The enzyme was determined to be 74.59 kD by Sephacryl S-200 gel filtration. It was glycoprotein.The enzyme(NAGase-E) was modified respectively by several chemical modification reagents,such as DL-Dithiothreitol(DTT),bromoacetic acid(BrAc),N-bromosuccinimide (NBS),Azodicarboxylate(EDC),acetic anhydride and acetyl acetone(AA) at certain condition,and the residue activity was assayed in normal reaction media.The results showed that the residues of histidine,tryptophan and carboxyl of acidic amino acids were necessary for the enzyme activity while the residues of disulfide bond,lysine and arginine were not necessary for the enzyme activity.The effects of water contamination(metal ions and organic solvent),Feed Component (amino acid and saccharide) and antibacterial medicaments on the enzyme(NAGase-E) activity had been studied.1.The effects of several metal ions on the enzyme activity had been studied.The results show that:Mg²⁺ had no effects on the activity of the enzyme,while Co²⁺ and Mn²⁺ had a little active effect on the enzyme.Al³⁺ could inhibit the enzyme activity by 44.4%when its concentration reached to 0.54 mmol/L.Pb⁺,Cu²⁺ and Zn²⁺ can inhibit the enzyme activity with the inhibitor concentration leading to 50%of enzyme activity lost(IC₅₀) were estimated to be 0.4 mmol/L,0.05 mmol/L and 0.04 mmol/L,respectively.2.The effects of several organic solvent on the enzyme activity had been studied.The results show that:methanol,ethanol,propanol,ethylene glycol,propanediol,glycerol,acetone, formaldehyde,dimethyl sulfoxide,1,4-dioxane and N,N-dimethylformamide inhibit the enzyme activity in different degree with the inhibitor concentration leading to 50%of enzyme activity lost(IC₅₀) were estimated to be 3.7 mol/L,2.3 mol/L,1.15 mol/L,2.4 mol/L,2 mol/L, 2.3 mol/L,0.80 mol/L,1.15 mol/L,0.68 mol/L,0.88 mol/L and 0.8 mol/L.3.The effects of several amino acid on the enzyme activity had been studied.The results show that:L-His and L-Asp had just a little effect on the enzyme activity,and L-Lys could inhibit the enzyme activity,obviously.4.The effects of several saccharides on the enzyme activity had been studied.The results show that:rha could active the enzyme and enhanced the enzyme activity by 15%when its concentration reached to 0.2 mol/L.0.8 mol/L concentration of fru,suc,glu and man could obtain inhibit rate 47.61%,57.54%,85.70%and 90.50%.0.6 mol/L and 0.3 mol/L concentration of ara and gal could obtain inhibit rate 41.62%and 37.71%,respectively.0.09 mol/L glcUA could make NAGase almost loose its activity.And NAG had obvious inhibit rate 83%,when its concentration reached to 80 nmol/L.5.The effects of several antibacterial medicaments on the enzyme activity had been studied.The results show that:benzylpenicillic,kanamycin,and streptomycin had no effects on the enzyme activity.The gentamicin had just a little active effects on the enzyme,while the inhibition effect of enroflxacin was most prominent.2.9 mg/ml enroflxacin could make the enzyme loose 50%activity.The changes of the activities of NAGase and chitinase from the mud crab during different growing stages were investigated in this contribution.The results exhibited that the activities of chitinase-E and NAGase-E showed a significant rhythm,namely decreasing gradually from stagel to stage5,and then keeping in a steady level.Contrarily,the activity changes of NAGase-H and chitinase-H were found to be random.But the change of NAGase and chitinase were synchronously.The optimum pH for NAGase-E and NAGase-H were 5.6-5.8, and the optimum temperatures for them were 42℃and 40±2℃,respectively.The pH stabilities for NAGase-E and NAGase-H were 5.0-10.0 and 5.0-9.0,respectively.Besides,the temperature which NAGase-E remained steady was below 45℃,while that for NAGase-H was below 42℃.The changes of the activities of NAGase and chitinase from the mud crab during different seasons had been studied.The results show that the activities of chitinase and NAGase from epidermis and hepatopanereas showed a synchronously significant rhythm.The lower activity was present in March,then decreasing gradually until November.During different season,the optimum pH for NAGase-E and NAGase-H were 5.6-5.8,and the optimum temperatures for them were 42℃and 40±2℃,respectively.The pH stabilities for NAGase-E and NAGase-H were 5.0-10.0 and 5.0-9.0,respectively.Besides,the temperature which NAGase-E remained steady was below 45℃,while that for NAGase-H was below 42℃.