Purification And Characterization Of E Gase From Glyptotermes Xiamenensis Li Et Huang

Endo-1,4-β-D-Glucanase (NGase, EC 3.2.1.4) was purified from Glyptotermes Xiamenensis Li et Huang by extraction with 0.01 mol/L PBS buffer (pH 6.7) and ammonium sulfate fraction, the chromatographyon DEAE-Sepharose Fast Flow Sephadex G-100, The purified enzyme was a single band on polyacrylamide gel electrophoresis and the specific activity was determined to be 458.7 U/mg. The enzyme was determined to be 44.8 kD by Sephadex G-100 gel filtration.The enzyme (EGase-E) was modified respectively by several chemical modification reagents, such as bromoacetic acid (BrAc), N-bromosuccinimide (NBS), Azodicarboxylate (EDC), acetic anhydride, p-chloromercuribenzoate (pCMB), P-Mercaptoethanol,and acetyl acetone (AA) ,Hydrogen peroxide(H₂O₂), Phenylmethanesulfonyl fluoride(PMSF),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 lysine and arginine were necessary for the enzyme activity while the residues of mercapto, disulfide bond, L-arginine guanidine, Sulfide, serine deformity,were not necessary for the enzymeactivity.The enzyme modified with N-bromosuccinimide is totally inactivated and itscharacteristic peak of UV-absorption spectrum at 278 ran also destroyed, the 334 nm peak of the fluorescence emission spectrum is gradually descended and eventually lost, indicating that the tryptophan resdues are essential to the enzyme function. By the method of C. L. Tsou's plot, the number of essential Trp was found only one.The effects of several metal ions on the enzyme activity had been studied. The results show that: Cr^-,Br^-,I^-and Li⁺,Na⁺,K⁺ had no effects on the activity of the enzyme, while Co²⁺ could inhibit the enzyme activity by 40.1% when its concentration reached to 0.45 mmol/L.Cu²⁺,Pb²⁺,Hg²⁺ and Cd²⁺ can inhibit the enzyme activity with the inhibitor concentration leading to 50% of enzyme activitylost(IC₅₀) were estimated to be 0.51,1.2,0.18 and 2.5 mmol/L,respectively.The inactivation effects in ions solvents (Cu²⁺,Pb²⁺,Hg²⁺) have been studied using the kinetic method of Lineweaver-Burk plot. And their inhibition types and inhibition constants have been determined.The results showed that the inactivation of the enzyme in these ions solvents was non- reversible reaction.The effects of several organic solvent on the enzyme activity had been studied. The results show that: methanol, ethanol, propanol, glycol, propylene, glycerin, acetone, dimethyl sulfoxide,1,4-dioxane and phenol, inhibit the enzyme activity in different degree with the inhibitor concentration leading to 50% of enzyme activity lost(IC₅₀) were estimated to be 10.1%,15.1%,16.5%,3.8%,14.5%,16.2%,0.85%,0.58 mol/L,0.48 mol/L,0.25 mol/L.The inhibition Mechanism of glycol and phenol are reversible,The inhibition constants(K_I) of glycol and phenol are 3.8% and 0.64 mol/L.