Purification And Characterization Of GOD From Penicillium Amagasakiense

Glucose oxidase(GOD, EC 1.1.3.4) has great application in the fields of food,medicine and biology etc. This research established a set of method to extract and purity the glucose oxidase from Penicillium amagasakiense. We research different time,temperature,stirring speed and different concentration of Ca²⁺ Tween 80 on the GOD activity units from fermentation liquor. Under the consdition of 24℃,200rpm,fermentation for 6 days,0.3 mol/L of Ca²⁺,3% of Tween 80, the total GOD activity from fermentation liquor are to maximize。After 6 days fermentation, the mycelium was destroyed to withdraw the intracellular glucose oxidase with buffer solution, while the fermentation liquor was the extracellular glucose oxidase. Then fermentation liquor by the concentration, 50mmol/L Tris-HCl buffer (pH 8.0) dialysis to pH 8.0, the chromatography on DEAE-32, then by gel filtration through Sephacryl S-200. The purified enzyme was a single band on polyacrylamide gel electrophoresis and the specific activity was determined to be 472 U/mg. The subunit of enzyme was determined to be 75 kD.The optimum pH is 5.6, and the optimum temperature is 40℃for the oxidase of glucose. Under the condition of pH 7.0, 37℃, Mchaelis-Menten contant (Km) is 122.6 mmol/L, and the maximum velocity (Vm) is 25.52μmol/(L·min). The enzyme is stable in range of pH from 5.5 to 8.5 and in temperature below 50℃. The enzyme (GOD) was modified respectively by several chemical modification reagents, The results showed that the residues of histidine, tryptophan and L-arginine guanidine were necessary for the enzyme activity, while the residues of mercapto, disulfide bond, Sulfide, serine deformity, carboxyl of acidic amino acids lysine were not necessary for the enzyme activity. The inactivation effects in chemical modification reagents (BrAc,NBS,acetyl acetone) have been studied using the kinetic method of Lineweaver-Burk plot. The inhibition constants(K₁) of BrAc, NBS and acetyl acetone are 26.5,19.2,7.35 mmol/L. The effects of several metal ions on the enzyme activity had been studied. The results show that: Cl^-,Br^-,I^- and Li⁺,Na⁺,K⁺,Ca²⁺,Mn²⁺ had no effects on the activity of the enzyme, white Ba²⁺,Co²⁺,Cd²⁺,have a slight inhibitory effect on the activity of the enzyme. The inhibition constants (K₁) of Cu²⁺,Ag⁺ and Hg²⁺ are 210.78μmmol/L, 0.065μmol/ and 83.1μmol/L. The effects of several organic solvent on the enzyme activity had been studied. The results show that: methanol, ethanol, propanol, glycol, propylene glycol and glycerol had no effects on the activity of the enzyme. Formaldehyde inhibit the enzyme activity in different degree with the inhibitor concentration leading to 50% of enzyme activity lost(IC₅₀) were estimated to be 81.25 mmol/L, The inhibition constants(K₁) of formaldehyde is 30 mmol/L. The effects of several denaturant on the enzyme activity had been studied. The results show that: EDTA,Urea have a slight inhibitory effect on the activity of the enzyme. The inhibition constants (K₁) of Gu·HCl,SDS are 1.75 mmol/L and 57.15 mmol/L.