Extraction And Purification Of Glucose Oxidase From Aspergillus Niger A9 And Study On The Properties Of The Enzyme

Glucose oxidase has great application in the fields of food medicine and biology etc. This research established a set of method to extract and purify the glucose oxidase from Aspergillus niger A9. This method is simple and easy in industrial application and then studied the properties of purified enzyme systematically.This experiment use a strain of Aspergillus niger A9 which preserved in the laboratory as the fermentation strain to ferment glucose oxidase in the 10L fermentation tank for four days. The mycelium of Aspergillus niger A9 was distroyed to withdraw the intracellular glucose oxidase with buffer solution. Glucose oxidase was purified by using ultrafilration, ammonium sulfate fractionation, Sephadex G-25, DEAE-cellulose ion-exchange chromatography and Sephadex G-100 gel filtration. The result indicated that by controlling the detail conditions of ion-exchange chromatography and molecular sieve chromatography, most miscellaneous protein had been removed. The purified glucose oxidase showed a single band in PAGE electrophoresis. The specific activity reached 349.84 U/mg and which was 15.83 folds higher than the crude fermenting liquor and the yield was 23.75%.In the base of obtaining of single component glucose oxidase from Aspergillus niger A9, the properies of purified glucose oxidase was studied. Measured by SDS-PAGE, the molecular weight of glucose oxidase was 138,200 Dalton, the glucose oxidase had two same subunit protein, and the molecular weight was 70,210 Dalton. The glycosylation of purified glucose oxidase was 2.67% based on sulfuric acid-phenol method. The specificity for substrate of glucose oxidase is better, and for glucose the Km is 35.74 mmol/L, Vmax is 35.71 μmol/min which is tested by dual-reciprocal graph. The optimum pH value and temperature were pH6.7 and 30℃ respectively. The range of pH value stability was wide and it was stable between pH value 4.0~8.0. Between pH value 5.0~5.5 the activity is almost unchanged and between pH value 6.0~8.0 it was also stable, the activity was always above 72%, but at pH value 4.0 the activity dropped to 60%. The thermal stability was very good. The activity is almost unchanged between 30~50°C, but dropped to 51% in 60℃. This showed that glucose oxidase could be used in high temperature and this hadgreat value in industry because high temperature could prevent other bacteria effectly. Metallic ions had influence on glucose oxidase. The glucose oxidase was inhibited strongly by Ag\ Hg2\ NaHSOs and was inhibited a little by Cu2+> Fe2+, but it was actived by Na\ Ca2\ Mg2\ Zn2+, Mn2+in some extent.This paper has done some research on the glucose oxidase including the extraction > purification and the enzyme properties and this will be a foundation for the study of its catalysed mechanisrrK clone and industrial application.