Production, Purification and Characterization of D-glucose Oxidase from Aspergilluse Niger

The glucose oxidase (GOD) catalyses oxidation of ~-D- glucose to D-gluconic acid and H202. Aspergilluse niger is one of the most fungal sources used for the GOD production. The present report deals with the optimization of culture conditions for the GOD enzyme production, it's purification from culture broth and cell extract, characterization of the purified enzyme and comparison of this enzyme with the standard enzyme. Seventeen samples of Aspergilluse niger were screened for the GOD activity. Samples were cultured in a non specific diagnostic medium. Then the GOD overproducer samples were detected in a specific diagnostic medium and the NRRL-3 strain was selected for the optimization of the GOD synthesis in the submerged culture optimized with the "ONE AT A TIME" method. After the optimization, the enzymatic specific activity reached to 0.22 (from 0.172). For the production of the GOD in large scale, NRRL-3 was cultured in the optimized medium in a bioreactor.Mycelium and postculture liquid medium were used for the enzyme purification. The GOD was purified by a combination of ion exchange and filtration chromatography with approximately a 90-fold enrichment in the specific activity and an enzyme recovery of 9%. The purified enzyme had an apparent native molecular weight of 160 KDa and a denatured molecular weight of 80 KDa determined by the SDS-PAGE. The optimum value of pH for the enzyme activity was between 5.6 and 5.8. The enzyme was severely by O-phtalate (10 mM) and partialy by Ag2 +, Cu2 +, Hg2 +, hydrazine and hydroxylamine (all in 10 mM). The enzyme Km value for glucose oxidase was 37mM.