Purification, Properties, and Molecular Features of Glucose Oxidase from Aspergillus niger

Abstract

Glucose oxidase [β-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4] from Aspergillus niger was purified by DEAE-cellulose chromatography and Sephadex G-200 gel filtration using phosphate buffer, pH 7.0. 1. The preparation has a specific activity of 172 μmoles oxygen consumed/min/mg of enzyme at 30° and pH 5.6, and is essentially catalase-free. The constituents of the enzyme are protein (74.0±2.8%), neutral sugar (16.4±0.3%), amino sugar (2.4± 0.5%), and 2 moles of iron per 160, 000 daltons, in addition to FAD. 2. Optical data for the newly purified holoenzyme show that the ratio A₂₈₀/A₄₅₀ is 11.1, and e for enzyme-bound FAD at 450 nm is 1.52x10⁴. 3. The holoenzyme (molecular weight, 160, 000) consists of two identical subunits with molecular weights of 79, 000±4, 000, and the molecular weight of the apoenzyme seems to be identical with that of the subunit, as shown by SDS-polyacrylamide gel electrophoresis and gel filtration on Sephadex G-200. 4. FHD (flavin-hypoxanthine dinucleotide) has coenzymatic activity equal to that of FAD.