Glucose oxidase is an enzyme extracted from the growth medium of Aspergillus niger (Schomburg, D; Witt, S; Wohlfahrt, G; Hecht, H; Kalisz, H M (2000) Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of glucose. Biochemical Journal, 347,553-559) (Gibson, Q H; Bright, H J (1967) The oxidation of 1-Deuterated Glucose by glucose oxidase Journal of Biological Chemistry, 242(15), 994-1003) (Kalisz HM, Hecht HJ, Schomburg D, Schmid RD. Effects of carbohydrate depletion on the structure, stability and activity of glucose oxidase from Aspergillus niger. Biochim Biophys Acta 1991;1080(2):138–42.)( Hatzinikolaou DG, Hansen OC, Macris BJ, Tingey A, Kekos D, Goodenough P, et al. A new glucose oxidase from Aspergillus niger characterization and regulation studies of enzyme and gene. Appl Microbiol Biotechnol 1996; 46:371–81.) and like many proteins act outside of cells.
The glucose oxidase enzyme is oxido-reductase that catalyses the oxidation of glucose to hydrogen peroxide and D-glucono-δ-lactone (Kalisz, H M; Hendle, J; Schmid, R D (1997) Structural and Biochemical Properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense Applied Microbiology and Biotechnology, 47, 502-507) (Hatzinikolaou DG, Macris BJ. Factors regulating production of glucose oxidase by Aspergillus niger. Enzyme Microb Technol 1995;17:530–4). In solution at pH 7 glucose exists in cyclic hemiacetal form as 63.6% β-D-glucopyranose and 36.4% α-D-glucopyranose where glucose oxidase binds to only β-D-glucopyranose due to the specificity of the enzyme. It converts all of the glucose in solution because the equilibrium between the α and β anomers is driven towards the β side as it is consumed in the reaction as time passed by. Glucose oxidase catalyzes the oxidation of β-D-glucose into D-glucono-1,5-lactone, which then hydrolyzes to gluconic acid. (Danneel HJ, Rossner E, Zeeck A, Giffhorn F. Purifcation and characterization of a pyranose oxidase from the basidiomycete Peniophora gigantea and chemical analyses of its reaction products Eur J Biochem 1993;214:795–802.) (Pluschkell S, Hellmuth K, Rinas U. Kinetics of glucose oxidase excretion by recombinant
Aspergillus niger. Biotechnol Bioeng 1996;51:215–20.)
GOx has a cofactor, flavin adenine dinucleotide (FAD- a common component in biological oxidation-reduction (redox reactions). As Redox reactions involve a gain or loss of electrons from a molecule, hence, in the GOx-catalyzed redox reaction, accepts electron and reduced to FADH2 (Witt S, Wohlfahrt G, Schomburg D, Hecht H, Kalisz H. Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of β-D-glucose. J Biochem 2000;347:553–9) . Then FADH2 is again regain FAD form by oxidized to molecular oxygen (O2) which converts O2 to hydrogen peroxide (H2O2) (Witteveen C, Veenhuis M, Visser J. Localization of glucose oxidase and catalase activities in Aspergillus niger. Appl Environ Microbiol 1992;58(4):1190–4)
Glucose oxidase reaction mechanism (Witt S, Wohlfahrt G, Schomburg D, Hecht H, Kalisz H. Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of β-D-glucose. J Biochem 2000;347:553–9)
Glucose oxidase is widely used coupled with peroxidase for the determination of free glucose in sera or blood plasma for diagnostics, using spectrometric assays manually or with automated procedures (Wilson, R; Turner, P F (1992) Glucose oxidase: an ideal enzyme, Biosensor and Bioelectronics, 7,165-185). In the glucose oxidase assay, the glucose is first oxidized by glucose oxidase to produce gluconate and hydrogen peroxide. The hydrogen peroxide is then oxidatively coupled with a chromogen to produce a colored compound which may be measured spectroscopically. For example, hydrogen peroxide together with 4 amino-antipyrene (4-AAP) and phenol in the presence of peroxidase yield a red quinoeimine dye that can be measured at 505nm. The absorbance at 505 nm is proportional to concentration of glucose in the sample.