Glucose oxidase (GOD) has a wide range of applications. It is used as biocatalyst, in biosensors or for diagnostic applications. Presently, industrial production of gluconic acid is performed by microbial fermentation [1]. Direct oxidation of glucose using GOD would simplify the process. A problem is the inactivation of the enzyme during reaction. Thus many efforts were made to stabilise the enzyme, e.g. by immobilisation, leading to a reduced activity, or addition of catalase in excess to destroy hydrogen peroxide. All these methods of avoiding rapid inactivation of glucose oxidase are performed by changing the environment of the enzyme.

A possible rational approach of molecular stabilisation starts with the analysis of observed covalent modifications of the enzyme during reaction. Inactivation was investigated simultaneously kinetically and by mass spectrometric analysis of enzyme modifications. Reaction and inactivation of glucose oxidase was monitored by measuring oxygen concentration during reaction with b-D-glucose. The reaction was modelled using the known ping-pong mechanism of the enzyme [2] and by a one step irreversible inactivation correlated with hydrogen peroxide formation.

Molecular changes of the enzyme during the inactivation process were determined by the analysis of proteolytic fragments using MALDI mass spectrometry indicating the oxidation of the enzyme at specific points.

[1] Roehr, M., Kubicek, C.P., Kominek, J., Biotechnology, Vol. 6, VCH Weinheim, 348-362, 1996.

[2] Gibson, Q.H., Swoboda, B.E.P., Massey, V., J. Biol. Chem., 239, 3927-3934, 1964.