Use of cell-free glucose-fructose oxidoreductase (GFOR)from Zymomonas mobilis in a continuous process for the simultaneous production of sorbitol and gluconic acid requires efficient stabilization of the enzyme. Whereas GFOR was found to be stable in the presence or absence of any of its substrates or produce at 25 degrees C, the enzyme was rapidly inactivated during the time course of its own catalytic action. The loss of activity was demonstrated to be strictly linked to catalysis, and consequently, partially inactivated GFOR remained stable when a total conversion of the substrates had been attained. The oxidation of cysteine residues seems to be involved in this unusual mechanism of enzyme inactivation, because dithiothreitol, reduced glutathione, cysteine, and thioglycolate were identified as being efficient protecting agents of GFOR. Inter- or intramolecular disulfide bond formation apparently does not occur during the inactivation process because, once inactivated, no GFOR activity could be recovered by means of a subsequent addition of reductive chemicals. When 5 mM dithiothreitol was used as a stabilizer during continuous operation with GFOR retained in an ultrafiltration membrane reactor, an equilibrium of glucose and fructose fed and converted could be maintained for >100 h at a productivity of 0.6 mmol IU GFOR h(-1).