Glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis is a NADP(H)-containing protein tetramer which catalyses the simultaneous conversion of D-glucose and D-fructose into, respectively, D-gluconolactone and D-sorbitol. Since both products are chemical commodities with many uses in food and chemical industries, the enzyme has drawn some attention for industrial application. A specific advantage of GFOR is the nondissociable enzyme-bound NADP(H) cofactor which makes unnecessary the exogenous addition of the expensive and unstable NADP(H) during the reaction. In addition, no coenzyme regeneration is required with GFOR.

Using GFOR as a soluble biocatalyst in biochemical reactors, we found a kinetic correlation between substrate turnover and irreversible enzyme inactivation, thus leading to a poor operational stability of the enzyme. The inactivation of GFOR is triggered by destabilising interactions with the D-gluconolactone product. The mechanism of inactivation proceeds as a sequence of reactions which affect the protein structure conformationally and chemically. The initial steps (which do not cause activity loss) are conformational realignments close to the NADP(H) site, leading to (i) the exposure of one reactive cysteine residue, and (ii) on deactivation of the cysteine, the evolution of increased surface hydrophobicity. Aggregation of the protein tetramer leads to the loss of enzyme activity and completes the inactivation.

The mechanism of GFOR inactivation provides a rationale for stabilising the enzyme activity, that is by protecting aggregation-prone hydrophobic protein surfaces. This can be done by using the molecular chaperone, GroEL. The chaperone confers long-term stability to GFOR under operational conditions by efficiently suppressing aggregate formation. GroEL provides optimal extra stability to GFOR when added in stoichiometric or greater concentrations. ATP is not required for efficient stabilisation although it was found to increase the stabilising effect of GroEL. GFOR appears to be recognized by GroEL in a native-like conformation with retained quarternary structure and enzyme activity. The binary protein complex GFOR-GroEL was isolated by gel permeation chromatography, and GroEL-bound GFOR was found to display enzyme activity. The results are used to illustrate potentials and limits of the use of chaperones such as GroEL in enzyme stabilisation.