Modelling enzymatic reduction of 2-keto-D-glucose by suspended aldose reductase

Batch experiments have been systematically carried out at 25°C, pH = 7, over 24-76 h reaction time in order to evaluate the activity of a commercial (recombinant human) aldose reductase (ALR) used to catalyze the reduction of 2-keto-D-glucose (kDG) to fructose using NADPH as cofactor, by employing various enzyme/reactants initial ratios. A kinetic model was proposed by extending the 'core' reaction mechanism proposed in literature for the reduction of several saccharides and keto-derivates (glucose, galactose, xylose, glyceraldehydes) by the human or animal ALR (wild or modified), or by similar aldo-keto reductases (e.g. sorbitol dehydrogenase, xylose reductase) in the presence of NAD(P)H. The reaction pathway assumes a very quick reversible formation of a stable ALR * NADPH complex, from which a small fraction is binding the substrate thus determining a succession of Bi-Bi reversible reactions leading to the final product (fructose). Model parameters have been estimated based on the recorded data sets of four observable key-species, being in concordance with the reported values in literature for similar processes. The results confirm the conformational change of E*NADP+ complex allowing the release of [NADP.sup.+] as being the rate-limiting step of the overall process. The results also underline the necessity to stabilize the fast deactivating enzyme by immobilization, as well as the requirement of a continuous in-situ regeneration of the cofactor. Key words: Keto-glucose reduction to fructose, aldose reductase, reaction mechanism, kinetic model