Mixtures of xylose, glucose and pyruvate were fed to a membrane bioreactor equipped with a charged NF membrane (NTR 7450). Value-added products were obtained in the reactor via enzymatic cofactor-dependent catalysis of glucose to gluconic acid and pyruvate to lactic acid, respectively. The initial cofactor (NADH) concentration could be decreased to 10% of the stoichiometric value (relative to glucose) without compromising process time and substrate conversion via i) efficient cofactor regeneration and high retention of cofactor (R=0.98) in the membrane bioreactor. Furthermore, accumulation of xylose (R < 0.1), lactic acid (R=0.38) and gluconic acid (R=0.63) was minimized. After separation of the cofactor in the membrane bioreactor, xylose, lactic acid and gluconic acid were separated based on charge repulsion and size exclusion in a sequence of two NF steps. Broad substrate specificity of glucose dehydrogenase (EC 1.1.1.47) (GDH) lead to partial conversion of xylose to xylonic acid, causing some loss of xylose, but the results obtained nevertheless showed that it is possible to build a robust system for conducting enzyme reactions by sequentially regenerating the cofactor and at the same time obtaining valuable products of high purity.