An integrated membrane system was investigated for the separation of mixtures of xylose and glucose. Separation of these sugars is extremely challenging due to their similar structure, size and charge. In order to enhance the xylose separation factor in nanofiltration (NF), we present an enzymatic process for converting glucose to gluconic acid followed by separation of xylose from gluconic acid by nanofiltration. Process conditions which favored the negative charge repulsions between gluconic acid and the NF270 membrane were examined. At the best conditions (9:1 feed molar ratio of xylose to gluconic acid, 0.15 M total feed concentration, pH 95,25 degrees C and 4 bar), we achieved a xylose separation factor of 34 and a throughput of 18.7 L m(-2) h(-1). In comparison, the separation factor was only 1.4 for solutions of xylose and glucose at the same process conditions, thus demonstrating the huge potential of the integrated system. Full conversion of glucose to gluconic acid assisted by glucose oxiclase (GOD) could be achieved by coupling a parallel reaction catalyzed by catalase (CAT), where H2O2 (GOD-inhibitor formed in the first reaction) was decomposed to water and oxygen. GOD has a high oxygen-demand and it was demonstrated that sufficient oxygen could be obtained by controlling the CAT-catalyzed reaction through initial H2O2 addition. The new strategy suggested in this study, integrating reaction and nanofiltration to enhance separation while obtaining another value-added stream, presents new options for separating compounds with similar molecular weights by nanoffitration. (C) 2015 Elsevier B.V. All rights reserved.