Cofactor immobilization for efficient dehydrogenase driven upgrading of xylose

In this study, the coupled immobilization on nanosilica of xylose dehydrogenase and alcohol dehydrogenase was accomplished with high efficiency, which was 90 %. Moreover, immobilization of the cofactors oxidized and reduced nicotinamide adenine dinucleotide, i.e., NAD⁺ and NADH, on silica material was examined and the impact on the effectiveness of the process was determined. The highest efficiency of NAD+ immobilization was found to be 56 %, which was obtained after 24 h of immobilization at 30 °C, pH 7 For NADH, the best immobilization efficiency was 53 % which was achieved after 24 h at 25 °C, pH 7. The K_M and V_max values were determined for various configurations of the biocatalytic systems showing, as expected, that immobilization of the enzymes decreased the catalytic rate (V_max) and slightly increased the K_M, but verifying that the immobilization of the cofactors did not significantly affect the kinetics, but would enable high conversion, and potentially continued enzymatic reaction. The use of the system configuration with co-immobilized enzymes, immobilized NAD⁺ and immobilized NADH thus allowed for obtaining over 90 % efficiency of xylose conversion in one batch, which was significantly higher than the systems with single free or only one immobilized cofactor. Using UV-Vis measurements, it was confirmed that effective cofactor regeneration occurred in the systems with immobilized components thus allowing for sustained enzyme catalyzed upgrading of xylose to xylonic acid.