Evaluation of cross-linkers in the design of immobilized multi isomerase cascade for the preparation of rare sugars.

The cascade of sugar isomerases is one of the most practical methods for producing rare sugars, and enzyme immobilization endows it with high economic efficiency, operational convenience and reusability. However, the most employed cross-linker glutaraldehyde (GA) has the disadvantages of enzyme deactivation and limitation of substrate binding. Herein, three compounds, glyoxal, GA, and 2,5-furandicarboxaldehyde (DFF) were evaluated within a previously developed cascade comprising ribose-5-phosphate isomerase and D-tagatose-3-epimerase to prepare D-ribulose form D-xylose. Analyses of surface morphology, element and chemical bond revealed that all compounds effectively cross-linked the isomerases. High concentration of the cross-linkers was generally beneficial for binding protein and preventing enzyme leak during reusing cycles. Glyoxal performed the highest immobilization rate, though it hadn't been employed as a cross-linker for enzyme immobilization. DFF mediated cross-linking revealed the highest activity recovery, substrate conversion and residual activity after reusing cycles, suggesting better biocompatibility than glyoxal and GA. After 8 rounds of recycling, the residual activity of enzyme immobilized by DFF was 61.4 %, ∼30 % higher than that of GA. This study proved a potential alternative cross-linker DFF for the immobilization of enzyme cascade with high activity recovery and reusability, which could promote the efficient production of high value-added products from biomass monosaccharides.