A two-enzyme cascade reaction consisting of two reaction pathways. Studies in bulk solution for understanding the performance of a flow-through device with immobilised enzymes

substrate solution was pumped. Comparison was made between (i) co-immobilised and (ii) sequentially immobilised enzymes (BCA first, HRP second). Significant differences for the two arrangements in terms of measured product yield (DCF) could be explained based on quantitative UV/vis absorption measurements carried out in bulk solution. We found that the lower DCF yield observed for sequentially immobilised enzymes originates from a change in one of the two possible reaction pathways due to enzyme separation, which was not the case for enzymes that were co-immobilised (or simultaneously present in the bulk solution experiments). The higher DCF yield observed for co-immobilised enzymes did not originate from a molecular proximity effect (no increased oxidation compared to sequential immobilisation).