Multi-reaction kinetic modeling for the peroxidase–aldolase cascade synthesis of a D-fagomine precursor

[Display omitted] •Multi-enzymatic reaction cascade evaluation for the synthesis of iminocyclitols.•A complex system composed of seven distinct reaction and two enzyme inactivation.•Kinetic modeling for elucidation of reaction mechanism.•Cross-inhibition and inactivation in peroxidase/aldolase reactions.•High improvement in reaction productivity through process intensification. The feasibility of a peroxidase–aldolase cascade reaction for the synthesis of therapeutically-valuable iminocyclitols is discussed herein. A two-enzyme system consisting of chloroperoxidase (CPO) and D-fructose-6-phosphate aldolase (FSA) was evaluated for the synthesis of a D-fagomine precursor (preFagomine) from a N-Cbz-3-aminopropanol. An in-depth, systematic, step-by-step kinetic modeling of seven reactions and two inactivation decays was proposed to elucidate the reaction mechanism, prepare suitable stabilized biocatalysts, and find the optimal conditions for its application. The model described accurately the data and predicted the outcome at different experimental conditions. The inactivation of FSA caused by CPO was identified as the main bottleneck in the reaction. A two-step reaction approach and the use of immobilized enzymes on magnetic nanoparticle clusters and functionalized agarose carriers increased the stability of FSA, with an 1839-fold higher preFagomine formation per mol of enzyme in comparison to a one-pot reaction using soluble enzymes.