Kinetic Modeling of Phosphate‐Based Enzyme Cascade for the Production of Fructose From Starch

In this work, we build a mechanistic kinetic model for a phosphate‐based enzyme cascade for high‐yield fructose production from waste starch, a potentially industrially impactful cascade whose kinetics have remained underexplored. The cascade proceeds through sugar phosphate intermediates catalyzed by alpha‐glucan phosphorylase (AGP), phosphoglucomutase (PGM), phosphoglucoisomerase (PGI), transaldolase (TRA), and 3‐phosphoglycerate phosphatase (3‐PGP), thus enabling a kinetically driven route that overcomes equilibrium limitations of conventional glucose isomerization. The intrinsic kinetic behavior of each enzyme was incorporated to build the overall deterministic kinetic model for the cascade. Thermodynamic and kinetic parameters were obtained or estimated from literature and theory, and a small number of highly sensitive parameters were fit to batch experimental data. Metabolic control analysis and net‐rate analysis revealed transaldolase as the primary driver of fructose yield. We also highlight the power of this model by providing microscopic details such as coverages of various species bound to enzymes and using these insights in proposing experimental conditions that can improve fructose yields. Through this work, we emphasize that this modeling framework not only has the power to propose experimental conditions that improve the yield of the desired fructose product but also provide fundamental insights into the behavior of enzyme cascades.