Bernd Nidetzky

One‐pot cascade reactions of coupled disaccharide phosphorylases enable an efficient transglycosylation via intermediary α‐ d ‐glucose 1‐phosphate (G1P). Such transformations have promising applications in the production of carbohydrate commodities, including the disaccharide cellobiose for food and feed use. Several studies have shown sucrose and cellobiose phosphorylase for cellobiose synthesis from sucrose, but the boundaries on transformation efficiency that result from kinetic and thermodynamic characteristics of the individual enzyme reactions are not known. Here, we assessed in a step‐by‐step systematic fashion the practical requirements of a kinetic model to describe cellobiose production at industrially relevant substrate concentrations of up to 600 mM sucrose and glucose each. Mechanistic initial‐rate models of the two‐substrate reactions of sucrose phosphorylase (sucrose + phosphate → G1P + fructose) and cellobiose phosphorylase (G1P + glucose → cellobiose + phosphate) were needed and additionally required expansion by terms of glucose inhibition, in particular a distinctive two‐site glucose substrate inhibition of the cellobiose phosphorylase (from Cellulumonas uda ). Combined with mass action terms accounting for the approach to equilibrium, the kinetic model gave an excellent fit and a robust prediction of the full reaction time courses for a wide range of enzyme activities as well as substrate concentrations, including the variable substoichiometric concentration of phosphate. The model thus provides the essential engineering tool to disentangle the highly interrelated factors of conversion efficiency in the coupled enzyme reaction; and it establishes the necessary basis of window of operation calculations for targeted optimizations toward different process tasks.