One-pot systems are an interesting proposal to carry out multi-enzymatic reactions, though this strategy implies establishing an optimal balance between the activity and operability of the involved enzymes. This is crucial for enzymes with marked differences in their operational sta-bility, such as one-pot production of lactofructose syrup from cheese whey permeate, which in-volves two enzymes—β-galactosidase (β-gal) and glucose isomerase (GI). The aim of this work was to study the behavior of one-pot sequential batch production of lactofructose syrup considering both enzymes immobilized individually, in order to evaluate and design a strategy of replacement of the catalysts according to their stabilities. To this end, the modelling and simulation of the process was carried out, considering simultaneously the kinetics of both reactions and the kinetics of inactivation of both enzymes. For the latter, it was also considered the modulating effect that sugars present in the medium may have on the stability of β-gal, which is the less stable enzyme. At the simulated reaction conditions of 40 °C, pH 7, and 0.46 (IUGI/IUβ-gal), the results showed that considering the stability of β-gal under non-reactive conditions, meaning in absence of the effect of modulation, it is necessary to carry out four replacements of β-gal for each cycle of use of GI. On the other hand, when considering the modulation caused by the sugars on the β-gal stability, the productivity increases up to 23% in the case of the highest modulation factor studied (η = 0.8). This work shows the feasibility of conducting a one-pot operation with immobilized enzymes of quite different operational stability, and that a proper strategy of biocatalyst replacement increases the productivity of the process.