This work presents an analysis on the suitability of the Simulated Moving Bed (and pseudo-SMB) technology on separating the mixture obtained from the lactose oxidation catalyzed by glucose-fructose oxidoreductase and glucono-α-lactonase enzymes. These enzymes from the Zymomonas mobilis bacteria are able to oxidize lactose in presence of fructose to its respective organic acid—lactobionic acid—and sorbitol. Some alternative arrays of chromatographic systems, as fixed bed column, SMB unit, 4 section pseudo-SMB, have been explored to separate the multi-component mixture, in such way to make possible the product recovery and the recycle of substrates to the enzymatic reactor. This study involved the definition of appropriate operating conditions and the prediction of the performance of the separation units, or arrangement of units, through modeling and simulations tools. To define the proper operating conditions, inequalities from equilibrium theory and the concept of the separation volume analysis have been considered. In this analysis, equilibrium and kinetic parameters for the compounds adsorbing on DOWEX 50W-X4 resin, in K+ and Ca+2 ion-loadings, have been obtained from chromatographic methods (pulse and adsorption-desorption techniques). The enzymatic kinetic of production of lactobionic acid and sorbitol using permeabilized cells of Z. mobilis is shown. The strategy of keeping the highest value of reaction rate by the integration of a chromatographic system proved to be viable when it was found the feasibility to apply the SMB system in cascade.