Considerations for the optimization of biocatalyst formulation in multi-enzymatic reactions: Co-immobilized enzymes advantages depend on enzyme kinetic properties

Abstract

The design of combi-biocatalyst for optimizing cascade reactions is challenging, particularly in terms of the choice of the biocatalyst formulations, including the relative amounts of enzymes to be used, and the experimental set-up strategy chosen for optimization. This paper demonstrates, via dynamic simulation, the optimization of a combi-biocatalyst designed for a multi-cascade two-reaction series (A → B, B → C) involving two enzymes (E1, E2) in free, individually immobilized, or co-immobilized formulations. This has been analyzed in three different scenarios: (1) KM is identical for both enzymes (i.e. KM1 = KM2), (2) KM1 > KM2, and (3) KM1 < KM2. The ratio of mass transport and reaction times was defined using a modified Thiele modulus, providing a useful metric to evaluate the relative magnitude of mass transport limitations. Global mass transport was considered. In the absence of mass transport limitations, the three catalyst formulations gave identical results. However, when (moderate) mass transport limitations were included, substrate (A) and intermediate (B) concentration gradients appear, meaning that the multi-enzyme catalyst formulation becomes critical. The results indicate that although enzyme co-immobilization always provides some kinetic advantages compared to the use of individually immobilized enzymes, these advantages increased when KM2 < KM1 and vice versa. The first situation also provides the most efficient combi-biocatalyst. The optimal enzyme ratio in all enzyme formulations, determined by using either initial rates or time to reach the target yield, is in some instances quite different. This suggests that, although more time-consuming, the latter parameter should be utilized in the design of the enzyme mass ratio for the combined biocatalysts. The optimal enzyme ratio depends on the enzyme formulation. Therefore, extrapolating the results achieved using individually immobilized enzymes to co-immobilized biocatalysts can lead to the preparation of a biocatalyst with sub-optimal efficiency.