Rational Co-Immobilization of Bi-Enzyme Cascades on Porous Supports and their Applications in Bio-Redox Reactions with In Situ Recycling of Soluble Cofactors

In bio-redox cascade reactions that are immobilized on porous supports, mass-transfer limitations may impede the effective concentration of the cofactor around the corresponding dehydrogenases. This main drawback has been addressed by the co-immobilization of both the main and recycling dehydrogenases. Herein, we report tailor-made co-immobilization procedures to assemble three different bio-redox orthogonal cascades in vitro (two selective reductions and one selective oxidation) with in situ cofactor-regeneration. However, the co-immobilization itself does not guarantee the success of the biotransformation because the same co-immobilization chemistry may not be suitable for the two enzymes that are involved in the bio-redox cascade. Therefore, our co-immobilization system was optimized for each bi-enzymatic cascade. In all cases, the optimized co-immobilization procedure was more efficient in the biocatalytic cascade than if the two dehydrogenases were immobilized on two different carriers. In one specific case (one thermophilic cascade), the co-immobilization of an optimal ratio of main/recycling dehydrogenases (1:5) on the same carrier resulted in a biocatalyst that was able to recycle NADH up to 9000 times per equivalent of substrate in 1 hour at 55?degrees C. Moreover, uniform distributions of both dehydrogenases across the porous surface also enhanced the recycling efficiency of the cofactor 1.5-fold versus cascades in which the enzymes were not uniformly distributed across the same porous surface, presumably because of vicinal cooperation effects. Hence, this system for the co-immobilization of bi-enzymatic systems may be extended to other biocatalytic cascades, thereby opening a window for the optimization of other multi-enzyme biotransformations in which cofactor-recycling is necessary.