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

Abstract

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 °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.

Immobilizing Systems Biocatalysis for the Selective Oxidation of Glycerol Coupled to In Situ Cofactor Recycling and Hydrogen Peroxide Elimination

“…[36] The TTN numbers are low probably because in this multienzyme cascade the limiting step is the product inhibition of GlyDH rather than the NAD + recycling. The observed TTN values for the co-immobilized system are comparable to those reported for DHA production catalyzed by GlyDH from Cellulomonas sp and Xylose reductase from Pichia stipitis immobilized on silica nanoparticles and GlyDH [23] from Citrobacter braakii and NOX from Thermus thermophius co-immobilized on agarose beads [10] under similar conditions. Moreover, the complete elimination of H 2 O 2 avoids enzyme inactivation by chemical oxidation and, importantly, avoids the unspecific oxidation of DHA that reduces the product yield of the biotransformation dramatically.…”

“…[6,9] We have paid attention to the preparation of immobilized multienzyme systems to catalyze tandem reactions. [10][11][12] The co-immobilization of multienzyme systems can improve: 1) the kinetics of the chemical cascade because of the spatial localization of the different biocatalytic modules, which avoids the accumulation of intermediates and increases the cofactor recycling efficiency [10] and 2) the stability of the biocatalyst because of the in situ elimination of toxic byproducts. [13] Nevertheless, to yield water and O 2 as innocuous products, which avoided the spontaneous DHA oxidation triggered by H 2 O 2 .…”

“…We have recently reported two examples in which the optimal design of the immobilization protocols allowed the co-immobilization of several enzymes on the same carrier through their optimal immobilization chemistries, which preserved both the global activity and stability of the multienzyme systems. [10,13] The success of this approach relies on the versatility of the surface chemistry given by the agarose beads that allows us to synthesize carrier surfaces activated with different reactive groups that specifically attach each enzyme through its optimal immobilization chemistry.…”

Selective oxidation of glycerol to 1,3-dihydroxyacetone by covalently immobilized glycerol dehydrogenases with higher stability and lower product inhibition

“…Such recycling guarantees available NAD + to be readily used by the immobilized GlyDH-Gs. This result agrees to other biotransformations where NOX has been used as redox cofactor recycling partner (Riebel et al, 2003;Rocha-Martín et al, 2012a).…”

“…Cross-linked agarose beads (4%) were from Agarose Beads Technology (Madrid, Spain). Glycerol dehydrogenase (GlyDH-Cb) from C. braakii was produced and purified as published elsewhere (Rocha-Martín et al, 2012a). The Coomassie (Bradford) protein assay kit was purchased from Pierce (Rockford, Illinois, USA).…”