Multi-enzyme co-immobilization on tri-heterofunctional supports

Multi-enzyme cascade biotransformations in one pot are gaining momentum since they have demonstrated enhanced catalytic performance than traditional step-by-step transformations requiring sequential pots. Although their evident advantages, the co-immobilization of several enzymes requiring different anchoring chemistries and stability conditions is still challenging. In this work, we exploited an heterofunctional support activated with three different chemical functionalities in order to immobilize a wide variety of different enzymes under mild conditions. This support is based on agarose microbeads activated with aldehyde, amino and cobalt-chelates moieties that allow a fast and irreversible immobilization of enzymes (5 to 30 min), making most of the heterogeneous biocatalysts highly thermostable (up to 21-fold higher than the soluble one). We also demonstrated the potential of this tri-functional support to efficiently co-immobilize a multi-enzyme system composed by an alcohol dehydrogenase, a NADH oxidase and a catalase. The confined multi-enzymatic system demonstrates higher performance than the soluble enzyme counterparts reaching and accumulated TTN of 1x104 during five batch consecutive cycles under operational conditions. Finally, we expanded the versatility of the described exploited heterogeneous chemistry to other frequently used immobilization supports such as cellulose microbeads and commercial methacrylate porous beads. We envision this solid material to be a reference platform for co-immobilizing multi-enzyme systems with enhanced properties to catalyze stepwise biotransformations.