Monitoring and control of the release of soluble O(2) from H(2)O(2) inside porous enzyme carrier for O(2) supply to an immobilized d‐amino acid oxidase

While O(2) substrate for bio‐transformations in bulk liquid is routinely provided from entrained air or O(2) gas, tailored solutions of O(2) supply are required when the bio‐catalysis happens spatially confined to the microstructure of a solid support. Release of soluble O(2) from H(2)O(2) by catalase is promising, but spatiotemporal control of the process is challenging to achieve. Here, we show monitoring and control by optical sensing within a porous carrier of the soluble O(2) formed by an immobilized catalase upon feeding of H(2)O(2). The internally released O(2) is used to drive the reaction of d‐amino acid oxidase (oxidation of d‐methionine) that is co‐immobilized with the catalase in the same carrier. The H(2)O(2) is supplied in portions at properly timed intervals, or continuously at controlled flow rate, to balance the O(2) production and consumption inside the carrier so as to maintain the internal O(2) concentration in the range of 100–500 µM. Thus, enzyme inactivation by excess H(2)O(2) is prevented and gas formation from the released O(2) is avoided at the same time. The reaction rate of the co‐immobilized enzyme preparation is shown to depend linearly on the internal O(2) concentration up to the air‐saturated level. Conversions at a 200 ml scale using varied H(2)O(2) feed rate (0.04–0.18 mmol/min) give the equivalent production rate from d‐methionine (200 mM) and achieve rate enhancement by ∼1.55‐fold compared to the same oxidase reaction under bubble aeration. Collectively, these results show an integrated strategy of biomolecular engineering for tightly controlled supply of O(2) substrate from H(2)O(2) into carrier‐immobilized enzymes. By addressing limitations of O(2) supply via gas‐liquid transfer, especially at the microscale, this can be generally useful to develop specialized process strategies for O(2)‐dependent biocatalytic reactions.