Abstract
While O2 substrate for bio-transformations in bulk liquid is routinely provided from entrained air or O2 gas, tailored solutions of O2 supply are required when the bio-catalysis happens spatially confined to the microstructure of a solid support. Release of soluble O2 from H2 O2 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 O2 formed by an immobilized catalase upon feeding of H2 O2 . The internally released O2 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 H2 O2 is supplied in portions at properly timed intervals, or continuously at controlled flow rate, to balance the O2 production and consumption inside the carrier so as to maintain the internal O2 concentration in the range of 100-500 µM. Thus, enzyme inactivation by excess H2 O2 is prevented and gas formation from the released O2 is avoided at the same time. The reaction rate of the co-immobilized enzyme preparation is shown to depend linearly on the internal O2 concentration up to the air-saturated level. Conversions at a 200 ml scale using varied H2 O2 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 O2 substrate from H2 O2 into carrier-immobilized enzymes. By addressing limitations of O2 supply via gas-liquid transfer, especially at the microscale, this can be generally useful to develop specialized process strategies for O2 -dependent biocatalytic reactions.