Monitoring and control of the release of soluble O2 from H2O2 inside porous enzyme carrier for O2 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 H2O2 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 H2O2. 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 H2O2 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 mu M. Thus, enzyme inactivation by excess H2O2 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 H2O2 feed rate (0.04-0.18 mmol/min) give the equivalent production rate from d-methionine (200 mM) and achieve rate enhancement by similar to 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 H2O2 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.

Automated summary

This study demonstrates the use of optical sensing for monitoring and controlling the release of soluble O-2 in the microstructure of a solid support during bio-catalysis. By precisely controlling the supply of H2O2, the internal O-2 concentration can be maintained within the desired range, preventing enzyme inactivation and gas formation. The implementation of this strategy results in a significant enhancement in reaction rate.