Co-immobilization of enzymes and electron mediator for efficient biocatalysis

Abstract

Oxidoreductases are enzymes that transfer electrons from an electron donor to electron acceptor. Since many reactions involve oxidation and reduction, studies on the biocatalytic use of oxidoreductase have been conducted. In biocatalysis, continuous-flow chemistry has advantages, and enzyme immobilization is a method for this. As oxidoreductase requires an electron mediator in many cases, it also requires immobilization of the electron mediator for continuous flow application. Electron mediators contain both natural and artificial cofactors. Immobilization of enzymes and electron mediators has advantages such as enabling their recycling and improving stability and activity. However, in previous studies, there are no case of artificial cofactor immobilization, and a support matrix with stronger affinity is required due to several limitations. Therefore, we proposed co-immobilization of enzymes and electron mediators by modeling the cascade reaction of ChCODH and MeFDH, which produces formate, a useful chemical from CO gas. Recombinant enzymes ChCODH and MeFDH with His tag were directly immobilized from cell lysate to Ni-NTA agarose. ELP-AMV with His tag was designed as a swing arm electron mediator for efficient electron transfer to the active site of the co-immobilized enzymes. Formate was produced in a system in which ChCODH, MeFDH, and ELP-AMV were co-immobilized on Ni-NTA agarose using His tag. In addition, formate was produced in a repeated batch, showing the possibility of application to continuous process. In conclusion, it was confirmed that the enzymes and electron mediator were co-immobilized and efficient biocatalysis was possible due to the swing arm electron mediator.