This study introduces a unique and universal method to construct direct electron transfer (DET)-type oxidoreductases complexed with cytochrome c, which is based on the SpyCatcher/SpyTag technology to overcome the inherent problem of heme c maturation during the recombinant preparation, to realize stable bioelectrochemical continuous monitoring system. Enzyme sensors employing DET-type enzymes are ideal for the continuous monitoring system, especially for the in vivo real time monitoring, considering their superior electrochemical properties. However, the availability of DET-type oxidoreductases has been the technological limitation to develop DET-type enzyme-based sensors. To overcome this issue, the authors have established the creation of DET-type oxidoreductases by recombinantly constructing chimeric proteins between oxidoreductases and heme molecules (heme b and heme c). However, due to the inherent biochemical properties of the de novo synthesis of heme c molecules, the creation of heme c containing fusion proteins are limited for those being secreted by specific bacterial machinery. Therefore, flavin dependent oxidoreductases, which are commonly folded in cytosol, are not the subjects to be genetically fused with heme c, which is the preferable electron transfer protein considering its stability suitable for continuous operation. In this research, to overcome this problem, we propose a universal method to construct direct electron transfer type oxidoreductases complexed with cytochrome c. This challenge was achieved by acknowledging the benefit of the SpyCatcher/SpyTag system, which known as covalently in vitro protein/protein complex formation system. By preparing cytochrome c–SpyCatcher fusion protein and SpyTag fused oxidoreductase, recombinantly and individually, and by mixing them in vitro, cytochrome c-SpyCatcher/SpyTag-oxidoreductase complexes were successively prepared. Thus formed complexes retained its original catalytic activities and redox properties of heme c, and the intra-complex electron transfer ability. The complexes also showed DET ability with electrode.

Development of a universal method to construct direct electron transfer type oxidoreductases complexed with cytochrome c to realize stable continuous bioelectrochemical monitoring system

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