Protein Engineering of the Soluble Metal-dependent Formate Dehydrogenase from Escherichia coli.

Rintaro Fuji, Koji Umezawa, Manami Mizuguchi, Masaki Ihara

Analytical sciences : the international journal of the Japan Society for Analytical Chemistry 2021 May 10

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Formate is the most targeted C1 building block and electron carrier in the post-petroleum era. Formate dehydrogenase (FDH), which catalyzes the production or degradation of formate, has acquired considerable attention. Among FDHs, a metal-dependent FDH that carries a complex active center, molybdenum-pterin cofactor, can directly transfer electrons from formate to other redox proteins without generating NAD(P)H. Previously, we reported an expression system for membrane-bound metal-dependent FDH from E. coli (encoded by the fdoG-fdoH-fdoI operon) and succeeded in its conversion to a soluble protein. However, this protein exhibited a too low stability to be purified and analyzed biochemically. In this study, we tried to improve the stability of heterologously expressed FDH through rational and irrational approaches. As a result, a mutant with the highest specific activity was obtained through a rational approach. This study not only yielded a promising FDH enzyme with enhanced activity and stability for industrial applications, but also offered relevant insights for the handling of recombinant large proteins.

Citation

Rintaro Fuji, Koji Umezawa, Manami Mizuguchi, Masaki Ihara. Protein Engineering of the Soluble Metal-dependent Formate Dehydrogenase from Escherichia coli. Analytical sciences : the international journal of the Japan Society for Analytical Chemistry. 2021 May 10;37(5):733-739