Analytical Sciences

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

Rintaro FUJI,* Koji UMEZAWA,*,** Manami MIZUGUCHI,* and Masaki IHARA*,**

*Department of Bioscience and Food Production Science, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 8304 Minami-minowa, Kamiina, Nagano 399-4598, Japan
**Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan

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.

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