Department of Bioscience and Food Production Science, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 8304 Minami-minowa, Kamiina, Nagano, 399-4598, Japan.
Institutes for Biomedical Sciences Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan.
Anal Sci. 2021 May 10;37(5):733-739. doi: 10.2116/analsci.20SCP15. Epub 2021 Jan 15.
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.
甲酸盐是后石油时代最具针对性的 C1 建筑块和电子载体。甲酸盐脱氢酶（FDH）能够催化甲酸盐的产生或降解，因此受到了广泛关注。在 FDH 中，一种带有复杂活性中心（钼喋呤辅因子）的依赖金属的 FDH 可以直接将电子从甲酸盐转移到其他氧化还原蛋白，而不会产生 NAD(P)H。此前，我们报道了一种来自大肠杆菌的膜结合型依赖金属的 FDH（由 fdoG-fdoH-fdoI 操纵子编码）的表达系统，并成功将其转化为可溶性蛋白。然而，该蛋白的稳定性太低，无法进行纯化和生化分析。在本研究中，我们尝试通过合理和不合理的方法来提高异源表达的 FDH 的稳定性。结果，通过合理的方法获得了具有最高比活性的突变体。这项研究不仅为工业应用提供了一种具有增强活性和稳定性的有前途的 FDH 酶，还为处理重组大型蛋白提供了相关见解。
