A high catalytic efficiency and chemotolerant formate dehydrogenase from Bacillus simplex

• Published in: Biotechnology journal 2024-01, Vol.19 (1), p.e2300330-n/a
• Main Authors: Boonkumkrong, Rattima, Chunthaboon, Paweenapon, Munkajohnpong, Pobthum, Watthaisong, Pratchaya, Pimviriyakul, Panu, Maenpuen, Somchart, Chaiyen, Pimchai, Tinikul, Ruchanok
• Format: Article
• Language: English
• Subjects: Bacillus; biocatalysis; Catalysis; chemotolerance; cofactor regeneration; formate dehydrogenase; Formate Dehydrogenases - metabolism; Formates; NAD - metabolism; solvent tolerance
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.202300330

NAD+‐dependent formate dehydrogenase (FDH) catalyzes the conversion of formate and NAD+ to produce carbon dioxide and NADH. The reaction is biotechnologically important because FDH is widely used for NADH regeneration in various enzymatic syntheses. However, major drawbacks of this versatile enzyme in industrial applications are its low activity, requiring its utilization in large amounts to achieve optimal process conditions. Here, FDH from Bacillus simplex (BsFDH) was characterized for its biochemical and catalytic properties in comparison to FDH from Pseudomonas sp. 101 (PsFDH), a commonly used FDH in various biocatalytic reactions. The data revealed that BsFDH possesses high formate oxidizing activity with a kcat value of 15.3 ± 1.9 s−1 at 25°C compared to 7.7 ± 1.0 s−1 for PsFDH. At the optimum temperature (60°C), BsFDH exhibited 6‐fold greater activity than PsFDH. The BsFDH displayed higher pH stability and a superior tolerance toward sodium azide and H2O2 inactivation, showing a 200‐fold higher Ki value for azide inhibition and remaining stable in the presence of 0.5% H2O2 compared to PsFDH. The application of BsFDH as a cofactor regeneration system for the detoxification of 4‐nitrophenol by the reaction of HadA, which produced a H2O2 byproduct was demonstrated. The biocatalytic cascades using BsFDH demonstrated a distinct superior conversion activity because the system tolerated H2O2 well. Altogether, the data showed that BsFDH is a robust enzyme suitable for future application in industrial biotechnology. Graphical and Lay Summary Bacillus simplex formate dehydrogenase (BsFDH) is a smaller NAD+‐dependent FDH enzyme that lacks the N‐terminal loop and C‐terminal elongation regions. It has the highest catalytic efficiency for formate oxidation, showing a 5‐fold higher catalytic efficiency than that of Psedomonas sp.101 (PsFDH). The high chemotolerance also demonstrated its suitability to be used as a cofactor degenerating enzyme in the biosynthesis process involving H2O2 byproducts.