Liquidi ionici quali mezzo di reazione e stabilizzanti enzimatici di reazioni catalizzate da formiato deidrogenasi per scopi di biocatalisi e fissazione di CO2.

Responsabili di progetto

Francesco Secundo, Vladimir Tishkov

Accordo

RUSSIA - RFBR-suspended - Russian Foundation for Basic Research

Bando

CNR/RFBR 2015-2017

Dipartimento

Scienze chimiche e tecnologie dei materiali

Area tematica

Scienze chimiche e tecnologie dei materiali

Stato del progetto

Rinnovo

Proposta di ricerca

Formate dehydrogenase (FDH, E.C. 1.2.1.2) is certainly one of the most widely applied cofactor regeneration systems used to promote NADH-dependent reductions and oxidations. Especially the quasi-irreversibility of the formate-driven NAD+ reduction reaction (yielding volatile carbon dioxide (CO2) as by-product) makes it an attractive cofactor regeneration system.

Beyond biocatalytic applications, formate dehydrogenases may play an important role to develop enzymatic processes able to reduce the concentration of CO2 in the atmosphere (e.g., transform CO2 to formic acid), which is a worldwide recognized challenge. In fact, further increases of carbon dioxide emissions are predicted to produce large and uncontrollable impacts on the world climate. Unfortunately, carbon is in its highest oxidation state in CO2 and its transformation into other valuable chemicals requires high energy input. Nevertheless, the use of carbon dioxide as C1 source to produce chemicals and fuels can be the basis of a greener and more sustainable chemical industry. In this regard, recently significant progress has been achieved in the reduction of carbon dioxide or bicarbonate using organometallic and enzyme catalysis.

Concerning this latter possibility, formate oxidation and CO2 reduction are interconvertible processes that are catalyzed by two main families of enzymes found in Eubacteria. In particular by i) iron-sulfur formate dehydrogenases (they catalyze NAD-independent formate oxidation and contain redox active molybdenum (Mo) or tungsten (W) prosthetic groups) and by ii) NAD+-dependent FDH enzymes that catalyze the concomitant reduction of NAD+ to NADH and formate oxidation to CO2.
Particularly interesting for CO2 reduction is FDH-Catalyzed Reduction. Carbon dioxide reductase activity has been successfully demonstrated in vitro with FDH enzymes containing a W-MGD cofactor instead of the Mo-MGD cofactor. Furthemore, also among NAD+-Dependent FDH, which are devoid of any metallic prosthetic groups, it has been recently reported the reversibility of these enzymes with NADH-dependent CO2 reduction to form methanol (Appel et al., Chem. Rev. 2013, 113, 66216658). For example, one enzyme originates from the anaerobic acetogen strain Clostridium carboxidivorans DSM15243. This FDH, recombinantly expressed in E. coli, was found to be oxygen-tolerant and highly active in the desired NADH oxidation. Furthermore, its binding affinity for formate was at least 30 times lower than that of the FDH from C. boidinii, making it much more useful for the formation of formate. Another enzyme is found in Clostridium autoethanogenum and it is an NADPH-dependent FDH containing selenocysteine- and tungsten as part of a complex composed of seven subunits. Interestingly, there is high sequence similarity between the formate-producing and formate-consuming FDH enzymes, which suggests that dioxide reduction occurs in microorganisms essentially by a reverse of mechanism compared to that of oxidation.

Because of the high potentialities of this group of enzymes both in biocatalysis and in CO2 fixation processes, there is the need to explore the oxido-reductive properties of as many as possible FDH from various origins (e.g., microbial and plants). Furthemore, it arises the problem of making these enzymes more stable in biocatalysis or in devices that aim to fix CO2, in particular as that described by Baeg and coworkers (J. Am. Chem. Soc. 2012, 134, 1145511461) of a photocatalyst-enzyme coupled artificial photosynthesis system for solar energy able to produce formic acid from CO2.

A possible strategy based on the use ionic liquids (ILs) to improve the stability of FDHs has been reported by Doumèche and coworkers (Journal of Molecular Catalysis B: Enzymatic 65 (2010) 73-78). They proved that the chemical modifications of formate dehydrogenase by ionic liquid-inspired cations improved the enzyme activity and stability in [MMIm][Me2PO4]. Other authors also have shown that ILs can act both as solvent and as enzyme stabilizers (for a review see Patel et al. , Applied Biochemistry and Biotechnology April 2014, Volume 172, Issue 8, pp 3701-3720).

ILs are purely ionic, salt-like materials which are composed of organic cations and various anions. The properties of an IL can be varied by changing the nature of the cation-anion combination. Compared to water and organic solvents, they exhibit a negligible volatility, non flammability, a wide electrochemical window, high thermal and chemical stability and strong solubility power. Due to their inimitable properties and large variety of applications, ionic liquids have been extensively used in enzyme catalysis and protein stability and separation. Therefore, because of the very high number of ionic liquids that can be prepared and that are commercially available nowadays, it is possible to design or choose an ionic liquid that can serve as a noble and selective solvent for any particular enzymatic reaction, protein preservation and other protein based applications. The use of ILs as solvents for chemical reactions also is due to their property to dissolve both polar and non-polar organic, inorganic and polymeric materials. Among ILs, imidazolium based ionic liquids can dissolve high amounts of CO2 and - depending of the anion - will be miscible with water or form a two phase system. Based on this, ionic liquids could be exploited not only as stabilizers of FDH but also as media where CO2 concentration could be increased compared to that in water, thus favoring its enzymatic reduction.

Obiettivi della ricerca

This proposal aims at developing modified FDH for improving the stability of this group of enzymes obtained from different sources (bacteria, yeasts and plants) . Modified FDHs will be prepared by chemical modification with ILs (Italian group) as well as by site-directed mutagenesis (Russian group) and by combination of both methods. Furthermore, at the same time this proposal aims to explore the use of FDH in/with ionic liquids (as neat solvent or in biphasic systems with water) for biocatalytic purposes and/or for fixation of CO2. The improvement of stability will be pursued especially in the contest of these applications.

The project will be implemented joining the competences of the Russian group coordinated by Prof. Tishkov of Moscow State University/Innotech MSU in Techno park of Moscow University, an expert of isolation and characterization of FDH from different sources, with those of the Italian group coordinated by Dr. Secundo of CNR, an expert of enzyme modification and their application in non-aqueous media.

The Russian group will suggest and provide some FDH that might be more promising for the purposes of the project. The Italian group will test activity and stability of the enzymes obtained from the Russian group and modified with ionic liquids. The Italian group will also consider the possibility to modify the enzyme cofactor (e.g., NAD+ linked to PEG) to evaluate if in the modified form could favor FDH stabilization and activity in neat ILs.

Ultimo aggiornamento: 20/04/2024