Harnessing the potential of deep eutectic solvents in biocatalysis: design strategies using CO2 to formate reduction as a case study.

Introduction: Deep eutectic solvents (DESs) have emerged as green solvents with versatile applications, demonstrating significant potential in biocatalysis. They often increase the solubility of poorly water-soluble substrates, serve as smart co-substrates, modulate enzyme stereoselectivity, and potentially improve enzyme activity and stability. Despite these advantages, screening for an optimal DES and determining the appropriate water content for a given biocatalytic reaction remains a complex and time-consuming process, posing a significant challenge.

Methods: This paper discusses the rational design of DES tailored to a given biocatalytic system through a combination of experimental screening and computational tools, guided by performance targets defined by solvent properties and process constraints. The efficacy of this approach is demonstrated by the reduction of CO₂ to formate catalyzed by NADH-dependent formate dehydrogenase (FDH). By systematically analyzing FDH activity and stability, NADH stability (both long-term and short-term stability after solvent saturation with CO₂), and CO₂ solubility in initially selected glycerol-based DESs, we were able to skillfully guide the DES screening process.

Results and discussion: Considering trade-offs between experimentally determined performance metrics of DESs, 20% solution of choline chloride:glycerol in phosphate buffer (ChCl:Gly_80%B) was identified as the most promising solvent system for a given reaction. Using ChCl:Gly as a co-solvent resulted in an almost 15-fold increase in FDH half-life compared to the reference buffer and stabilized the coenzyme after the addition of CO₂. Moreover, the 20% addition of ChCl:Gly to the buffer improved the volumetric productivity of FDH-catalyzed CO₂ reduction in a batch system compared to the reference buffer. The exceptional stability of the enzyme in this co-solvent system shows great potential for application in continuous operation, which can significantly improve process productivity. Additionally, based on easily measurable physicochemical solvent properties and molecular descriptors derived from COSMO-RS, QSAR models were developed, which successfully predicted enzyme activity and stability, as well as coenzyme stability in selected solvent systems with DESs.