Solvents For Biocatalysis Research Articles

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

IntroductionDeep 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.MethodsThis 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 discussionConsidering trade-offs between experimentally determined performance metrics of DESs, 20% solution of choline chloride:glycerol in phosphate buffer (ChCl:Gly80%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.

High-Value Bioconversion of Ginseng Extracts in Betaine-Based Deep Eutectic Solvents for the Preparation of Deglycosylated Ginsenosides.

Deep eutectic solvents (DES), as a green alternative to traditional organic solvents in biocatalysis, not only activate proteins but even increase the efficiency of enzymatic reactions. Here, DES were used in a combinatorial enzyme-catalyzed system containing β-glucosidase BGLAt and β-galactosidase BGALAo to produce deglycosylated ginsenosides (De-g) from ginseng extracts (GE). The results showed that DES prepared with betaine and ethylene glycol (molar ratio, 1:2) could significantly stimulate the activity of the combinatorial enzymes as well as improve the acid resistance and temperature stability. The DES-based combinatorial enzyme-catalyzed system could convert 5 g of GE into 1.24 g of De-g (F1, F2, 20 (S)-PPT, and CK) at 24 h, which was 1.1 times that of the buffer sample. As confirmed by the spectral data, the changes in the conformations of the combinatorial enzymes were more favorable for the binding reaction with the substrates. Moreover, the constructed DES-based aqueous two-phase system enabled the recovery of substantial amounts of DES and De-g from the top phase. These results demonstrated that DES shows great application as a reaction solvent for the scale-up production of De-g and provide insights for the green extraction of natural products.

Inhibitory Effect of DMSO on Halohydrin Dehalogenase: Experimental and Computational Insights into the Influence of an Organic Co-solvent on the Structural and Catalytic Properties of a Biocatalyst.

Although the application of organic solvents in biocatalysis is well explored, in-depth understanding of the interactions of solvent with proteins, in particular oligomeric ones, is still scant. Understanding these interactions is essential in tailoring enzymes for industrially relevant catalysis in nonaqueous media. In our study, the homotetrameric enzyme halohydrin dehalogenase (HHDH) from Agrobacterium radiobacter AD1 (HheC) was investigated, as a model system, in DMSO/water solvent mixtures. DMSO, the most commonly used co-solvent for biocatalytic transformations, was found to act as a mixed-type inhibitor with a prevalent competitive contribution. Even 5 % (v/v) DMSO inhibits the activity of HheC by half. Molecular dynamics (MD) simulations showed that DMSO keeps close to Ser-Tyr catalytic residues forming alternate H-bonds with them. Stability measurements paired with differential scanning calorimetry, dynamic light scattering methods and MD studies revealed that HheC maintains its structural integrity with as much as 30 % (v/v) DMSO.

Eutektische Lösungsmittel in der Biokatalyse

Perfect solvents in biocatalysis have to fulfill a large number of requirements, such as high enzyme activity and stability, and high substrate solubility. Deep eutectic solvents (DES) have recently been evaluated as new solvents in different biocatalytic reactions. They can provide improvements in substrate supply, conversion and stability. The best results were obtained when the DES is formed by the substrates of an enzymatic reaction.

Deep eutectic solvents in biocatalysis

Deep eutectic solvents in biocatalysis

Compartmentalized Aqueous-Organic Emulsion for Efficient Biocatalysis.

The design and construction of polymeric compartmentalized structures in water have been intensively explored for controllable catalysis, but there is still the challenge of setting up catalytic compartments in organic media. Here, we designed a simple block copolymer, PCL-b-PEG-b-PCL, to construct a stable and multi-compartmentalized emulsion in an organic solvent by hand-shaking. This gentle emulsion preparation allowed a successful encapsulation of vulnerable biocatalysts such as benzaldehyde lyase (BAL) and alcohol dehydrogenase (ADH). The compartmentalization provided the emulsion with an exceptionally large interfacial area that could enhance BAL activity up to 225 times as compared to the traditional biphasic system. Moreover, the system could be easily scaled up due to its facile preparation with low cost. Therefore, our results pave the way for developing compartmentalized structures in solvents for biocatalysis in large-scale synthetic chemistry.

Towards understanding directed evolution: more than half of all amino acid positions contribute to ionic liquid resistance of Bacillus subtilis lipase A.

Ionic liquids (ILs) are attractive (co-)solvents for biocatalysis. However, in high concentration (>10 % IL), enzymes usually show decreased activity. No general principles have been discovered to improve IL resistance of enzymes by protein engineering. We present a systematic study to elucidate general engineering principles by site saturation mutagenesis on the complete gene bsla. Screening in presence of four [BMIM]-based ILs revealed two unexpected lessons on directed evolution: 1) resistance improvement was obtainable at 50-69 % of all amino acid positions, thus explaining the success of small sized random mutant libraries; 2) 6-13 % of substitutions led to improved resistance. Among these, 66-95 % were substitutions by chemically different amino acids (e.g., aromatic to polar/aliphatic/charged amino acids), thus indicating that mutagenesis methods introducing such changes should, at least for lipases like BSLA, be favored to improve IL resistance.

Methods for stabilizing and activating enzymes in ionic liquids—a review

AbstractIonic liquids (ILs) have evolved as a new type of non‐aqueous solvents for biocatalysis, mainly due to their unique and tunable physical properties. A number of recent review papers have described a variety of enzymatic reactions conducted in IL solutions; on the other hand, it is important to systematically analyze methods that have been developed for stabilizing and activating enzymes in ILs. This review discusses the biocatalysis in ILs from two unique aspects (1) factors that impact the enzyme's activity and stability, (2) methods that have been adopted or developed to activate and/or stabilize enzymes in ionic media. Factors that may influence the catalytic performance of enzymes include IL polarity, hydrogen‐bond basicity/anion nucleophilicity, IL network, ion kosmotropicity, viscosity, hydrophobicity, the enzyme dissolution, and surfactant effect. To improve the enzyme's activity and stability in ILs, major methods being explored include the enzyme immobilization (on solid support, sol–gel, or CLEA), physical or covalent attachment to PEG, rinsing withn‐propanol methods (PREP and EPRP), water‐in‐IL microemulsions, IL coating, and the design of enzyme‐compatible ionic solvents. It is exciting to notice that new ILs are being synthesized to be more compatible with enzymes. To utilize the full potential of ILs, it is necessary to further improve these methods for better enzyme compatibility. This is what has been accomplished in the field of biocatalysis in conventional organic solvents. Copyright © 2010 Society of Chemical Industry

Stability and activity of immobilised penicillin G amidase in ionic liquids at controlled aw

Despite the great interest in ionic liquids as novel solvents for biocatalysis, there is still no clear idea of their influence on the stability and the activity of enzymes. Here we analysed the activity and stability of PGA in six different ionic liquids, having different cations ([bmim] and [omim]) and anions (CH3OSO3−, PF6− and BF4−). To be active in ionic liquids, PGA-450 requires an acceptable hydration (aw > 0.60), as in organic solvents. PGA is highly stable in [bmim][PF6] and [bmim][BF4], and catalytic activity, assayed by studying the synthesis of the amide of L-phenylglycine methyl ester with methyl phenylacetate, in these ILS is comparable to that obtained in toluene.

Thermodynamics of the Lipase-Catalyzed Esterification of 1-Dodecanoic Acid with (−)-Menthol in Organic Solvents

The thermodynamics of the lipase-catalyzed stereospecific esterification of 1-dodecanoic acid with (−)-menthol in seven organic solvents have been investigated. Equilibrium measurements were performed using hexane, heptane, cyclohexane, toluene, 2,2,4-trimethylpentane, acetonitrile, and 2-methyl-2-butanol at the temperature T = 298.15 K. The equilibrium constants have been correlated with the (octanol + water) partition coefficients and the relative permittivities of the solvents. The saturation molalities and the (hexane + water) partition coefficients of (−)-menthol, 1-dodecanoic acid, and (−)-menthyl dodecanoate have also been determined at T = 298.15 K. The equilibrium constant of the esterification reaction in aqueous media has been calculated. An important finding is that the molality of water plays an important role in determining the extent of ester formation. In acetonitrile and 2-methyl-2-butanol, which have a very high molality of water, the formation of (−)-menthyl dodecanoate is considerably smaller. However, in hydrophobic solvents with lower molalities of water, the formation of (−)-menthyl dodecanoate is favored in the reaction mixture. The value of the equilibrium constant varies from 3 to 26. However, the molality of (−)-menthyl dodecanoate changes from 0.034 mmol kg-1 in acetonitrile to 16 mmol kg-1 in heptane. The change in product yield is a factor of ≈500 and is a striking example of the utility of organic solvents in biocatalysis.