As biomanufacturers slowly transition from aqueous to non-aqueous solvents for biocatalysis, deep eutectic solvents (DES) are getting more attention as sustainable, tunable options. Their primary uses in biocatalysis are to enhance substrate solubility and enzyme activity, modulate enzyme stability, and tune stereo- and regioselectivity.
Furthermore, research shows DES can increase titer and productivity compared to traditional catalysts, and enzymes catalyzed in DES are thermally stable without the need for protein engineering.
But the choice of solvent isn’t a straightforward decision. Scientists are learning that the solvent itself is a key process parameter with spillover effects that can even affect stereochemistry.
The choice of solvent can have profound effects on product quality, according to a recent literature review by Xiamen University scientists Yuxuan Hou, a student, and professors Jinyu Meng, PhD; Binghui Chen, PhD; Jifeng Yuan, PhD; and Shizhen Wang, PhD.
“DES composition and water content shape the enzyme-specific microenvironment and thereby affect catalytic performance,” they note. Changing it affects the DES hydrogen- bond network, viscosity, diffusivity, polarity, and solvent distribution around the protein surface.
They cited studies in which the residual activity of cellulase and pectinase rose and then fell as water quantities increased, and a hydrogenase/TiO2 photocatalytic system in which increasing the solvent quantity reduced dioxygen transport, and increasing the water content increased activity.
Additionally, they write, “Accumulating evidence indicates that DES can enhance enzyme thermostability while preserving catalytic activity.” The choice of hydrogen bond donors and acceptors was a key determinant.
For example, in the case of Candida rugosa lipase, the hydrogen bond donor enabled the enzyme’s half-life to increase from one hour to more than nine hours using DES. For Humicola insolens cutinase, the hydrogen bond acceptor was the key determinant, increasing half-life from 22 minutes to 62. The benefit is that modulating thermostability through the solvent rather than through bioengineering is faster and more cost-effective.
“In addition, the aqueous fraction can affect the interactions between DES components and proteins, and alter hydration, thereby further modulating enzyme thermostability,” the scientists point out.
Changing the solvent can also affect the stereoselectivity and regioselectivity of an enzyme. Hou and colleagues cite one study in which baker’s yeast was used to convert arylpropanones [high value intermediates used to manufacture some active pharmaceutical ingredients]. With water as a solvent, the arylpropanones were largely converted to the “S” version, but when DES was used, the conversion was predominantly to the “R” version.
This solvent-modulated selectivity significantly enhances flexibility, but it also means quality must be finely controlled to ensure the choice of solvent doesn’t negatively affect the product.
Clearly, the choice of solvent has serious consequences for product quality that biomanufacturers may not fully appreciate. The challenge is a lack of data regarding enzyme behavior in various solvents.