Using Deep Eutectic Solvents to Overcome Limited Substrate Solubility in the Enzymatic Decarboxylation of Bio-Based Phenolic Acids

Phenolic acid decarboxylase from Bacillus subtilis (BsPAD) converts several p-hydroxycinnamic acid derivatives into the corresponding p-hydroxystyrenes, which are considered to be promising bio-based aromatic chemicals. Despite the enzymes high activity and stability, the low solubility of its substrates presents severe limitations for the establishment of industrial processes. Accordingly, deep eutectic solvents (DESs) have emerged as interesting alternatives to aqueous or organic solvents and biphasic systems as they offer unique reaction conditions while remaining biocompatible and biodegradable. Herein, we show that BsPAD could tolerate choline chloride (ChCl)-based eutectic solvents with 0-50% water content, which allowed conversion to the corresponding p-hydroxystyrene derivatives (>99%) at substrate loadings of up to 300 mM due to the exceptional solubilizing properties of this solvent. As the enzyme showed some remarkable reactivity differences in DES and water, we further explored the substrate scope of the enzyme and a mutant with increased space in the active site. The comparison of substrates with different substituents on the aryl group indicated that the substrate preference is determined by steric, rather than electronic effects. Furthermore, we report that the choice of the solvent influences the acceptance of different substrates as evidenced by the fact that DES strongly favored the conversion of caffeic acid, which is only poorly converted in aqueous media.