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

Abstract

Phenolic acid decarboxylase from Bacillus subtilis (BsPAD) converts several p-hydroxycinnamic acid derivatives into the corresponding phydroxystyrenes, which are considered to be promising bio-based aromatic chemicals. Despite the enzyme's 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.

A chemo-enzymatic tandem reaction in a mixture of deep eutectic solvent and water in continuous flow

“…We recently showed the excellent compatibility of BsPAD with DESs. 36 Choline chloride (ChCl)based eutectic mixtures were tested neat and in dilution with potassium phosphate buffer (50 mM, pH 6.0) for the decarboxylation of a variety of phenolic acid derivatives. Our results proved that not only the free enzyme, but also immobilized PAD shows excellent activity in deep eutectic solvents.…”

“…Recent findings showed that PAD is highly active in DES. 36 Experiments on interactions of the used DES and the biocatalyst were not coducted so far and are not scope of this work. A series of recently investigated DES-protein systems can be found in literature.…”

Non‐Conventional Media as Strategy to Overcome the Solvent Dilemma in Chemoenzymatic Tandem Catalysis

“…The addition of 30 % the cosolvent ethanol for the Heck reaction further diluted the reaction solution. PAD from Bacillus subtilis shows very good activity in a mixture of choline chloride:glycerol (1 : 1) with 50 % (v/v) water, which allowed to increase the substrate solubility dramatically . Due to the high viscosity of pure DES‐solutions, a 50 % mixture with water appeared to be highly suitable for a continuous approach.…”

“…PAD from Bacillus subtilis shows very good activity in a mixture of choline chloride: glycerol (1 : 1) with 50 % (v/v) water, which allowed to increase the substrate solubility dramatically. [28] Due to the high viscosity of pure DES-solutions, a 50 % mixture with water appeared to be highly suitable for a continuous approach. In a first, enzymatic step, a 70 mM solution of coumaric acid was decarboxylated by PAD encapsulated in alginate beads at 30°C.…”

A Phenolic Acid Decarboxylase-Based All-Enzyme Hydrogel for Flow Reactor Technology

“…The resulting styrene derivatives are of great industrial relevance since the terminal alkene provides a versatile handle for further chemical functionalization or polymerization reactions [16][17][18]. In contrast to other (de)carboxylases, several PADs found in bacteria [19][20][21][22] and plants [23] act on an acid-base mechanism via a quinone methide intermediate [24] and do not require a cofactor, therefore eliminating the need of a laborious and expensive cofactor regeneration system [25,26].…”