Spontaneous formation of highly concentrated phenylacetylcarbinol via in situ phase separation in deep eutectic solvents using Candida magnoliae whole cells.

Deep eutectic solvents (DESs) are promising green media for biocatalysis, yet their application in pyruvate decarboxylase (PDC)-mediated phenylacetylcarbinol (PAC) biotransformation with product separation remains limited. This study screened nine DESs using frozen-thawed whole cells (FT-WHC) of Candida magnoliae (C. mag.), followed by evaluation of water content and DES to phosphate buffer ratios. Glycerol-based DES resulted in significantly higher (p ≤ 0.05) volumetric PDC activity and PAC productivity than other DESs. Choline chloride: glycerol (ChCl: Gly (1:2) 20% (v/v) H2O) achieved the highest overall [PAC] of 148.2 ± 0.3 mM. Adding 100% (v/v) water decreased stability of volumetric PDC activity, but doubled PAC productivity, and reduced solvent cost compared with a 20% (v/v) H2O addition. The DES-to-phosphate buffer (Pi buffer) ratio at 1:1 was optimal, while lower ratios reduced PAC production. Spontaneous formation of PAC-enriched droplets (~ 2.5 M) as a top phase indicated simplified downstream recovery. This indicates DES-based self-separation driven by polarity and product partitioning. Overall, ChCl: Gly (1:2) with 100% (v/v) H2O at a 1:1 DES: Pi buffer ratio was selected for future PAC biotransformation. These findings indicate that DESs enhance biocatalytic performance and enable integrated reaction and product separation in a single step, offering a sustainable route to biochemical production.