Thiol‐Containing Deep Eutectic Solvents as 2‐In‐1 Cofactor Regeneration Media For Oxidoreductases: Understanding Non‐Innocent Solvents

ABSTRACT Oxidoreductases exhibit high enantio‐ and chemoselectivity, but their industrial use is often limited by cofactor dependence and poor water‐solubility of substrates. Deep eutectic solvents (DESs) can address these issues by acting as enzyme‐compatible media, substrate solubilizers, and cofactor‐regenerating systems. This work presents a new class of thiol‐containing DESs for biocatalysis that can function as 2‐in‐1 solvents and cofactor regenerators. Thermophilic thioredoxin reductase/thioredoxin‐1 (TR/Trx‐1) from Thermus thermophilus regenerates NADPH using dithiothreitol (DTT). DTT‐based DESs prepared with choline chloride, betaine, or glycerol enable higher DTT concentrations while maintaining enzyme compatibility. However, effective DES design requires balancing multiple parameters, particularly water content and final pH, which strongly influence enzyme stability and activity. Preadjusted buffer pH is essential to achieve optimal conditions in DES–aqueous systems. The reduction of acetophenone to ( R )‐phenylethanol using Lactobacillus brevis alcohol dehydrogenase, in combination with TR/Trx‐1, was evaluated in DTT‐based DESs. Optimized conditions (20 vol.% DES, final pH 7.5) enabled higher substrate loading (75 mM) and yield (82%), though enzyme stability remained limited. Integrating media engineering with protein engineering will be crucial to fully exploit these tunable solvent systems.