How Medium Affects the Activity and Stereopreference of Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase Catalyzed Reduction of Ketones

The activity and selectivity of W110G Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase were altered by cosubstrate and cosolvent at varying temperatures. A sharp drop in the enantiomeric excess (ee) was observed at 60°C in the first 3 h, suggesting increased selectivity mistakes in the reduction of 4-phenyl-2-butanone to the expected (S)-4-phenyl-2-butanol using 5% v/v of 2-propanol as a cosubstrate. The ee increased exponentially with cosubstrate concentration, reaching ≥94% with 30-70% v/v 2-propanol. However, a decrease in enzyme activity was noticed at ≥30% v/v 2-propanol by a sharp drop in conversion. The lowest ee (<3%) was registered using 5% v/v 2- propanol at 30-40°C, which prolonged enzyme life that allowed reversible redox reaction with selectivity mistakes to give the R-alcohol compared to ≥18% ee at 50-60°C, where faster reaction rates promoted selectivity mistake, but enzyme life was shortened by protein denaturation at the elevated temperatures. Water-miscible and immiscible organic cosolvents (25% v/v) increased enzyme selectivity. For methanol, ethanol, ethylene glycol and tert-butanol, the activity/conversion decreased with an increase in pKa and log P while the stereoselectivity/ee increased.