The ratio of substrate to catalyst (S/C) is a prime target for the application of asymmetric production of enantiomerically enriched intermediates by whole-cell biocatalyst. In the present study, an attractive increase in S/C was achieved in a natural deep-eutectic solvent (NADES) containing reaction system under microaerobic condition for high production of (S)-1-[3,5-bis(trifluoromethyl)phenyl]ethanol ((S)-3,5-BTPE) with Candida tropicalis 104. In PBS buffer (0.2 M, pH 8.0) at 200 rpm and 30 °C, 79.5 g (Dry Cell Weight, DCW)/L C. tropicalis 104 maintained the same yield of 73.7% for the bioreduction of 3,5-bis(trifluoromethyl)acetophenone (BTAP) under an oxygen-deficient environment compared with oxygen-sufficient conditions, while substrate load increased 4.0-fold (from 50 mM to 200 mM). Furthermore, when choline chloride:trehalose (ChCl:T, 1:1 molar ratio) was introduced into the reaction system for its versatility of increasing cell membrane permeability and declining BTAP cytotoxicity to biocatalyst, the yields were further increased to 86.2% under 200 mM BTAP, or 72.9% at 300 mM BTAP. After the optimization of various reaction parameters involved in the bioreduction, and the amount of biocatalyst and maltose co-substrate remained 79.5 g (DCW)/L and 50 g/L, the S/C for the reduction elevated 6.3 times (3.8 mM/g versus 0.6 mM/g). By altering the respiratory pattern of the whole-cell biocatalyst and exploiting the ChCl:T-containing reaction system, the developed strategy exhibits an attractive potential for enhancing catalytic efficiency of whole-cell-mediated reduction, and provides valuable insight for the development of whole-cell catalysis.
Keywords: cofactor regeneration; natural deep-eutectic solvents (NADESs); oxygen-deficient environment; ratio of substrate/catalyst (S/C).