Effect of Deep Eutectic Mixtures in Hydroxylation of Fatty Acids: A Correlation between Water Activity and Thermostability of FA-HY1

To validate the performance and application of fatty acid hydratases in green and sustainable deep eutectic solvents (DESs), a robust system with the combination of fatty acid hydratase-1 (FA-HY1) from Lactobacillus acidophilus and DESs for the biocatalytic hydroxylation of fatty acids was developed. As the cosubstrate of hydroxylation reactions, water molecules have been proven to be a key factor for the thermostability of FA-HY1 in DES systems. We found that FA-HY1 displayed improved thermostability at lower water activity. In particular, the half-life time of FA-HY1 increased 6-fold in a choline chloride/sorbitol system with water activity (a = 0.84) compared to an aqueous buffer system. Moreover, the thresholds of a for regulating the synthesis of hydroxy fatty acids (HFAs) in FA-HY1/DES systems were determined. We further investigated the recyclability of FA-HY1 in a choline chloride/sorbitol system, where significantly, after three rounds of recycling, a high hydroxylation efficiency of 83.2% was still observed. Monitoring the secondary structure of FA-HY1 using synchrotron radiation circular dichroism analysis revealed that the DESs appear to delay the change in confirmation of FA-HY1. In addition, molecular dynamics simulations were performed in DES and aqueous systems, which revealed the mechanistic features of the thermostability of FA-HY1 in DESs.