Natural deep eutectic solvents as green and biocompatible reaction medium for carbonic anhydrase catalysis

Easy deactivation of free enzymes under non-native condition has become a stumbling block to the industrial application of biocatalysis. Natural deep eutectic solvent (NADES) has been exploited as a novel reaction medium for improving enzyme stability. The present work focused on preserving and enhancing the activity of carbonic anhydrase (CA) in a more economical and biocompatible NADES system. We synthesized six choline chloride/betaine-based NADES and analyzed the effects of compositions and concentrations of NADES on their physicochemical properties. The Bet-Gly (1: 2) NADES (55%) was proved to be more suitable as reaction medium for CA by analyzing enzyme activity in the presence of NADES. The enhancement in the stability of CA was found to be as a result of a three-dimensional hydrogen bonding network, rather than the individual or the synergistic effect of betaine and glyceride. The conformational change of CA to become more compact was confirmed both by fluorescence spectrum analysis and circular dichroism analysis. It is worth mentioning that a remarkable thermal stability was maintained when CA was incubated at temperature below 60 °C, and about 96% of activity was still restored in 55% NADES at 60 °C for 12 h.