Mechanism of deep eutectic solvents enhancing catalytic function of cytochrome P450 enzymes in biosynthesis and organic synthesis.

Indigo is an insoluble blue dye, which generates serious pollution in its production process. Increasing focus has come to the biosynthesis of indigo that are more environment-preserved and high-efficient. Hence, this study was designed to explore the specific role of various deep eutectic solvents (DESs) on cytochromeP45-BM-3 catalyzing indole to produce indigo. DESs were synthesized by heating and stirring. The structure of the solvent was analyzed by nuclear magnetic resonance (NMR) and fourier transform infrared spectrum (FT-IR), and the relationship between the viscosity, density and refractive index of the solvent, and the water content of the solvent was examined. Circular dichroism spectrometer was used to detect the tertiary structure of the enzyme protein. The effect of solvent type, concentration, pH, temperature, and water content on the catalytic activity and stability of P450 BM-3 was measured using an ultraviolet spectrophotometer. A new solvent biphasic system was established using DESs and buffers, and indigo was prepared using recombinant E. coli-biocatalyzed indole. DESs were low-melting eutectics formed by molecules interaction of components through hydrogen bonding. The physical properties of DESs such as density, viscosity, and refractive index varied with water content and temperature of the solvent. The pH, water content, and temperature of DESs were positively correlated with the catalytic activity of P450 BM-3. To sum up, DESs can improve the catalytic activity and thermal stability of P450 BM-3. Indigo can be efficiently prepared using the DESs-buffer biphasic system.