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.

靛蓝是一种不溶性蓝色染料，在其生产过程中会产生严重的污染。人们越来越关注更加环保和高效的靛蓝生物合成。因此，本研究旨在探索各种深共熔溶剂 (DESs) 对细胞色素 P45-BM-3 催化吲哚产生靛蓝的具体作用。DESs是通过加热和搅拌合成的。通过核磁共振(NMR)和傅里叶变换红外光谱(FT-IR)分析了溶剂的结构，考察了溶剂的粘度、密度和折射率与溶剂的含水量之间的关系。圆二色光谱仪用于检测酶蛋白的三级结构。溶剂类型、浓度、pH值、温度的影响，使用紫外分光光度计测定水分含量对 P450 BM-3 催化活性和稳定性的影响。使用DESs和缓冲液建立新的溶剂双相系统，并使用重组制备靛蓝大肠杆菌-生物催化的吲哚。DESs是由组分通过氢键相互作用形成的低熔点共晶。DESs 的物理性质，如密度、粘度和折射率，会随着溶剂的含水量和温度而变化。DESs的pH值、含水量和温度与P450 BM-3的催化活性呈正相关。综上所述，DESs可以提高P450 BM-3的催化活性和热稳定性。使用 DESs 缓冲​​液双相系统可以有效地制备靛蓝。