Design And Synthesis Of Betaine-based Deep Eutectic Solvent And Its Application In Biocatalysis And Extraction Of Bioactive Substances

In the economic era when the scale of the solvent industry is expanding,the birth of green solvents is quietly occupying the mainstream market of the solvents industry.Some solvents that can produce "three wastes" in production or application are gradually eliminated,which caused the increasingly appearance of some new solvents.Deep eutectic solvent(DES)is a new solvent with similar properties of ionic liquids that developed after it.On the other hand,deep eutectic solvent is gradually replacing some of Ionic liquid with high cost and complex synthesis process for its low cost,easy availability of raw materials and simple synthesis.The research of DES is still in its infancy at this stage,and the most researched of which is the DES system based on choline chloride.The design,synthesis and application of DES with different physical and chemical properties are crucial to the development of this new green solvent.This experiment focuses on the development of betaine-based DES system,and explores the application of this new solvent system in two directions: biocatalysis and extraction of biologically active substances.The specific research contents are as follows:(1)Experimental design and synthesis of six kinds of natural deep eutectic solvents(Natural deep eutectic solvent,NADES)based on betaine and choline chloride were performed,and the stability of bovine erythrocyte carbonic anhydride(Ⅱ)was evaluated in two NADES solvent systems.Our experiments discussed in detail the effects of NADES composition and concentration on its physical and chemical properties,as well as CA activity and structure.At the same time,the effects of metal ions,anions and high temperature on CA activity were analyzed to evaluate the stability of CA in the NADES solvent systems.The results showed that Bet-Gly(1:2)NADES with a mass fraction of 55% is more suitable as a solvent system for carbonic anhydrase.Carbonic anhydrase still maintained 96% of the initial enzyme activity after incubated in 55% NADES at 60 ℃ for 12 hours,showing the excellent thermal stability of Carbonic anhydrase.In addition,the experiment also proved by fluorescence spectroscopy and circular dichroism analysis,combined with theoretical calculations,that the carbonic anhydrase molecules existed in the NADES hydrogen bond network in a more compact conformation.The protective effects of the three-dimensional supramolecular network structure improved the carbonic anhydrase stability.(2)The experiment designed 14 deep eutectic solvents based on betaine for the extraction of flavonoids from chrysanthemum.After that we analyzed the physical and chemical properties of DES by means of viscosity and conductivity characterization.In addition,the formation of hydrogen bonds in DES was proved by the characterization of infrared spectroscopy.The effects of DES types,material-to-liquid ratio,extraction time and extraction temperature on the extraction volume of total flavonoids,quercetin and quercetin were specifically discussed.The experimental results showed that Bet:1,2-Prop(1:3)-DES,Bet:U(1:2)-DES and Bet:EG(1:2)-DES were the best DES systems for extracting total flavonoids.Under the optimized conditions,the content of quercetin extracted by Bet:U(1:2)-DES was the highest,and the final extraction amount was 11.57 mg/g,which was better than that of 70% methanol system.The extraction of total flavonoids and quercetin with Bet:1,2-Prop(1:3)-DES under optimized conditions was better than that of Bet:U(1:2)-DES and Bet:EG(1:2).)-DES,and the extraction amount of total flavonoids and quercetin was 84.60 mg/g and 10.14 mg/g respectively.Antioxidant activity experiments showed that the Bet:1,2-Prop(1:3)-DES,Bet:U(1:2)-DES and Bet:EG(1:2)-DES systems had good biological properties Compatibility,and can replace traditional organic solvents for the extraction of flavonoids from plants.