Study Of The Synthesis Of [C_nPy]Cl(n=2,4,6) Pyridine Ionic Liquid And The Extraction Of Papain Using Aqueous Two-Phase System

As an environmentally friendly green solvent,the application fields of ionic liquids range from synthetic chemistry to separation and purification,functional materials and many other fields.Papain has the characteristics of high temperature resistance,good stability and is widely used in food,medicine and other fields.Traditional methods of papain extraction have many disadvantages such as the enzyme activity is not stable,so it can not meet the demands of modern industry.Therefore,a new method for preparing high-purity and high-activity papain is extremely needed.The synthesis of N-alkyl pyridine chloride salt ionic liquid was studied in this paper and applied to[CnPy]Cl(n=2,4,6)-K2HPO4 aqueous two-phase system.The phase behavior of the system and the partition behavior of papain in the system were explored,and the partition model of papain in this kind of aqueous two-phase system was established.The optimal extraction conditions were determined by single factor and response surface experiments.The purification results were verified by SDS-PAGE electrophoresis.The results are shown as follows:(1)Synthesis of three N-alkyl pyridine chloride salt ionic liquids.The N-ethyl pyridine chlorine salt(EPyCl),N-butyl pyridine chlorine salt(BPyCl),N-hexyl pyridine chlorine salt(HPyCl)were synthesized by one-step method.The purity of three products were characterized by Differential Scanning Calorimetry(DSC).These products yield were 92.42%,90.07%,85.67%,respectively.The three products were characterized by DSC and the purity of them were ideal.The L9(34)orthogonal table was used to optimize the synthesis conditions and the optimal synthesis conditions were reaction temperature 80?.72 h,n(pyridine):n(chloralkane)=1.0:1.2 mol and the dosage of ethyl acetate solvent 100 mL.(2)Study of phase diagrams.The binodal curve data of the[CnPy]Cl(n=2,4,6)-K2HPO4 system was determined by the clear-cloud point assist method at a temperature of 298.15 K and then drew the triangle phase diagrams.The data fitting of binodal curve was based on Merchuk and the fitting degree was above 0.996,which was ideal.(3)Study of liquid/liquid/solid boundary lines.The effects of the side alkyl chain length of the ionic liquid and the temperature of the system on the slope of the liquid/liquid/solid boundary were investigated and linearly fitted.The slope was negatively correlated with the length of the side alkyl chain,temperature and phase-forming ability.The order of phase-forming ability is:[C6Py]Cl>[C4Py]CI>[C2Py]Cl.(4)Study of Liquid/liquid equilibrium.The content of the top and bottom phases in the system was determined by refractive index method and acid-base neutralization method at T=298.15 K,and the two empirical equations Othmer-Tobias and Bancroft were used to correlate the data.The linear correlation coefficient was higher than 0.995.The equations are well correlated and provide data support for the establishment of the model.(5)Study of model.The correlation between the logarithm of papain partition coefficient K and the concentration difference of the top and bottoM phase components was higher.Thus,the model Was found by Matlab at T=298.1 5 K.The relative deviation of the model was less than 4%.The model is shown as follows:LnK=AL+BL2+CAmong them,L=(wil2+ws2)1/2,wil and ws are the concentration differences in the top and bottom phases of the ionic liquid and the salt,respectively.(6)Study of partition behavior.The effects of the concentration of K2HPO4,concentration of ionic liquid,dosage of papain,pH and temperature on the partition behavior of papain were investigated by the single factor experiment.Box-Behnken Design of RSM was developed to optimize the extraction conditions.The optimized extraction conditions were 0.35 g/mL K2HPO4,0.25 g/mL[C4Py]Cl,pH 7.87,dosage of papain 2.17 mg/mL,temperature 30?.The purification fold reached 1.88 compared with that of commercial enzyme.It was obviously that through the SDS-PAGE electrophoresis experiment,the miscellaneous lane of the purified enzyme decreased.In this paper,the synthesized ionic liquids were successfully applied to the aqueous two-phase system to extract papain,and a high-purity enzyme was obtained.This paper provides theoretical guidance for promoting the industrial preparation of high-purity and high-activity papain.