Partition Behavior Of Papain In Aqueous Two-Phase PEG/Ammonium Sulfate And PPG/ion Liquid Systems

As a result of strong protein hydrolysis ability and has wide adaptability at pH and temperature, papain has a wide application in food, medicine and so on. Traditional extraction method of papain can not meet the needs of high quality papain, so, it has important application value to find a new way of preparation of high quality, high active papain. Aqueous two-phase system (ATPS) as an environmental and efficient separation has been widely used in biologically active substances separation and purification. For most of the aqueous two-phase system researches have focused on optimization system, it has less reported on the correction of PEG/ammonium sulfate and ion liquid aqueous two phase system on extraction of papain. The liquid-liquid equilibrium dates are studied by cloud point measurement. The experimental data of the aqueous two-phase systems were fitted by empirical nonlinear expression. With papain as a model protein, many factors affecting on partitioning behavior have been studied. The experiment studied the correlation between logarithm of the partition coefficient and the concentration of each component, and established an empirical equation. The correlation method is not available of the PPG400/[Amim]Cl aqueous two-phase system.The detailed information of the paper is summarized as follows:(1) Liquid-liquid equilibrium of the polyethylene glycol (PEG)/ammonium sulfate or ionic liquids aqueous two-phase systems. Phase diagrams were determined by the cloud point method. We investigated the effect of molar mass of PEG, the type of ionic liquids and temperature on the liquid-liquid equilibrium for aqueous two phase systems. The bimodal curves were fitted using an empirical equation. The tie lines were correlated using the Othmer-Tobisa and Bancrof equations. The resulted showed that the ability of PEG and ionic liquid for two phase formation followed the order:PEG6000>PEG4000>PEG2000;[Amim]Cl>[C4mim]Ac>[C4mim]Cl. It was observed that the two phase area of PEG/ammonium sulfate was expanded with increasing temperature. The correlation coefficients (were>0.99) demonstrated the reliability of the equation. The objects of this work are to provide the basic data and the necessary theoretical guidance.(2) PEG/(NH4)2SO4aqueous two-phase system is used to extract papain. The influence of PEG with different molecular weights and concentration, the mass faction of (NH4)2SO4, pH value and enzyme added on distribution behavior in aqueous two-phase system was discussed. The separation condition of papain in aqueous two-phase system was optimized by response surface method. The results showed that the best extraction conditions are:20.16%(w/w) PEG6000,18.11%(w/w)(NH4)2SO4, pH7.34, the dosage of papain is2mg/g. In this condition, the partitioning coefficient of papain in the two-phase system up to2.79, papain activity recovery of77.49%can beobtained.(3) PPG400/[Amim]Cl aqueous two-phase system is used to extract papain. The influence of the concentration of PEG, the concentration of [Amim]Cl, pH value, the temperature and enzyme added on distribution behavior in aqueous two-phase system was discussed. The separation condition of papain in aqueous two-phase system was optimized by response surface method. The calculated ΔtrG°m values are negative for all systems evaluated reflecting the spontaneous and preferential preferential partitioning of papain for IL-rich phase and as indicated by the K>1. ΔtrH°m values are negative indicating that the transference of papain from PPG400-rich phase to the IL-rich phase is an exothermic process. The results showed that the best extraction conditions are:40%(w/w)[Amim]Cl,20%(w/w)PPG400, pH7.61,the dosage of papain is2.24mg/g. In this condition, the partitioning coefficient of papain in the two-phase system up to3.24, papain activity recovery of90.78%can be obtained. Comparaed with the partitioning coefficient of papain, the PPG400/[Amim]Cl aqueous two-phase system is more suitable for the separation and purification of papain.(4) Based on above experiments, the experiment studied the correlation between logarithm of the partition coefficient and the concentration of each component, and established an empirical equation. Due to the correlation less than0.7, the correlation method is not available of the PPG400/[Amim]Cl aqueous two-phase system. The mean relative deviations between the experimental and calculated of papain partition coefficient were<10%and less than other similar models. Experimental results demonstrated the reliability of the model. The result provided a solid foundation for further research and engineering calculation.