| Papain is widely found in the roots,stems,leaves,and fruits of the papaya(Carica papaya),which is the most abundant in immature milk.The active center of papain contains cysteine,which belongs to the thiol-containing(-SH)peptide endopeptidase,it has the characteristics of high enzyme activity,good thermal stability,natural health and safety,and so on,so in food,medicine,feed,day Chemical,leather and textile industries are widely used.In this experiment,a novel,green extraction technique using ionic liquid biphasic phase was used to seperate papain.It combines the advantages of both ionic liquids and aqueous two-phases system,effectively combining mild,high-efficiency,and designability.In order to study the partitioning behavior of papain in the aqueous two-phase system of imidazole ionic liquids,two ionic liquids of imidazole diamide amine salt and imidazole acetate were selected in this paper.Their phase forming ability and phase equilibrium data were investigated.Optimization of papain extraction conditions by single factor analysis and response surface experiments.The ionic liquid two-phase system with the best effect was selected,the distribution model in the system was established and verified,and the results were verified by SDS-PAGE electrophoresis.The main contents are as follows:(1)Two kinds of ionic liquids and different inorganic salts were used to determine the binodal lines of the two-phase aqueous system.The imidazole diamide amine salt was found to have a stronger ability to form two aqueous phases than imidazolium acetate.The phase forming ability of inorganic salt and imidazole diamide amine salt is:Na2CO3>K2C03>K2HPO4>K3PO4>MgS04>(NH4)2S04>NaH2P04.[Cnmim]Ac(n=2,4,6,8)can only phase with K2CO3,K2HP04(only[C2mim]Ac and K2HPO4 cannot phase).The Merchuk equation was used to correlate the double-tap data,and the correlation coefficient was substantially greater than 0.99.(2)Select a suitable point from the phase diagram to prepare a dual aqueous system and determine the content of the upper and lower phase components.Using Othmer-Tobias and Bancroft empirical equations to correlate the data,the correlation coefficients are all greater than 0.99,and the standard deviations are all less than 6.75%,indicating that the test selected equation is applicable to correlate the phase equilibrium data of[Cnmim]Ac(n=4,6,8)-K2HPO4 and[Cnmim]N(CN)2-K2HPO4 ionic liquid aqueous two phase(3)Determine the effect of inorganic salt and its concentration on papain activity.The changes in the concentrations of MgSO4.K2HPO4 and NaH2PO4 did not cause large changes in enzyme activity,but also increased the activity of the enzyme to some extent.The(NH4)2SO4.K3PO4.Na2CO3.K2CO3 will inhibit the activity of the enzyme as the concentration increases,and even at high concentrations will destroy the spatial structure of the enzyme.(4)By examining each single factor,the extraction effect of[Cnmim]Ac(n=4,6,8)-K2HPO4 and[Cnmim]N(CN)2-K2HPO4 systems on papain was found.The effects of ionic liquid concentration,salt concentration and pH on the partitioning behavior of papain in the aqueous two-phase system were investigated.The optimum extraction conditions were:mass fraction of[C8mim]Ac 27.79%,mass fraction of K2HPO420.09%,pH = 8.88,partition coefficient and enzyme activity recovery can reach the maximum,the partition coefficient is 11.65,and the enzyme activity recovery rate is 88%.(5)Based on the above tests,determine the concentration of each component in the upper and lower phases and the partition coefficient K,and examine the correlation between the concentration of each component and the distribution coefficient.The correlation coefficient between the partition coefficient and the difference between the upper and lower phase ionic liquid concentrations and the salt concentration difference is greater than 0.84,which can be used to establish the distribution model.The difference between the concentration of InK and the upper and lower phases is:The correlation coefficient of model was greater than 0.9757,and the relative deviation was less than 5%.Through verification,this model can accurately predict the distribution behavior of papain in this system.The research on this topic provides a new method and theoretical basis for the preparation of high-purity and high-activity papain,provides theoretical guidance for establishing the distribution model of bioactive substances in ionic liquid biphasic phase.It is helpful for solving the key problems that restrict the purification technology of papain. |
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