| Papain is a specific proteolytic enzyme containing sulfhydryl groups in papaya,which is widely used in food,medicine and other fields.However,the traditional method of separation and purification of papain can not obtain high activity and high purity papain,therefore,it has a very important practical significance for the study of the separation method of high quality papain.As a simple and efficient separation and purification technology,aqueous two-phase system(ATPS)has been widely used in the separation and purification of protein or enzyme,but at present,most of the research on ATPS focuses on PEG/salt and ionic liquid(IL)/salt ternary ATPSs.However,There are few reports on quaternary ATPSs.Ionic liquid ATPSs are mainly concentrated in imidazole and pyridine ionic liquid ATPSs,but quaternary ammonium ionic liquid ATPSs are rarely reported.In this paper,the effect of quaternary ammonium ionic liquids assembled by different anions and cations on the phase behavior of PEG/salt ATPSs was studied,and the liquid-liquid equilibrium data of the ATPSs were determined.The ATPS with the best phase forming ability and papain separation and purification effect was selected.The process conditions for the separation and purification of papain were optimized by response surface,and the structure of papain before and after extraction was characterized.Finally,the experimental results were verified by SDS-PAGE electrophoresis.The contents are as follows:(1)Synthesis of[N2222]BF4 and its effect on phase behavior of PEG-NaH2PO4·2H2O ATPSsQuaternary ammonium ionic liquid[N2222]BF4 was synthesized and characterized by 1H NMR and its purity was verified.The phase diagrams of PEG/NaH2PO4·2H2O and PEG/NaH2PO4·2H2O/[N2222]BF4 ATPSs were determined by cloud point method,and the two-node data were correlated by merchuk equation.The effect of[N2222]BF4 on the phase behavior of PEG/NaH2PO4·2H2O ternary ATPSs was investigated,the results showed that the fitting degree of the two-node curve data was above 0.997,and the correlation degree was very good.The area of aqueous two-phase region from large to small is as follows:PEG6000+4wt%IL(IL stands for[N2222]BF4)>PEG4000+4wt%IL>PEG2000+4wt%IL>PEG6000>PEG4000>PEG2000>PEG600+4wt%IL>PEG600>PEG400+4wt%IL>PEG400,this chapter lays a theoretical foundation for the subsequent selection of ATPSs.(2)Study on Separation and purification of papain by PEG/sodium dihydrogen phosphate dihydrate/quaternary ammonium ionic liquids ATPS.PEG/NaH2PO4·2H2O ternary ATPSs and PEG/NaH2PO4·2H2O/quaternary ammonium ionic liquid quaternary ATPSs were constructed.The phase diagrams were drawn by cloud point method,and the two-node curve data were correlated by merchuk equation.The optimum PEG4000/NaH2PO4·2H2O/[N2222]BF4 ATPS was selected from the enzyme separation and purification effect,and the experimental results showed that papain tended to be distributed to the bottom phase rich in phosphate,and the separation and purification conditions of papain were optimized by response surface methodology.The optimum conditions for the separation and purification of papain by aqueous two-phase separation were obtained as follows:PEG4000 16.4 wt%,NaH2Po4·2H2O 13.7 wt%,pH 6.22,temperature 60?,enzyme concentration 12.0 mg/ml,using the total normalized value(OD)as the evaluation index.Under these conditions,the purification factor of papain increased from 1.331(without ILs)to 3.380(containing 4 wt%[N2222]BF4).The total OD value was 0.9979,the maximum predicted OD value was 0.9994,and the deviation rate was-0.15%.Therefore,the model established by the experiment can predict the experimental results very well.In order to verify the practical application effect of this model,the crude enzyme from papaya latex was separated and purified by the same ATPS.the results showed that the purification factor of crude enzyme from papaya milk was increased from 3.517 to 12.04.(3)Study on separation and purification of papain by PEG/ammonium sulfate/quaternary ammonium ionic liquid ATPSs.PEG/(NH4)2SO4 ternary ATPS and PEG/(NH4)2SO4/quaternary ammonium ionic liquid quaternary ATPSs were constructed.the phase diagram was drawn by cloud point method,and the effects of different structures on ionic liquids on the phase behavior of PEG/(NH4)2SO4 ternary ATPSs were studied.The composition and length of(TL)in ATPS were determined.the PEG400/(NH4)2SO4/[N1111]Br two-phase system with the best separation and purification effect was screened out by single factor,and the conditions of papain separation and purification were optimized by response surface methodology(RSM).Papain was distributed in the top phase of ATPSs.The optimum separation and purification conditions were as follows:salt concentration 20.20 wt%,PEG400,concentration 20.25 wt%,pH 7.0.under these conditions,by comparing the separation and purification effect of crude papain in the two systems,the purification factor of crude enzyme from papaya latex increased from 2.13 times(without IL)to 13.51 times(containing 4wt%[N1111]Br).The recovery of papain activity increased from 64.2%to 96.46%.SDS-PAGE analysis showed that the impurity protein bands of purified crude papain from papaya latex were significantly reduced.The concentration of papain bands were purified by PEG/(NH4)2SO4/[N1111]Br ATPS,this ATPS was significantly higher than that purified by PEG/(NH4)2SO4 ternary ATPS.The effect of papain separation and purification was verified.(4)Study on Separation and purification of papain by quaternary ammonium ionic liquids/salts ATPSs.On the basis of the first three chapters,the quaternary ammonium ionic liquids/salts ATPSs was constructed.the content of the top and bottom phase components and the line length of the ATPSs were determined.The best[N4444]Br/(NH4)2SO4 ATPS was screened out by separation and purification effect.The optimum conditions for separation and purification of papain were obtained by response surface methodology(RSM):(NH4)2SO4 16.00 wt%,[N4444]Br 19.55 wt%,pH 6.94.Under these conditions,commercial enzyme and crude papain extract were added respectively.Comparing the separation and purification results of the two enzymes,the protein recovery,activity recovery of papain and purification factor of the ATPS with commercial enzyme were 87.65%,96.04%and 2.870,respectively.The protein recovery,enzyme activity recovery and purification factor of papaya latex in ATPS were 79.32%,95.73%and 13.96 respectively.In conclusion,the ATPS had a better purification effect on the crude enzyme extracted from papain latex.the UV-vis absorption spectrum and FT-IR absorption spectrum of papain before and after extraction showed that the characteristic absorption peak of papain did not change.The results showed that the extraction conditions of the ATPS were mild and the conformation of papain was not changed.Papain was separated and purified by PEG/salts/quaternary ammonium ionic liquids and quaternary ammonium ionic liquids/salts ternary ATPSs,respectively.The partition behavior of papain in phosphate-rich bottom phase and PEG-rich or quaternary ammonium ionic liquids was discussed respectively.the effects of the addition of ionic liquids with different structures on the phase behavior of aqueous two-phase were compared.The optimum separation and purification system was screened out,which was innovative to a certain extent. |
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