Preparation And Application Of Affinity-based Aqueous Two-phase Mixed Micellar System

Aqueous two-phase micellar system(ATPMS)is an advantageous liquid-liquid extraction technique in biochemistry,which has been proposed as an attractive and useful method for the separation and purification of biomaterials.In order to improve the specificity of ATPMS,a novel affinity-based ATPMS composed of mixed micelles was constructed by introducing a metal-chelated surfactant Triton X-114-IDA-Cu(II)(TX-Cu(II))into an aqueous solution of hydrophobically modified ethylene oxide polymer(HM-EO),and the potential application of this HM-EO/TX-Cu(II)system was investigated.A novel copper-chelated surfactant Triton X-114-IDA-Cu(II)(TX-Cu(II))was fabricated by successive modifications of nonionic surfactant Triton X-114(TX).Briefly,the hydroxyl group of TX was activated by epichlorohydrin and then was coupled with IDA.Finally,the TX-IDA was chelated with copper ions to prepare TX-Cu(II).The synthesis conditions on the TX-Cu(II)were investigated and optimized systematacially.The results indicated that the yield of TX-epoxide above 79.1%was obtained in the ice-water bath composed of n(ECH):n(TX)=1.5:1,n(BF3):n(TX)=1:20 for about 18 h,and then 10 mol/L NaOH was added at n(NaOH):n(TX)=2:1 for about 4 h.Subsequently,The optimal conditions for preparing TX-IDA were explored,and the yield of TX-IDA about 74.5%was achieved at pH 12.5,n(IDA):n(TX)=1.5:1,55 ?,20 hours.The following chelation of copper ion was a relatively easy reaction procedure.When the n(Cu2+):n(TX)=1:1,and the solution was stirred at room temperature for 6h,the reaction yield of TX-Cu(II)obtained was 65.2%with a purity of 90.5%.The affinity-based binary aqueous two-phase micellar system(ATPMS)was constructed by introducing the synthetic surfactant TX-Cu(II)into an aqueous solution of HM-EO.The phase diagram of the HM-EO/TX-Cu(II)system was measured.Experimental results indicated that the HM-EO was capable of forming mixed micellar aggregates with TX-Cu(?),and the mixed micellar system could spontaneously separate into two phases at a lower temperature(4 ?).The rise of temperature enabled the area between two coexistence curves enlarge significantly,whereas the decrease of solution pH was favorable to improving the phase separation of mixed micellar system.It was also found that the addition of salt made the mixed micellar network formation and phase separation easier.The ability of anions to promote phase separation followed the order SO42->Cl>Br>I-,and that of cations was K+>Na+.The BSA and lysozyme were selected as model proteins,through measuring the partition coefficients of them in the HM-EO/TX-Cu(?)system,the effect of various factors,such as the concentration of phase-forming surfactants,pH,temperature and conductive were investigated.It was found that both the BSA and lysozyme mainly partitioned to the micelle-poor phase due to the excluded-volume effects,and the partition coefficients of them increased with the increase of HM-EO content.The pH of the solution could affect the partitioning behavior of a protein primarily by modifying the charge distribution over the biomolecule surface.The increase in the ionic strength of the solution could reduce the interaction between the protein and surfactant head groups,hence changing the protein partition coefficient.With the increase of temperature,there was an increase of the excluded-volume effect in the micelle-rich phase,which effects the partition of BSA and lysozyme into the micelle-poor phase.However,the rise of temperature could also lead to changing of protein structure or denaturation that was not favorable for the enzyme recovery of lysozyme.In addition,the total enzyme recovery of YND decreased slightly with the TX-Cu(?)content increased from 0 to 0.1%,indicating no obvious loss of enzyme activity occurred in the HM-EO/TX-Cu(?)system.The affinity partitioning of hexahistidine-tagged Yeast 3',5'-bisphosphate nucleotidase(YND)in the HM-EO/TX-Cu(?)system was performed to learn its potential.It was found that the selectivity of the ATPMS enhanced with increasing TX-Cu(?)content.The YND was partitioned preferentially to the micelle-rich phase due to affinity binding of it to TX-Cu(?)and was stable in the system,while the protein impurities were removed to the micelle-poor phase,where they experienced fewer excluded-volume effects.Finally,the HM-EO/TX-Cu(II)was used directly to extract YND from the fermentation broth.In the forward extraction experiment,the recovery yield in the micelle-rich phase was 63.58%with a purification factor of 1.42.After back extraction,YND was extracted successfully into the micelle-poor phase with a recovery yield of 49.23%,corresponding to a purification factor of 2.63.The results indicated that the new affinity-based HM-EO/TX-Cu(II)system had high partitioning performance which is promising for effectively separation of the histidine-tagged proteins.According to the above,a novel affinity-based ATPMS composed of HM-EO and metal-chelated surfactant TX-Cu(II)was constructed,and used for the separation of intracellular enzyme histidine-tagged YND.The liquid-liquid phase-equilibrium features of the HM-EO/TX-Cu(II)system was investigated,and the partitioning behavior of model proteins(BSA,lysozyme)were studied by using this new system.On this basis,the possibility of this novel HM-EO/TX-Cu(II)system for separating histidine-tagged proteins from the fermentation broth was demonstrated by the affinity partitioning of YND.The affinity-based ATPMS would have potential applications in the separation and purification of biomolecules.