The Research On The Synthesis Of Novel Green Solvents And Their Aqueous Two Phase Systems For Green Separation Of Proteins

Proteins are components of living organism that play critical roles in phenomenalike metabolism, gene expression, signal transduction, cellular and extracellularstructures, and the like. Proteomics is about studying organisms in protein levelquantitatively, dynamically and integrally, which mainly about the characteristics ofprotein, including the expression, translation, modification and conformation ofproteins, the interaction between proteins and so on. With the great progress ofproteomics research, separation, enrichment and rapid detection of pro teins havereceived a great attention in the field of biological technology. Protein purification isto require target proteins from a mixture using downstream biological engineeringtechnology. Traditional methods to purify proteins involve precipitation,electrophoresis, dialysis, chromatography, over-speed centrifugal and so on. Some ofthese separation techniques are time and cost consuming. So it is particularly importantto look for a protein separation method that is high efficient, low consumption, andenvironment protecting.In recent years, ionic liquids (ILs) have emerged as promising alternative mediafor the replacement of conventional organic solvents. As an environmentally friendlyreaction medium and green solvents，ionic liquids have their―green‖characteristicssuch as negligible volatility and non-flammability under ambient conditions, largeliquid range, high thermal and chemical stability, strong solubility power and a numberof possible variations in cation and anion features which allow fine-tuning of the ILproperties. However, the synthesis process of IL is complex, high-cost and difficult inpurification. In addition, pyridinium or imidazolium-based ionic liquids are notcompletely "green". These shortcomings have limited its large-scale industrialapplications and development. Therefore, finding a simply synthetic and greeneralternative solvent has very important practical significance.In this work, two kinds of novel green solvents were synthesized includingguanidine ILs and DESs, combined with aqueous two-phase extraction (ATPE) toextract proteins. The effects of various factors on the protein extraction wereresearched, the optimum extraction conditions of protein were determined, and themechanism of protein extraction was studied systematically. It is proved that theaggregation phenomenon is the virtual governing force controlling the BSA extraction.The major content of the present work is as follows: 1、Synthesis and characterization of novel green solventsSynthesis and characterization of guanidinium ILs: eight kinds of ILs consistingof1,1,3,3-tetramethylguanidinium cation and different anions were synthesized byneutralization of1,1,3,3-tetramethylguanidine (TMG) and eight different kinds ofacids (acrylic acid, methacrylic acid, acetic acid, DL-lactic acid, maleic acid, itaconicacid, sorbic acid and trans-cinnamic acid). The structures of all the synthetic ILs wereconfirmed by FT-IR,1HNMR and13CNMR spectra.Synthesis and characterization of DESs: four kinds of DESs were synthesizedby stirring two eutectic mixtures at80℃until a homogeneous, colorless liquid wasformed. The investigatived DESs were based on quaternary ammonium salts (cholinechloride, tetramethylammonium chloride and tetrapropylammonium bromide) andhydrogen-bond donors (HBD)(urea and methylurea) with the certain molar ratio (1:2)of quaternary ammonium salt to HBD. Then, the structures of all the synthetic DESswere confirmed by FT-IR.2、Extraction of proteins in guanidinium ILs-based ATPSsA method of combining guanidinium ionic liquids with aqueous two-phaseextraction to extract protein including bovine serum albumin (BSA), ovalbumin (OVA)and bovine hemoglobin (BHb) was established. The concentrations of proteins weredetermined by measuring the absorbance at278nm for bovine serum albumin (BSA)and ovalbumin (OVA), and at404nm for bovine hemoglobin (BHb) using a UV2450UV-vis spectrophotometer. The standard curve were Y=-0.00291+0.61535X(R2=0.99984), Y=0.01607+0.89953X (R2=0.99971), Y=-0.00874+6.38159X(R2=0.99945). The linearity for analyzing BSA, OVA and BHb were in theconcentration range of0.05-1.00mg/mL,0.10-1.00mg/mL and0.05–0.175mg/mLwith the correlation coefficient0.99984,0.99971and0.99945, respectively. The phasediagrams were prepared by turbid titration method to investigate the phase equilibrium.Single factor experiments proved that the extraction efficiency of bovine serumalbumin (BSA) was influenced by the mass of IL, K2HPO4and BSA, also related to theseparation time and temperature. The optimum conditions (IL:3.0mmol, K2HPO4:0.5g/mL, BSA:25mg, T:25℃, t:30min) were determined through orthogonalexperiment by the five factors described above. The results showed that under theoptimum conditions, the extraction efficiency could reach up to99.62%. UV-visspectra, FT-IR spectra, DLS and TEM were combined to investigate the microstructureof the top phase and the possible mechanism for the extraction. Methodological study showed that the proposed method had good precision and reproducibility, providing anew way of green separation for proteins.3、Extraction of proteins in DESs-based ATPSsILs was replaced by DESs which are simple synthesized, low cost and greener. Amethod of combining DESs with ATPE to extract protein including bovine serumalbumin (BSA), ovalbumin (OVA) and trysin (Try) was set up. The concentrations ofproteins were determined by measuring the absorbance at278nm for BSA, OVA andTry using a UV2450UV-vis spectrophotometer. The standard curve werey=-0.00291+0.61535x, R2=0.99984(BSA), y=0.01607+0.89953x, R2=0.99971(OVA),y=0.00154+0.13881x, R2=0.99993(Try). The linearity for analyzing BSA, OVA andTry were in the concentration range of0.05-1.00mg/mL,0.10-1.00mg/mL and0.05-1.00mg/mL with the correlation coefficient0.99984,0.99971an d0.99993,respectively. Single factor experiments proved that the extraction efficiency of BSAwas influenced by mass of DES, concentration of K2HPO4solution, separation timeand extraction temperature. The optimum conditions (DES:1.6g, K2HPO4:0.6g/mL,T:25℃, t:10min) were determined through orthogonal experiment by the fourfactors described above. The results showed that under the optimum conditions, theextraction efficiency could reach up to99.90%. UV-vis spectra, FT-IR spectra, CD,DLS and TEM were combined to investigate the microstructure of the top phase andthe possible mechanism for the extraction. Studies showed that the proposed methodhad good precision and reproducibility, and made some breakthrough in the field ofextraction solvent.