Preparation Of Phenylboronic Acid Functionalized Temperature-sensitive Block Polymer And Its Application In Separation And Purification Of Polysaccharide And Glycoprotein

As common natural products,polysaccharides and glycoproteins have been applied to all aspects of human life,and they have become a research hotspot because of their unique biological activities.Common separation and purification methods for polysaccharides and glycoproteins are complicated in operation,and the product purity is not high.Meanwhile,the separation and purification process will destroy the structure and activity of the product.Based on this,in this study,the phenylboronic acid-functionalized thermosensitive block polymer was designed and prepared,and anew methods for separating and purifying polysaccharides（astragalus polysaccharide）and glycoproteins（HRP）were proposed.The details are as follows:（1）A new phenylboronic acid functionalized temperature-sensitive block polymer PEG₁₁₃-b-PVBA₄₉-b-PNIPAM₁₀₅05 was synthesized by reversible addition fragmentation chain transfer（RAFT）polymerization reaction,by infrared spectroscopy（FT-IR）,ultraviolet-visible,¹H NMR and gel permeation chromatography（GPC）characterized the temperature-sensitive block polymers PEG₁₁₃-b-PNIPAM₁₀₅05 and PEG₁₁₃-b-PVBA₄₉-b-PNIPAM₁₀₅05 Structure,degree of polymerization and molecular weight;measure the UV-visible light transmittance of the polymer solution at different temperatures,study the change of the cloud point of the temperature-sensitive block polymer,by comparing PEG₁₁₃-b-PNIPAM₁₀₅05 and PEG₁₁₃-b-PVBA₄₉-b-PNIPAM₁₀₅05 LCST of verified the successful synthesis of phenylboronic acid functionalized temperature-sensitive block polymer.And by controlling the temperature,the clear-turbidity phenomenon of the solution was intuitively observed;dextran with different molecular weights will have different effects on the cloud point of the PEG₁₁₃-b-PVBA₄₉-b-PNIPAM₁₀₅05 solution,and the three dextran reduce the temperature sensitivity The molecular weight order of the LCST of the block polymer is 100,000<40,000<6000.（2）Based on the influence of the different molecular weights of polysaccharides on the cloud points of the phenylboronic acid-functionalized thermosensitive block polymer,a new method for fractional precipitation of astragalus polysaccharides by controlling temperature was developed.Due to the different effects of astragalus polysaccharides with different molecular weights on the cloud point of the polymer,the conditions of PEG₁₁₃-b-PVBA₄₉-b-PNIPAM₁₀₅05 temperature-controlled precipitation of astragalus polysaccharides were explored.The experimental resultsshowed that PEG₁₁₃-b-PVBA₄₉-b-PNIPAM₁₀₅05 and astragalus The optimal binding pH of polysaccharide was 8,the optimal dissociation pH was 4,both the time of binding and dissociation were 30 min.Different fractions of astragalus polysaccharides（APS1,APS2,APS3 and APS4）precipitated at different temperatures.A series of structural tests were carried out on the four fractions of astragalus polysaccharides,including FT-IR,SEM,GPC,GC and Congo red experiments,and their antioxidant and antitumor activities were studied.The Congo Red experiment can determine that APS1 and APS3 contain a triple helix structure,APS2 and APS4 do not have a triple helix structure or the triple helix structure is specific.The monosaccharide composition and relative content of astragalus polysaccharides were analyzed by gas chromatography,and the biological activity experiment can be draw the conclution that the molecular weight of the polysaccharide will affect the biological activity of the polysaccharide fraction in a certain extent.（3）A two-step method combining aqueous two-phase extraction and affinity temperature-controlled precipitation was constructed to separate and purify glycoproteins.The aqueous two-phase extraction process was conducted for the primary separation and purification purpose,and the temperature-controlled precipitation based on the phenylboronic acid-functionalized thermosensitive block polymer was carried out for the further purification of horseradish peroxidase（HRP）.For liquid-liquid phase equilibrium of a series of PEG-salt aqueous phase systems（ATPS）,the empirical equation with the highest fitting degree of the double-bar data isw₁（28）exp(a（10）bw₂^0.5（10）cw₂（10）dw₂²).The equation was coupled with the"lever principle",and the liquid-liquid phase equilibrium data of PEG-salt ATPS at room temperature was quickly calculated by calculation software,and a complete ATPS was drawn.The PEG4000-（NH₄）₂SO₄ of the ATPS was screened with 9 kinds of targets.The best combination of the ATPS was PEG4000 with a mass fraction of 0.2and（NH₄）₂SO₄withamassfractionof0.18.The use of this ATPS successfully achieved the preliminary purification of HRP.Subsequently,phenylboronic acid functionalized temperature-sensitive block polymer PEG₁₁₃-b-PVBA₄₉-b-PNIPAM₁₀₅05 was added to various protein mixing systems to further purify HRP.The two-step separation method constructed above was applied to the actual horseradish crude enzyme solution to illustrate the effectiveness of the method.