本文選題：膜色譜　切入點：膜色譜介質(zhì)　出處：《中國科學院大學(中國科學院過程工程研究所)》2017年博士論文　論文類型：學位論文

【摘要】：膜色譜結(jié)合了膜過濾高通量和液相色譜高選擇性的特點,能夠高效地從復(fù)雜料液如發(fā)酵液、血漿、乳清中獲得純度較高的生物活性分子,因此在生物產(chǎn)品制備領(lǐng)域具有廣闊的應(yīng)用潛力。然而,現(xiàn)階段膜色譜介質(zhì)(基膜材料)存在穩(wěn)定性較差、機械性能欠佳及其制備工藝復(fù)雜、不易控制等問題。本研究采用簡單易操作的聚多巴胺(PDA)涂覆對機械強度較高的微濾膜進行活化,然后以PDA為中間功能層偶聯(lián)膜色譜配基構(gòu)建多種膜色譜介質(zhì),并應(yīng)用于蛋白分離純化和酶的固定化。(1)通過將PDA涂覆于親水性聚醚砜膜(PES)形成中間功能層,然后偶聯(lián)三種不同的配基(聚乙烯亞胺、十二硫醇和組氨酸)制備陰離子交換、疏水和親和膜色譜介質(zhì)。處理免疫球蛋白G/人血清白蛋白(IgG/HSA)混合液時,通過陰離子交換膜色譜分離可獲得純度為96.7%的HSA;經(jīng)過疏水膜色譜分離可獲得純度為94.6%的IgG;通過親和膜色譜分離,可獲得的純度接近100%的IgG。同時經(jīng)PDA改性,可有效提高膜介質(zhì)的親水性并降低其非特異性吸附。(2)通過將PDA涂覆于疏水性聚偏二氟乙烯膜(PVDF),對其進行親水改性(膜表面接觸角由原來的116°下降至61.3°),然后偶聯(lián)聚丙烯胺配基制備得到耐鹽型陰離子(STAE)交換膜色譜介質(zhì),可用于高鹽環(huán)境下單克隆抗體的高效精制。自制STAE膜色譜介質(zhì)在含有150 mM NaCl的緩沖液中可以保留75%的蛋白吸附容量。另外,Langmuir等溫吸附模型比Freundlich吸附模型更適合模擬蛋白在STAE膜表面的吸附。當流速為10-100膜體積(MV)/min時,膜色譜介質(zhì)的蛋白吸附容量基本不受流速的影響。而且所制備STAE膜色譜介質(zhì)的機械性能、穩(wěn)定性、分離效率和重復(fù)使用性均優(yōu)于商業(yè)化產(chǎn)品。(3)以涂覆于疏水膜的PDA為平臺,可實現(xiàn)陰離子交換配基的快速匹配,構(gòu)建適用于特定分離體系的膜色譜介質(zhì)。同時通過優(yōu)化流速、洗脫鹽濃度、緩沖液pH、進樣體積和配基密度等條件,實現(xiàn)了一步法從血漿沉淀Ⅳ中分離得到純度為94.6%的α1-抗胰蛋白酶(AAT),其質(zhì)量回收率和活性收率分別為94.2%和96.6%,該純化效果優(yōu)于采用商業(yè)化產(chǎn)品獲得的結(jié)果以及文獻報道結(jié)果。本研究建立了一種通過設(shè)計膜色譜介質(zhì)實現(xiàn)復(fù)雜料液中快速純化高純度生物藥物的新方法。(4)采用自制的陰離子交換膜色譜介質(zhì)和金屬螯合膜色譜介質(zhì),對發(fā)酵料液中的漆酶進行同步純化和固定化以構(gòu)建酶膜反應(yīng)器。兩種膜色譜介質(zhì)上固定的漆酶均具有較高的純度。膜表面漆酶的活力和比活力不受固定化過程中操作流速的影響,流穿式固定化模式比傳統(tǒng)的浸泡模式更高效。所制備的酶膜反應(yīng)器具有較好的穩(wěn)定性、可重復(fù)性和良好的雙酚A(BPA)去除能力。由于金屬離子與漆酶結(jié)合更牢固,因此處理BPA料液時,用金屬螯合膜色譜介質(zhì)構(gòu)建的酶膜反應(yīng)器沒有蛋白泄漏。研究發(fā)現(xiàn)當堆疊四層膜處理(BPA)時,用金屬螯合膜色譜介質(zhì)構(gòu)建的酶膜反應(yīng)器對BPA的去除率可提高至99.2%;當料液處理通量提高至50L/m~2h時,BPA的去除效率(473.0 rmg/m~2h)高于文獻中報道的結(jié)果。
[Abstract]:Membrane chromatography combined with the characteristics of the high selectivity of the membrane filtration flux and liquid chromatography, which can efficiently plasma from the complex liquid such as liquid fermentation, and obtain bioactive molecules with high purity whey, so in the field of preparation of biological products with a wide range of potential applications. However, the present stage membrane chromatography medium (basal material) there is poor stability, poor mechanical properties and the preparation process is complex, not easy to control. This research adopts a simple and easy operation of the polydopamine (PDA) microfiltration membrane coating on the mechanical strength of high activation, then using PDA as the intermediate function layer coupling membrane chromatography ligand to construct various membrane chromatography media, immobilization and application in the separation and purification of protein and enzyme. (1) by PDA coated with hydrophilic polyether sulfone (PES) to form an intermediate function layer, and then coupling three different ligands (polyethyleneimine, twelve thiol and histidine) preparation Anion exchange and hydrophobic membrane affinity chromatography medium. Treatment of immunoglobulin G/ in human serum albumin (IgG/HSA) mixture, separation can be obtained with purity of 96.7% HSA by anion exchange membrane chromatography; through the hydrophobic membrane chromatography separation can be obtained with purity of 94.6% IgG; isolated by pro and membrane chromatography, the purity can get close to 100% IgG. and modified by PDA, which can effectively improve the hydrophilicity of the membrane medium and reduce the nonspecific adsorption. (2) the PDA is coated on the hydrophobic polyvinylidene fluoride membrane (two PVDF), the hydrophilic (membrane surface contact angle decreased from 116 degrees to 61.3 then the coupling degree), polypropylene amine ligands were prepared by salt type anion exchange membrane chromatography medium (STAE), can be used in high salt environment single clone antibody, purification. Homemade STAE membrane chromatography medium in buffer containing 150 mM NaCl can retain 75% of the protein The adsorption capacity of Langmuir. In addition, the adsorption isotherm model than the Freundlich adsorption model is more suitable for the simulation of proteins on the surface of the STAE film. When the film is 10-100 volume flow rate (MV) of /min, the effect of protein adsorption capacity of membrane chromatography medium is not affected by flow rate. The mechanical properties, and the preparation of STAE film color spectrum medium stability, the separation efficiency and repeatability are better than commercial products. (3) is coated with a hydrophobic film on the PDA platform, which can realize fast matching of anion exchange membrane chromatography medium ligand, applicable to the construction of specific separation system. At the same time by optimizing the elution velocity, salt concentration, buffer pH, sample volume and the ligand density and other conditions, the one-step separation is obtained with a purity of 94.6% alpha 1- antitrypsin IV in plasma from precipitation (AAT), the mass recovery and activity recovery were 94.2% and 96.6%, the purification effect is better than that of the commercial The product obtained by the results and literature reports. This study established a new method for rapid purification of high purity biological drugs through the design of complex liquid membrane chromatography medium. (4) using anion exchange membrane chromatography media and metal chelating membrane chromatography medium, simultaneous purification and immobilization of laccase fermentation material solution to the construction of the enzyme membrane reactor. Two kinds of membrane chromatography medium immobilized laccase were with high purity. The operation effect of membrane surface velocity of laccase activity and specific activity of the immobilized in the process flow, wear type fixed model than the traditional immersion mode more efficient. The enzyme membrane reaction the equipment has good stability, good repeatability and bisphenol A (BPA) removal capacity. Because the metal ions are firmly combined with laccase, so BPA liquid, enzyme membrane constructed by metal chelating membrane chromatography medium reaction Is no protein leakage. The study found that when stacked four layer membrane (BPA), constructed by metal chelating membrane chromatography medium enzyme membrane reactor on the removal rate of BPA can be increased to 99.2%; when the feed processing flux increased to 50L/m~2h, the removal efficiency of BPA (473 rmg/m~2h) is higher than the results reported in literature.

【學位授予單位】：中國科學院大學(中國科學院過程工程研究所)
【學位級別】：博士
【學位授予年份】：2017
【分類號】：TQ028.8;O652.63

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