醋酸纤维素接枝小分子多肽的膜色谱制备及分离性能研究

发布时间：2018-03-11 20:46

本文选题：醋酸纤维素　切入点：亲和膜色谱　出处：《北京化工大学》2015年硕士论文　论文类型：学位论文

【摘要】：随着社会对生物活性大分子的需求越来越高,很多领域如医药、生物等对分离技术的纯度要求也越来越高。膜色谱技术被认为是一种高效节能环保的分离技术,且具有易装填和放大的优点,工业化前景很好。本研究旨在制备一种亲和膜色谱,通过在膜上接枝适当的配基以亲和吸附相应的生物大分子,以达到分离纯化该物质的目的。首先,本文探索了相转化法制备醋酸纤维素膜色谱的可行性。通过优化浓度、溶剂配比、添加剂、蒸气浴湿度与温度等条件,醋酸纤维素膜的纯水通量从283 Lm-2h-1提高到1598Lm-2h-1,相同方法制得的醋酸纤维素接枝p-环糊精膜纯水通量为639 Lm-2h-1,但该接枝膜填装膜色谱时最多仅可添加3层,且随着压力增大膜内部孔道被完全压塌,因此认为相转化法制得的醋酸纤维素膜不适合作为亲和膜色谱的底膜使用。其次,本文尝试用静电纺丝法制备醋酸纤维素膜,该膜具有较高的孔隙率和机械性能,通过优化浓度、溶剂配比、接收距离、流速、电压等条件制备出的醋酸纤维素膜纤维直径在264nm到5.121μm之间,且具有良好形态。醋酸纤维素膜去乙酰化后接枝p-环糊精所制得的接枝物膜可装填25层膜色谱且连续使用24h,葛根素粗品经过该膜色谱处理后,葛根素纯度由42.52%提高到84.1%,由此可证明静电纺丝法制得的醋酸纤维素膜可作为膜色谱底膜使用。最后,本文制备了可特异性识别蛋白SH3结构域的小分子多肽LPPLPLPPLP,将静电纺丝法制得的醋酸纤维素膜经去乙酰化、氧化处理后接枝小分子多肽LPPLPLPPLP,接枝率可达15.1μg/mg,把BSA做杂质与磷脂酶A2混合成磷脂酶粗品,考察膜色谱分离性能。结果表明,将接枝小分子多肽LPPLPLPPLP的亲和膜填装成20层膜色谱,粗品溶液经过该膜色谱的处理,可有效将磷脂酶A2固定在膜色谱上,饱和吸附率为7.2μg/mg,再通过收集膜色谱洗脱液并浓缩,由此达到纯化磷脂酶A2的目的。
[Abstract]:With the increasing demand for bioactive macromolecules in society, many fields, such as medicine, biology and so on, require higher purity of separation technology. Membrane chromatography technology is considered to be a kind of high efficiency, energy saving and environmental protection separation technology. It has the advantages of easy loading and amplification, and has a good prospect of industrialization. The aim of this study was to prepare an affinity membrane chromatography, and to adsorb the corresponding biomolecules by grafting appropriate ligand on the membrane. First of all, the feasibility of preparing cellulose acetate membrane chromatography by phase inversion method was explored. By optimizing concentration, solvent ratio, additive, vapor bath humidity and temperature, etc. The pure water flux of cellulose acetate membrane was increased from 283Lm-2h-1 to 1598Lm-2h-1.The pure water flux of cellulose acetate grafted with p-cyclodextrin was 639Lm-2h-1, but only three layers could be added in the filling membrane chromatography. With the increase of pressure, the inner pore of the membrane is completely collapsed, so it is considered that the cellulose acetate membrane prepared by phase transformation method is not suitable for use as the bottom membrane of affinity membrane chromatography. Secondly, this paper attempts to prepare cellulose acetate membrane by electrostatic spinning method. The film has high porosity and mechanical properties. The fiber diameter of cellulose acetate membrane is between 264 nm and 5.121 渭 m by optimizing concentration, solvent ratio, receiving distance, flow rate, voltage and so on. After deacetylation of cellulose acetate membrane, the graft film prepared by grafting pcyclodextrin can be loaded with 25 layers of membrane chromatography and used continuously for 24 hours. The crude product of puerarin can be treated by the membrane chromatography. The purity of puerarin increased from 42.52% to 84.1, which proved that the cellulose acetate membrane prepared by electrospinning can be used as the bottom film of membrane chromatography. In this paper, a small molecular peptide, LPPLPLPPLP, which can specifically recognize the domain of protein SH3, was prepared. The cellulose acetate membrane prepared by electrospinning was deacetylated. After oxidation treatment, the graft rate of LPPLPLPPLP was 15.1 渭 g / mg. BSA was mixed with phospholipase A2 to form phospholipase crude product. The results showed that the affinity membrane of LPPLPLPPLP was filled into a 20-layer membrane chromatography. The crude solution was treated by the membrane chromatography and phospholipase A2 was effectively immobilized on the membrane chromatography. The saturated adsorption rate was 7.2 渭 g / mg. then the phospholipase A2 was purified by collecting the eluent and concentrating it.
【学位授予单位】：北京化工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TQ051.893;O652.63

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