疏水色谱膜对混合蛋白的分离性能研究

发布时间：2018-03-23 20:42

  本文选题：木质纤维　切入点：接枝改性　出处：《北京化工大学》2015年硕士论文

【摘要】：膜色谱兼具传统柱色谱的高选择性,以及膜分离技术高效率、低压降、易放大实施的优越性,相比于柱色谱更适合用于大规模蛋白分离纯化。疏水作用膜色谱利用不同蛋白分子疏水性的差异导致与分离膜的作用力不同达到分离纯化目的,分离条件温和,有利于保持蛋白的活性。本研究以纸纤维为基材,通过接枝改性获得纸基疏水色谱分离膜,并考察所得分离膜对混合蛋白的分离性能。主要研究工作如下：1)采用紫外光引发,将异丙基丙烯酰胺(NIPAM)及NIPAM与丙烯酸丁酯(BA)共聚物大分子单体(P(NIPAM-BA),平均分子量5276Da, BA含量为5.96%)接枝至滤纸上,得到平均接枝率为4.5%、6.7%、12.2%的纸基温敏色谱膜。通过红外光谱测试,证明滤纸上成功接枝了温敏支化聚合物。2)考察了温敏膜色谱纯化脂肪酶粗酶的适宜条件。选用接枝率12%左右的温敏膜,在流动相无盐条件下考察色谱膜的分离性能。膜组件于30℃逐渐升温至40℃并维持加热10min后往流动相注入脂肪酶粗酶溶液进行高温吸附,于4℃进行低温淋洗脱附,收集淋洗流出蛋白溶液。SDS-PAGE测试结果表明,经温敏膜色谱的纯化,脂肪酶粗酶中的杂蛋白全部在吸附阶段得以分离,脱附淋洗液中含有高纯度日标蛋白,纯化效果良好；橄榄油乳化液水解滴定法测定表明,经温敏色谱膜纯化后的脂肪酶酶的比活提高了10.9倍。3)采用丝光处理、环氧开环聚合方法制备的聚氧化苯乙烯接枝改性木质纤维制备了纸基疏水色谱膜(PSO-g-PF膜),考察了该疏水色谱膜的性能。光学显微镜及SEM对其形态图像观测表明,PSO-g-PF膜的微观形貌与商品化滤纸及未改性纸浆纤维网类似,具备纸纤维膜所特有的孔形态结构和高通透性。膜组件分离实验结果显示,在流动相为1.4M硫酸铵盐溶液时,PSO-g-PF膜对BSA的吸附量达3.7mg·g-1,为未接枝改性的纸浆纤维膜的1.8倍,表明PSO-g-PF膜适合用作疏水色谱蛋白分离膜,而且重复使用次数可达3-4次。4)研究了PSO-g-PF疏水色谱膜对混合蛋白的分离性能。对于BSA/溶菌酶混合蛋白,吸附阶段流动相为0.9M硫酸铵盐溶液时,可将二者成功拆分；分离黑曲霉脂肪酶／溶菌酶混合蛋白时,吸附阶段流动相为1.0M硫酸铵盐溶液时,可将二者成功拆分；分离黑曲霉脂肪酶/BSA混合蛋白时,吸附阶段流动相为0.7M硫酸铵盐溶液时,可从混合蛋白中去除黑曲霉脂肪酶,得到纯BSA；纯化脂肪酶粗酶时,吸附阶段流动相为1.1M硫酸铵盐溶液时,能去除杂蛋白,得到高纯度的脂肪酶,其比活提高了12.1倍。
[Abstract]:Membrane chromatography has the advantages of high selectivity of traditional column chromatography, high efficiency of membrane separation technology, low pressure drop and easy amplification. The hydrophobic interaction membrane chromatography is more suitable for the separation and purification of large scale proteins than column chromatography. The hydrophobicity of different protein molecules leads to the separation and purification of the membrane with different forces, and the separation conditions are mild. The paper hydrophobic chromatographic separation membrane was obtained by graft modification, and the separation performance of the separation membrane to the mixed protein was investigated. The main research work is as follows: 1) UV light was used to initiate the separation. The copolymer of isopropylacrylamide (NIPAM) and butyl acrylate (NIPAM) was grafted onto the filter paper with an average molecular weight of 5276Daand BA content of 5.96. the paper based thermochromatographic film with an average grafting rate of 4.56.7and 12.2% was obtained. It was proved that the temperature sensitive branched polymer. 2) the suitable conditions for the purification of lipase by thermo-sensitive membrane chromatography were investigated. The temperature sensitive membrane with grafting rate of about 12% was selected. The separation performance of chromatographic membrane was investigated under the condition of mobile phase without salt. The membrane module was heated up to 40 鈩，

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