【作者】 王峥；

【导师】 姜涌明； 袁中一； 郭敏亮；

【作者基本信息】 扬州大学 ， 生物化学与分子生物学， 2001， 硕士

【摘要】 疏水作用色谱（Hydrophobic Interaction Chromatography，HIC）是二十世纪七十年代初发展起来的一种重要的生化分离技术，在生物大分子的分离中应用广泛。HIC填料常用载体为琼脂糖，以壳聚糖为载体的研究未有报道。本文合成了以壳聚糖（Chitosan，CTS）为载体、以烷基C₄为疏水配基、以戊二醛交联提高机械强度的新型HIC填料，对其合成条件的优化进行了研究，考查了其部分性质，并将其初步应用于α-淀粉酶的分离纯化。1对填料的合成条件进行了优化：（1）0.1～0.15％戊二醛先交联，可产生适当的机械强度。（2）壳聚糖合成浓度为0.2～1％（W/V），即2～10g/L时，填料具有较好的应用性。（3）在CTS浓度为5g/L时，控制丁醛浓度为0.13～0.26％（V/V），即醛氨摩尔比为0.5∶1～1∶1，填料具有较好的应用性。（4）希夫氏反应温度40～70℃均可，反应时间随温度升高而缩短。（5）间歇搅拌为宜。2对填料性质进行了研究：（1）（NH₄）₂SO₄作为流动相盐类，分离效果优于Na₂SO₄。（2）选择0.5～1hr进行吸附。吸附时间过短，疏水吸附来不及平衡；吸附时间过长，易造成生物大分子的变性失活。（3）吸附温度在5～70℃，吸附温度提高，填料对BSA的吸附容量增加；超过70℃，吸附容量迅速下降，蛋白质发生变性。α-淀粉酶则在40℃有最大吸附。一般宜选择20～25℃进行疏水分离。（4）pH对蛋白质与填料间相互作用的影响较为复杂。3新型疏水色谱填料在酶分离纯化上的应用： 用优化条件合成的CTS CL-C₄装柱色谱，一步纯化α-淀粉酶，比活提高1.7倍，总蛋白回收94.67％，酶活收率为72.62％。更多还原

【Abstract】 Hydrophobic Interaction Chromatography (HIC) is a new Chromatographic technique developed in the 1970’s to separate biopolymers. Generally, Sepharose is used as the carrier, and using chitosan as a carrier has not been reported. In this paper, a new packing material of HIC-Butyl-Cross linked Chitosan (CTS CL-C4) has been synthesized which was prepared with Chitosan as the carrier, butyl as the hydrophobic ligand and gluteraldehyde as the cross-linker to raise the mechanical intensity. The control of the synthetic conditions has been researched and series of characteristics and application of CTS CL-C4 have been determined. The results are shown as the follows: 1. The concentration of gluteraldehyde, reactant proportion and reactant consistency determinate the hydrophobic nature of the packing material. For good operation and practical nature of guaranty, the more suitable synthetic conditions are selected. The last concentration of gluteraldehyde is 0.1 --0.15% (V/V); the concentration of Chitosan is 0.2-1.0%(W/V), that is 2-10g/L; the concentration of butyraldehyde is 0.13%-0.26%(V/V); mol-proportion of aldehyde and aniido is O.5:l-1:l; synthetic temperature is in 40℃-70℃; stirring intermittently. Concrete protein is defined the corresponding synthetic conditions on the basis of its hydrophobic nature. 2. Characteristics of CTS CL-C4 have been determined using bovine serum albumin (BSA) and C?-Amylase as HIC target: the adsorption of BSA increases with the increasing of salt concentration, (NH4)2SO4 is often used to get better effect; selection of adsorption time and temperature is 0.5-1hr and 20℃-25℃; effect of pH is complex and varied and under more research. 3. Crude ci -Amylase from Bacillus Subtilis can be effectively purified on this packing material with the enzyme activity recovered in 72.62% yield, protein recovered in 94.67% yield and a 1.7-fold purification.更多还原