固定化脂肪酶色谱柱的研究

发布时间：2018-05-08 18:24

  本文选题：复合载体 + 固定化酶　； 参考：《兰州大学》2017年硕士论文

【摘要】：酶作为一种天然催化剂,以其高效、温和、绿色的独特优势成为生物、化工、医药等领域的重要研究对象。脂肪酶来源广泛,能够在水相和有机相中催化酯化反应、水解反应、酯交换反应等,并且具有化学选择性、区域选择性、对映体选择性,是一种具有巨大潜力的工业用酶。但是,在使用过程中游离酶对外界环境敏感、成本高等缺点限制了其应用,因而推动了酶固定化技术的发展。经过固定化之后,酶的性能得到优化,扩大了酶催化的适用范围,有效降低了生产成本,提高生产产率。反应色谱技术是将化学反应和色谱分离集合在一个单元的技术,不仅能够简化化学过程,降低生产成本,而且能够突破可逆反应化学平衡的限制,提高产率。高效液相色谱能够很大程度上降低实验过程的复杂程度。将反应色谱技术和固定化酶技术结合使用后,将固定化酶装填为具有分离和催化功能的固定化酶色谱柱,实现生物催化与色谱技术的结合,深入反应色谱技术的研究。本论文中选择硅胶和纤维素组成的复合载体分别用物理吸附法和交联法制备了两种固定化酶,装填成固定化酶色谱柱后对其在线催化水解反应效果进行了评价。脂肪酶的底物选择对硝基苯酚棕榈酸酯,反应体系由正己烷、异丙醇、水组成。吸附法固定化酶的制备中,乙基纤维素涂覆硅胶是最适宜的载体,吸附固定化酶的温度耐受范围为15℃-45℃。乙基纤维素硅胶固定化酶色谱柱能实现对硝基苯酚棕榈酸酯的在线水解和分离,具有反应色谱的性质,稳定性很高,在温度为18℃-50℃范围内具有较高的催化活性,半衰期长达160 h。交联法固定化酶具有良好的操作稳定性,对对硝基苯酚具有良好的耐受性,经过极性底物抑制或者活化后可恢复活性。交联固定化酶色谱柱亲水性很强,对对硝基苯酚的吸附强于对对硝基苯酚棕榈酸酯的吸附。在水含量为10%时,固定化酶色谱柱的催化活性(转化率50%以上)高于游离酶。流动相中水含量升高时底物转化率上升,水含量超过20.7%时逐渐达到最大值。正己烷含量的上升会抑制固定化酶色谱柱的催化活性。经过160 h反应后交联固定化色谱柱活性几乎没有衰减,十分稳定。
[Abstract]:As a kind of natural catalyst, enzyme has become an important research object in biology, chemical industry, medicine and other fields with its unique advantages of high efficiency, mild and green. Lipases can catalyze esterification, hydrolysis and transesterification in aqueous and organic phases, and have chemical selectivity, regioselectivity and enantioselectivity. Lipase is a kind of industrial enzyme with great potential. However, in the process of using free enzyme is sensitive to the external environment, high cost and other shortcomings limit its application, thus promoting the development of enzyme immobilization technology. After immobilization, the performance of the enzyme was optimized, the application range of enzyme catalysis was expanded, the production cost was reduced effectively, and the production yield was increased. Reaction chromatography is a technique of separating chemical reaction and chromatography into one unit. It can not only simplify chemical process, reduce production cost, but also break through the limit of chemical equilibrium of reversible reaction and increase yield. High performance liquid chromatography (HPLC) can greatly reduce the complexity of the experimental process. After the combination of reaction chromatography and immobilized enzyme technology, the immobilized enzyme was packed into an immobilized enzyme chromatographic column with separation and catalytic functions. The combination of biocatalysis and chromatography technology was realized, and the study of reaction chromatography technology was carried out. In this paper, two kinds of immobilized enzymes were prepared by physical adsorption method and cross-linking method respectively on the composite carrier composed of silica gel and cellulose. The effect of on-line catalytic hydrolysis reaction was evaluated by filling the immobilized enzyme chromatographic column. The lipase substrate was p-nitrophenol palmitate. The reaction system consisted of n-hexane, isopropanol and water. In the preparation of immobilized enzyme by adsorption, ethyl cellulose coated with silica gel is the most suitable carrier, and the temperature tolerance range of adsorption immobilized enzyme is from 15 鈩，

本文编号：1862509