【作者】 王旭朋；

【导师】 高保娇；

【作者基本信息】 中北大学 ， 应用化学， 2006， 硕士

【摘要】 本文通过γ-氯丙基三甲氧基硅烷的媒介,将聚乙烯亚胺（PEI）化学偶联在硅胶微粒表面,制备了新型复合载体 PEI/SiO₂,最终制得了活性高且稳定性好的固定化青霉素酰化酶。通过红外光谱、电导滴定对复合载体表面的化学结构与组成进行了表征;通过测定复合载体表面 PEI 的偶合量,考察了各种反应条件对复合载体制备的影响规律,实验所得偶联反应的适宜条件为:反应温度 90⁹4℃;反应时间 5h;PEI 水溶液浓度为45g/100mL⁵0g/100mL;聚乙烯亚胺水溶液与氯丙基化硅胶质量比为 5:1。在实施酶的固定化过程中,考察了戊二醛用量、pH 值、固定化温度、固定化时间以及给酶量等条件对固定化青霉素酰化酶表观活力、活力回收率等性能的影响,实验所得最优固定化条件为:0.5g 表面键合有 9%PEI 的复合载体,使用 1%的戊二醛,在 pH 值为 7.92 缓冲溶液中,酶液用量 0.05mL,于 30℃下进行 14～15h 的固定化反应,所制固定化酶的活力为 118U/g,活力回收率为 46%。结合复合载体在固定化酶前的ξ电位及酶固定化反应过程的特点,探索了复合载体 PEI/SiO₂固定化酶的作用机理。研究结果表明:复合载体PEI/SiO₂ 表面带有大量的胺基,通过化学键合（胺基的 Schiff 反应）与物理吸附（质子化胺基与酶分子的静电吸引作用）两种作用将酶分子快速、高效地固定在载体上,充分地保持了酶分子的构象,获得了较高活性的固定化酶。为了考察所制固定化酶的性能,测定了游离酶与固定化酶催化反应的最适 pH 值、最适温度、固定化酶的连续操作稳定性、二者对金属离子的耐受性能及米氏常数。实验结果表明:游离酶和固定化酶催化反应的最适 pH 值分别为 9.32 和 7.34,最适温度分别为 50℃和 40℃,固定化酶具有良好的连续操作稳定性,且比游离酶具有较好的抗金属离子性能;二者的米氏常数分别为2.38×10^-1mol/L 和 5.44×10^-2mol/L。更多还原

【Abstract】 Polyethyleneimine （PEI） was coupled chemically to surface of silica gel particlesthrough the intermedium effect of γ-chlopropyltrimethoxysilane, the novel compositecarrier of PEI/SiO₂ was prepared, and finally immobilized penicillin acylase with higheractivity and stability was obtained. The chemical composition and structure of the surface ofcomposite carrier were characterized by using FITR and conductmetric titration method. Theinfluences of various factors on the preparing of the carrier were examined via measuringcoupled amount of PEI on the composite carrier. The favourable reaction conditions are: thereaction temperature of 90℃⁹4℃, the reaction time of 5h and the concentration of45g/100mL ⁵0g/100mL for PEI solution, the ratio of weight of PEI solution to CP-SIO₂ is5:1. When the penicillin acylase were immobilized on the composite carrier of PEI/SiO₂, theeffects of various immobilizing on the apparent activity and recovery ratio of activity ofimmobilized penicillin acylase were examined, such as the amount of glutaraldehyde、pH、time、temperature and the used amount of enzyme. The appropriate immobilization conditionsfor 0.5g of the composite support PEI/SiO₂ to which 9% of PEI was linked are as follows: 1%of glutaraldehyde, 7.92 of pH, 30℃ of temperature, 14～15h of time and 0.05mL of originalenzyme solution. Under these conditions, the obtained immobilized penicillin acylase possess118U/g of activity and 46% of recovery ratio of activity. we explored the immobilizationmechanism of the composite carrier for penicillin acylase with the zeta electric potentials ofthe carrier before enzyme were determined and the characters of immobilization process. Theexperiment results showed: Owing to a large quantity of amido contained on themacromolecular chains of PEI, there is not only chemical linkage（schiff reaction of amido）,but also is a great deal of physical adsorption（electrostatic adsorption between electriferousamido and enzyme molecular）during enzyme immobilization on the surface of PEI/SiO₂. Thecombination of a spot of chemical covalent and a great deal of physical adsorption results infast and stable immobilization and excellent maintenance of the molecular conformation ofenzyme, and leads to high activity of the immobilized enzyme. For examining theproperties of immobilized penicillin acylase, suitable pH and temperature of free enzymeand immobilized enzyme catalysis reaction was examined, continued operation stability andstability in metal ionic solutionan was studied, at the same time, michaelis-menten constant offree enzyme and immobilized enzyme were also measured. The experiment results showed:the optimum pH of enzyme catalysis reaction were 9.32 and 7.34, the optimum temperaturewere 50 ℃ and 40 ℃ for free enzyme and immobilized enzyme, respectively. Theimmobilized enzyme is more stable than free enzyme to the metal ionic solutionan andmichaelis-menten constant of immobilized enzyme and free enzyme were 2.38×10-1mol/Land 5.44×10-2mol/L,respectively.更多还原