固定化青霉素酰化酶新型载体PEI/SiO2的制备及其特性

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, 制备了固定化青霉素酰化酶的新型复合载体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; 通过红外光谱与电导滴定法测定, 对复合载体表面的化学结构与组成进行了表征; 为探索复合载体PEI/SiO2固定化酶的作用机理, 测定了复合载体在固定化酶前的ζ电位. 研究结果表明, 通过氯丙基硅烷偶联剂的媒介, 聚胺大分子PEI可以充分地被化学偶联在SiO2表面, 键合量可达到15%. 偶联反应的适宜条件: 反应温度90-94 ℃; 反应时间5h; PEI的质量浓度0.45-0.50 g/mL. 由于PEI分子链中含有大量氨基, 少量的共价键联与大量的物理吸附相结合, 既可使青霉素酰化酶被快速稳定地固定化, 又能很好地保持酶的构象, 使其具有较高的催化活性与活力回收率, 而且具有良好的连续操作稳定性, 重复使用15次, 固定化酶的活性可稳定地保持在初活性的87.5%水平上.

Preparation and Characters of Novel Support PEI/SiO2 for the Immobilization of Penicillin Acylase

Polyethyleneimine(PEI) was coupled chemically to the surface of silica gel particles through the intermedium effect of γ\|chloropropyltrimethoxysilane, novel complex carrier PEI/SiO2 for immobilizing penicillin acylase was prepared, and finally an immobilized penicillin acylase with a higher activity and stability was obtained. The influences of various factors on the preparation of the carrier were examined via measuring coupling amount of PEI on the compisite carrier. The chemical composition and structure of the surface of the composite carrier were characterized via IR and conductimetric titration. In order to explore the immobilization mechanism of the complex carrier for the penicillin acylase, the ζ potential of the support was determined before the enzyme was immobilized. The results show that the polyamine PEI can fully be coupled chemically to the surface of silica gel particles by the agency of γ-chloropropyltrimethoxysilane, and the linkage amount can reach 15%. The favorable reaction conditions are the reaction temperature is 90-94℃, the reaction time 5 h and the mass concentration 45-50 g/100 mL for PEI. Owing to a large quantity of amido contained on the macromolecular chains of PEI, there is not only chemical linkage, but also a great deal of physical adsorption during enzyme immobilization on the surface of PEI/SiO2. The combination of a spot of chemical covalent linkage and a great deal of physical adsorption results in fast and stable immobilization, and excellent maintenance of constellation for penicillin acylase, so that the activity and activity recovery ratio of the immobilized enzyme are all higher. Moreover, the immobilized enzyme has a better continuous operating stability and its activity can keep at a level of 87.5% of fresh one all along after having been used for fifteen time repeatedly.