青霉素酰化酶在新型复合载体上的固定化研究

Studies on immobilization of penicillin acylase on novel complex carrier PEI/silica gel

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／silica gel，然后通过双官能团试剂戊二醛的作用，将青霉素酰化酶固定在复合载体上；考察了戊二醛用量、pH值、固定化温度、固定化时间及给酶量等条件对固定化青霉素酰化酶表观活力、活性回收率等性能的影响；并通过测定复合载体在固定化前的ζ电位，探索了复合载体PEI／silica gel固定化酶的作用机理。研究结果表明，由于PEI分子链中含有大量胺基，共价键联与物理吸附相结合，使青霉素酰化酶被快速稳定地固定化，并具有高的催化活性与活力回收率。复合载体PEI／silica gel（0．5g）固定青霉素酰化酶的适宜固定化条件为：固定化温度为30℃；固定化时间为14-15h；戊二醛用量为1、2mmol／g；pH=7．92；给酶量为0、1mL／g。

The novel complex cartier, PEI/silica gel, for immobilization of penicillin acylase was prepared, as polyethyleneinime （PEI） was coupled chemically to surface of silica gel particles by the intennedium of γ-chlopropyltrimethoxysilane. On which penicillin acylase was immobilized with the cress-linking of glutaraldehyde then. The main factors impacting the apparent activity and recovery rate, such as the amount of glutaraldehyde, pH, immobilization time, temperature and the amount of enzyme were investigated. The Zeta electric potentials of the cartier were determined before immobilization of penicillin acylase in order to explore the immobilization mechanism. The experiment results showed that there were a lot of amidocyanogens in macromolecular chains of PEI linked by covalent bonds and physical adsorption to the surface of the cartier, so that penicillin acylase could be immobilized fast and stably keeping high catalysis activity and high recovery rate of activity. The appropriate immobilization conditions with： 0.5 g of the complex cartier PEI/silica gel （ in which 9% of PEI） were as follows： glutaraldehyde 1.2 mmol/g, pH 7.92, at 30 ℃, immobilization time 14 - 15 h, and original enzyme solution 0.1 mL/g.