Enzyme distribution derived from macroscopic particle behavior of an industrial immobilized penicillin-G acylase
Enzyme distribution derived from macroscopic particle behavior of an industrial immobilized penicillin-G acylase
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DOI：
10.1021/bp0340638
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发表时间：
2003-09-01
影响因子：
2.9
通讯作者：
Beeftink, HH
中科院分区：
文献类型：
--
作者：
van Roon, JL;Joerink, M;Beeftink, HH
工业上使用的固定化青霉素-G酰基酶的宏观动力学行为，称为组装酶，形成了一些简单的颗粒内生物催化模型分布的讨论基础。组装酶催化合成广泛使用的半合成抗生素头孢氨苄。尽管固定化具有明显的优点，但由于固定化酶的扩散限制，得到的头孢氨苄较少，不需要的副产物D-(-)-苯甘氨酸较多。为了合理优化集合酶，在宏观观察的基础上，对影响其宏观性能的主要参数--颗粒大小、酶载量和酶分布进行了研究。激光衍射测量表明，组装酶中的颗粒尺寸变化高达100倍。通过初始速率D-(-)-苯甘氨酰胺水解、头孢氨苄合成实验和活性中心滴定，测定了不同大小的组装酶及其级分的相对酶载量和总酶载量。这些实验表明，青霉素-G酰化酶在组装酶中的载量与其粒径呈负相关。除了酶的负载量，对颗粒内酶分布的估计来自头孢氨苄合成实验，其中存在质量传输限制。虽然这种方法不能提供特定标记实验的详细程度，但它简单、快速、廉价。在一组简单的模型预测中，大多数生物催化剂存在于粒子的外部区域(在外部100微米内)的异质性酶分布给出了对观察到的行为的最佳描述，尽管没有建立确切的关联。对颗粒内酶分布的非常详细的测定必须来自免疫标记。
The macroscopic kinetic behavior of an industrially employed immobilized penicillin-G acylase, called Assemblase, formed the basis for a discussion on some simple intraparticle biocatalytic model distributions. Assemblase catalyzes the synthesis of the widely used semisynthetic antibiotic cephalexin. Despite the obvious advantages of immobilization, less cephalexin and more of the unwanted byproduct D-(-)-phenylglycine are obtained due to diffusional limitations when the immobilized enzyme is employed. To rationally optimize Assemblase, the parameters particle size, enzyme loading, and enzyme distribution, which severely determine the macroscopic particle performance, were studied on the basis of macroscopic observations. Laser diffraction measurements showed that the particle sizes in Assemblase vary as much as 100-fold. The relative and total enzyme loadings in Assemblase and fractions thereof of different sizes were determined by initial-rate D-(-)-phenylglycine amide hydrolysis, cephalexin synthesis experiments, and active-site titration. These experiments revealed that the loading of penicillin-G acylase in Assemblase was inversely correlated with the particle diameter. Apart from enzyme loadings, estimates on the intraparticle enzyme distribution came from cephalexin synthesis experiments, where mass-transport limitations were present. Although this method cannot provide the level of detail of specific labeling experiments, it is simple, fast, and cheap. Within the set of simple model predictions, a heterogeneous enzyme distribution with most biocatalyst present in the outer region of the particle (within the outer 100 mum) gave the best description of the observed behavior, although no exact correlation was established. Highly detailed determination of intraparticle enzyme distributions must come from immunolabeling.