van Roon Jeroen L, Joerink Michiel, Rijkers Marinus P W M, Tramper Johannes, Schroën Catharina G P H, Beeftink Hendrik H
Food and Bioprocess Engineering Group, Department of Agrotechnology and Food Science, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands.
Biotechnol Prog. 2003 Sep-Oct;19(5):1510-8. doi: 10.1021/bp0340638.
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 microm) 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.
一种工业上使用的固定化青霉素G酰化酶（称为组装酶）的宏观动力学行为，为讨论一些简单的颗粒内生物催化模型分布奠定了基础。组装酶催化广泛使用的半合成抗生素头孢氨苄的合成。尽管固定化具有明显优势，但使用固定化酶时，由于扩散限制，头孢氨苄的产量较低，而不需要的副产物d-（-）-苯甘氨酸的产量较高。为了合理优化组装酶，基于宏观观察研究了严重影响宏观颗粒性能的参数，即粒径、酶负载量和酶分布。激光衍射测量表明，组装酶中的颗粒大小相差多达100倍。通过初始速率d-（-）-苯甘氨酸酰胺水解、头孢氨苄合成实验和活性位点滴定，确定了组装酶及其不同大小部分的相对和总酶负载量。这些实验表明，组装酶中青霉素G酰化酶的负载量与粒径呈负相关。除了酶负载量外，颗粒内酶分布的估计来自存在传质限制的头孢氨苄合成实验。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、快速且便宜。在一组简单的模型预测中，一种非均相酶分布（大多数生物催化剂存在于颗粒的外部区域，即在外部100微米内）对观察到的行为给出了最佳描述，尽管没有建立确切的相关性。颗粒内酶分布的高度详细测定必须来自免疫标记。
