Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, Iowa 52242, USA.
J Biol Eng. 2007 Oct 10;1:4. doi: 10.1186/1754-1611-1-4.
Efficient functioning of enzymes inside liposomes would open new avenues for applications in biocatalysis and bioanalytical tools. In this study, the entrapment of amyloglucosidase (AMG) (EC 3.2.1.3) from Aspergillus niger into dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs) was investigated. Negative-stain, freeze-fracture, and cryo-transmission electron microscopy images verified vesicle formation in the presence of AMG. Vesicles with entrapped AMG were isolated from the solution by centrifugation, and vesicle lamellarity was identified using fluorescence laser confocal microscopy. The kinetics of starch hydrolysis by AMG was modeled for two different systems, free enzyme in aqueous solution and entrapped enzyme within vesicles in aqueous suspension. For the free enzyme system, intrinsic kinetics were described by a Michaelis-Menten kinetic model with product inhibition. The kinetic constants, Vmax and Km, were determined by initial velocity measurements, and Ki was obtained by fitting the model to experimental data of glucose concentration-time curves. Predicted concentration-time curves using these kinetic constants were in good agreement with experimental measurements. In the case of the vesicles, the time-dependence of product (glucose) formation was experimentally determined and simulated by considering the kinetic behavior of the enzyme and the permeation of substrate into the vesicle. Experimental results demonstrated that entrapped enzymes were much more stable than free enyzme. The entrapped enzyme could be recycled with retention of 60% activity after 3 cycles. These methodologies can be useful in evaluating other liposomal catalysis operations.
酶在脂质体中的有效功能将为生物催化和生物分析工具的应用开辟新的途径。在这项研究中，研究了从黑曲霉中提取的淀粉葡糖苷酶（AMG）（EC 3.2.1.3）包封到二棕榈酰磷脂酰胆碱（DPPC）多层囊泡（MLVs）和大单室囊泡（LUVs）中的情况。负染色、冷冻断裂和冷冻电子显微镜图像证实了 AMG 存在时囊泡的形成。通过离心从溶液中分离出包封 AMG 的囊泡，并使用荧光激光共聚焦显微镜鉴定囊泡的层状结构。使用两种不同的系统（水溶液中的游离酶和水溶液中囊泡内的包封酶）对淀粉水解的 AMG 动力学进行了建模。对于游离酶系统，通过具有产物抑制的米氏动力学模型描述了本征动力学。通过初始速度测量确定动力学常数 Vmax 和 Km，通过将模型拟合到葡萄糖浓度-时间曲线的实验数据来获得 Ki。使用这些动力学常数预测的浓度-时间曲线与实验测量值吻合良好。对于囊泡，通过考虑酶的动力学行为和底物向囊泡的渗透，实验测定并模拟了产物（葡萄糖）形成的时间依赖性。实验结果表明，包封酶比游离酶稳定得多。在 3 个循环后，包封酶的回收率为 60%，保持 60%的活性。这些方法可用于评估其他脂质体催化操作。
