Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, P.O. Box 94, Grahamstown, South Africa.
Enzyme Microb Technol. 2011 Jul 10;49(2):146-52. doi: 10.1016/j.enzmictec.2011.05.011. Epub 2011 May 27.
Enzyme immobilization is an ever-growing research-area for both analytical and industrial applications. Of critical importance in this area are the effects of immobilization procedures upon the functionality of the immobilized biomolecules. Both beneficial and detrimental effects can be conferred through the selection and tuning of the immobilization procedure. Quartz-crystal microbalance with dissipation (QCM-D) has been previously used to great effect in tracking alterations to thin films of biomolecules immobilized onto quartz transducers. In this study, we investigate the ability of QCM-D to track and monitor film parameters of a monolayer of laccase immobilized on a series of self-assembled monolayers (SAMs), differing in lateral density of binding residues on the SAM and height of the SAM from the quartz surface. Both mass gains and rheological parameters for these varying surfaces were measured and trends later compared to the apparent enzyme kinetics of the immobilized laccase films, assessed electroanalytically (Paper II in this two part study). For covalent attachment of proteins, both shear and viscosity were increased relative to physically adsorbed proteins. An increase in lateral density of protein-binding surface of the SAM components was shown to increase the shear/viscosity of the resultant film while an increase in distance from the electrode (through incorporation of lysine linkers) was shown to decrease the shear/viscosity while simultaneously increasing the wet mass gain of the films. Shear and viscosity may be indicative of both enzyme denaturation and increased lateral protein packing within the film structure hence it is assumed that less distortion occurs with the inclusion of linkers which allow for more optimal protein immobilization.
酶固定化是分析和工业应用中一个不断发展的研究领域。在这个领域中，固定化过程对固定化生物分子的功能的影响至关重要。通过选择和调整固定化程序，可以产生有益和有害的影响。石英晶体微天平（QCM-D）以前在跟踪固定在石英换能器上的生物分子薄膜的变化方面效果显著。在这项研究中，我们研究了 QCM-D 跟踪和监测一系列自组装单层（SAM）上固定化的漆酶单层膜的膜参数的能力，这些 SAM 在 SAM 上的结合残基的横向密度和 SAM 与石英表面的高度上有所不同。这些不同表面的质量增益和流变参数都进行了测量，并与固定化漆酶膜的表观酶动力学进行了比较，这是通过电化学评估的（这两部分研究中的论文 II）。对于蛋白质的共价附着，与物理吸附的蛋白质相比，剪切和粘度都增加了。SAM 成分的蛋白质结合表面的横向密度增加表明，所得膜的剪切/粘度增加，而与电极的距离增加（通过掺入赖氨酸接头）表明剪切/粘度降低，同时增加膜的湿质量增益。剪切和粘度可能表明酶变性和膜结构中蛋白质的侧向堆积增加，因此可以假设包含允许更优化蛋白质固定化的接头时，扭曲程度更小。
