Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, P.O. Box 94, Grahamstown, South Africa.
Enzyme Microb Technol. 2011 Jul 10;49(2):153-9. doi: 10.1016/j.enzmictec.2011.05.004. Epub 2011 May 17.
The method of immobilization of a protein has a great influence on the overall conformation, and hence, functioning of the protein. Thus, a greater understanding of the events undergone by the protein during immobilization is key to manipulating the immobilization method to produce a strategy that influences the advantages of immobilization while minimizing their disadvantages in biosensor design. In this, the second paper of a two-part series, we have assessed the kinetic parameters of thin-film laccase monolayers, covalently attached to SAMs differing in spacer-arm length and lateral density of spacer arms. This was achieved using chronoamperometry and an electroactive product (p-benzoquinone), which was modeled in a non-linear regressional fashion to extract the relevant parameters. Finally, comparisons between the kinetic parameters presented in this paper and the rheological parameters of laccase monolayers immobilized in the same manner (Part I of this two paper series) were performed. Improvements in the maximal enzyme-catalysed current, i(max), the apparent Michaelis-Menten constant, K(m) and the apparent biosensor sensitivity were noted for most of the surfaces with increasing linker length. Decreasing the lateral density of the spacer-arms brought about a general improvement in these parameters, which is attributed to the decrease in multiple points of immobilization undergone by functional proteins. Finally, comparisons between rheological data and kinetics data showed that the degree of viscosity exhibited by protein films has a negative influence on attached protein layers, while enhanced protein hydration levels (assessed piezoelectrically from data obtained in Paper 1) has a positive effect on those surfaces comprising rigidly bound protein layers.
蛋白质的固定方法对其整体构象，进而对其功能有很大影响。因此，深入了解蛋白质在固定化过程中经历的事件是操纵固定化方法的关键，以制定一种策略，在最小化生物传感器设计中固定化缺点的同时，发挥其优点。在这篇由两部分组成的系列论文的第二篇中，我们评估了通过共价键附着在 SAM 上的薄膜漆酶单层的动力学参数，这些 SAM 的间隔臂长度和间隔臂的横向密度不同。这是通过计时安培法和电活性产物（对苯醌）来实现的，该产物以非线性回归方式建模，以提取相关参数。最后，本文中呈现的动力学参数与以相同方式固定化的漆酶单层的流变学参数（本两部分论文系列的第一部分）进行了比较。对于大多数具有增加的连接子长度的表面，观察到最大酶催化电流 i(max)、表观米氏常数 K(m)和表观生物传感器灵敏度的提高。减少间隔臂的横向密度会导致这些参数的普遍提高，这归因于功能蛋白经历的固定化多点减少。最后，流变学数据和动力学数据之间的比较表明，蛋白膜表现出的粘度程度对附着的蛋白层有负面影响，而增强的蛋白水合水平（通过从第 1 篇论文中获得的数据进行压电评估）对那些包含刚性结合蛋白层的表面有积极影响。
