Department of NanoBiotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria.
Biointerphases. 2011 Jun;6(2):63-72. doi: 10.1116/1.3589284.
Methods for organizing functional materials at the nanometer scale are essential for the development of novel fabrication techniques. One of the most relevant areas of research in nanobiotechnology concerns technological utilization of self-assembly systems, wherein molecules spontaneously associate into reproducible supramolecular structures. For this purpose, the laccase of Bacillus halodurans C-125 was immobilized on the S-layer lattice formed by SbpA of Lysinibacillus sphaericus CCM 2177 either by (i) covalent linkage of the enzyme to the natural protein self-assembly system or (ii) by construction of a fusion protein comprising the S-layer protein and the laccase. The laccase and the S-layer fusion protein were produced heterologously in Escherichia coli. After isolation and purification, the properties of the proteins, as well as the specific activity of the enzyme moiety, were investigated. Interestingly, the S-layer part confers a much higher solubility on the laccase as observed for the sole enzyme. Comparative spectrophotometric measurements of the enzyme activity revealed similar but significantly higher values for rLac and rSbpA/Lac in solution compared to the immobilized state. However, rLac covalently linked to the SbpA monolayer yielded a four to five time higher enzymatic activity than rSbpA/Lac immobilized on a solid support. Combined quartz crystal microbalance with dissipation monitoring (QCM-D) and electrochemical measurements (performed in an electrochemical QCM-D cell) revealed that rLac immobilized on the SbpA lattice had an approximately twofold higher enzymatic activity compared to that obtained with the fusion protein.
用于在纳米尺度上组织功能材料的方法对于开发新型制造技术至关重要。纳米生物技术中最相关的研究领域之一涉及自组装系统的技术利用，其中分子自发地缔合成可重复的超分子结构。为此，将嗜盐杆菌 C-125 的漆酶通过（i）酶与天然蛋白质自组装系统的共价连接或（ii）构建包含 S 层蛋白和漆酶的融合蛋白，固定在由溶壁微球菌 CCM 2177 的 SbpA 形成的 S 层晶格上。漆酶和 S 层融合蛋白在大肠杆菌中异源生产。分离和纯化后，研究了蛋白质的性质以及酶部分的比活性。有趣的是，与单独的酶相比，S 层部分赋予漆酶更高的溶解度。对酶活性的比较分光光度测量表明，与固定化状态相比，rLac 和 rSbpA/Lac 在溶液中的相似但明显更高的值。然而，与固定在固体载体上的 rSbpA/Lac 相比，共价连接到 SbpA 单层上的 rLac 产生的酶活性高四到五倍。结合石英晶体微天平与耗散监测（QCM-D）和电化学测量（在电化学 QCM-D 细胞中进行）表明，与融合蛋白相比，固定在 SbpA 晶格上的 rLac 的酶活性大约高出两倍。
