Kamathewatta Nilan J B, Deay Dwight O, Karaca Banu Taktak, Seibold Steve, Nguyen Tyler M, Tomás Brandon, Richter Mark L, Berrie Cindy L, Tamerler Candan
Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, United States.
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, United States.
Langmuir. 2020 Oct 13;36(40):11908-11917. doi: 10.1021/acs.langmuir.0c01986. Epub 2020 Sep 28.
Flavin oxidases are valuable biocatalysts for the oxidative synthesis of a wide range of compounds, while at the same time reduce oxygen to hydrogen peroxide. Compared to other redox enzymes, their ability to use molecular oxygen as an electron acceptor offers a relatively simple system that does not require a dissociable coenzyme. As such, they are attractive targets for adaptation as cost-effective biosensor elements. Their functional immobilization on surfaces offers unique opportunities to expand their utilization for a wide range of applications. Genetically engineered peptides have been demonstrated as enablers of the functional assembly of biomolecules at solid material interfaces. Once identified as having a high affinity for the material of interest, these peptides can provide a single step bioassembly process with orientation control, a critical parameter for functional immobilization of the enzymes. In this study, for the first time, we explored the bioassembly of a putrescine oxidase enzyme using a gold binding peptide tag. The enzyme was genetically engineered to incorporate a gold binding peptide with an expectation of an effective display of the peptide tag to interact with the gold surface. In this work, the functional activity and expression were investigated, along with the selectivity of the binding of the peptide-tagged enzyme. The fusion enzyme was characterized using multiple techniques, including protein electrophoresis, enzyme activity, and microscopy and spectroscopic methods, to verify the functional expression of the tagged protein with near-native activity. Binding studies using quartz crystal microbalance (QCM), nanoparticle binding studies, and atomic force microscopy studies were used to address the selectivity of the binding through the peptide tag. Surface binding AFM studies show that the binding was selective for gold. Quartz crystal microbalance studies show a strong increase in the affinity of the peptide-tagged protein over the native enzyme, while activity assays of protein bound to nanoparticles provide evidence that the enzyme retained catalytic activity when immobilized. In addition to showing selectivity, AFM images show significant differences in the height of the molecules when immobilized through the peptide tag compared to immobilization of the native enzyme, indicating differences in orientation of the bound enzyme when attached via the affinity tag. Controlling the orientation of surface-immobilized enzymes would further improve their enzymatic activity and impact diverse applications, including oxidative biocatalysis, biosensors, biochips, and biofuel production.
黄素氧化酶是用于多种化合物氧化合成的有价值的生物催化剂，同时将氧气还原为过氧化氢。与其他氧化还原酶相比，它们利用分子氧作为电子受体的能力提供了一个相对简单的系统，该系统不需要可解离的辅酶。因此，它们是作为具有成本效益的生物传感器元件进行改造的有吸引力的目标。它们在表面的功能固定为将其应用扩展到广泛的领域提供了独特的机会。基因工程肽已被证明是生物分子在固体材料界面功能组装的促成因素。一旦确定对感兴趣的材料具有高亲和力，这些肽可以提供具有取向控制的单步生物组装过程，这是酶功能固定的关键参数。在本研究中，我们首次探索了使用金结合肽标签对腐胺氧化酶进行生物组装。对该酶进行基因工程改造以掺入金结合肽，期望有效展示肽标签以与金表面相互作用。在这项工作中，研究了功能活性和表达，以及肽标记酶结合的选择性。使用多种技术对融合酶进行了表征，包括蛋白质电泳、酶活性以及显微镜和光谱方法，以验证具有接近天然活性的标记蛋白的功能表达。使用石英晶体微天平（QCM）进行结合研究、纳米颗粒结合研究和原子力显微镜研究，以确定通过肽标签结合的选择性。表面结合原子力显微镜研究表明，这种结合对金具有选择性。石英晶体微天平研究表明，与天然酶相比肽标记蛋白的亲和力大幅增加，而与纳米颗粒结合的蛋白质的活性测定提供了证据，表明该酶在固定化时保留了催化活性。除了显示选择性外，原子力显微镜图像显示通过肽标签固定时分子高度与天然酶固定时相比存在显著差异，表明通过亲和标签附着时结合酶的取向存在差异。控制表面固定酶的取向将进一步提高其酶活性，并影响包括氧化生物催化、生物传感器、生物芯片和生物燃料生产在内的各种应用。
