Department of Biology, University of Naples Federico II, 80126, Naples, Italy.
Department of Molecular Chemistry, CNRS, University Grenoble Alpes, 38000, Grenoble, France.
Sci Rep. 2021 Feb 4;11(1):2991. doi: 10.1038/s41598-021-82648-9.
The correct immobilization and orientation of enzymes on nanosurfaces is a crucial step either for the realization of biosensors, as well as to guarantee the efficacy of the developed biomaterials. In this work we produced two versions of a chimeric protein, namely ArsC-Vmh2 and Vmh2-ArsC, which combined the self-assembling properties of Vmh2, a hydrophobin from Pleurotus ostreatus, with that of TtArsC, a thermophilic arsenate reductase from Thermus thermophilus; both chimeras were heterologously expressed in Escherichia coli and purified from inclusion bodies. They were characterized for their enzymatic capability to reduce As(V) into As(III), as well as for their immobilization properties on polystyrene and gold in comparison to the native TtArsC. The chimeric proteins immobilized on polystyrene can be reused up to three times and stored for 15 days with 50% of activity loss. Immobilization on gold electrodes showed that both chimeras follow a classic Langmuir isotherm model towards As(III) recognition, with an association constant (K) between As(III) and the immobilized enzyme, equal to 650 (± 100) L mol for ArsC-Vmh2 and to 1200 (± 300) L mol for Vmh2-ArsC. The results demonstrate that gold-immobilized ArsC-Vmh2 and Vmh2-ArsC can be exploited as electrochemical biosensors to detect As(III).
正确地将酶固定在纳米表面上并使其定向是实现生物传感器的关键步骤，同时也能保证所开发的生物材料的功效。在这项工作中，我们制备了两种嵌合蛋白，即 ArsC-Vmh2 和 Vmh2-ArsC，它们分别结合了蕈状侧耳（Pleurotus ostreatus）来源的疏水蛋白 Vmh2 和嗜热砷酸盐还原酶 TtArsC 的自组装特性；这两种嵌合体均在大肠杆菌中异源表达，并从包涵体中纯化。对它们将 As(V)还原为 As(III)的酶促能力以及与天然 TtArsC 相比在聚苯乙烯和金上的固定化特性进行了表征。固定在聚苯乙烯上的嵌合蛋白可重复使用 3 次以上，在活性损失 50%的情况下可储存 15 天。在金电极上的固定化表明，两种嵌合体均遵循经典的 Langmuir 等温吸附模型来识别 As(III)，其与固定化酶的结合常数（K），对于 ArsC-Vmh2 为 650（±100）L mol，对于 Vmh2-ArsC 为 1200（±300）L mol。结果表明，金固定化 ArsC-Vmh2 和 Vmh2-ArsC 可被用作电化学生物传感器来检测 As(III)。
