Self-assembling thermostable chimeras as new platform for arsenic biosensing.

Affiliation

Puopolo R(1), Sorrentino I(2), Gallo G(1), Piscitelli A(3), Giardina P(3), Le Goff A(4), Fiorentino G(5).
Author information:
(1)Department of Biology, University of Naples Federico II, 80126, Naples, Italy.
(2)Department of Molecular Chemistry, CNRS, University Grenoble Alpes, 38000, Grenoble, France.
(3)Department of Chemical Sciences, University of Naples Federico II, 80126, Naples, Italy.
(4)Department of Molecular Chemistry, CNRS, University Grenoble Alpes, 38000, Grenoble, France. [Email]
(5)Department of Biology, University of Naples Federico II, 80126, Naples, Italy. [Email]

Abstract

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 (KAsIII) between As(III) and the immobilized enzyme, equal to 650 (± 100) L mol-1 for ArsC-Vmh2 and to 1200 (± 300) L mol-1 for Vmh2-ArsC. The results demonstrate that gold-immobilized ArsC-Vmh2 and Vmh2-ArsC can be exploited as electrochemical biosensors to detect As(III).