Periodic Reporting for period 1 - MELBA (MEMBRANE PROTEINS OF ELECTROACTIVE BACTERIA PROBED AT LIPID LAYERS ONTO MODIFIED ELECTRODES)
Okres sprawozdawczy: 2017-04-01 do 2019-03-31
The overall objective of MELBA is to develop an efficient and versatile electrochemical platform for probing electroactive bacteria membrane proteins incorporated in artificial lipid deposits supported on modified carbon electrode. In a first approach, the electrodes are modified with pH-responsive electrophores such as quinone units and then covered by lipid layers or deposits creating an artificial supported lipid membrane in which membrane proteins are incorporated and electrochemically probed. Indeed, the intrinsic electroactivity of the protein (if any) can be probed directly with the lipid modified electrode and possible proton transport occurring across the lipid layer by the membrane protein can in principle be detected with the grafted pH-responsive redox probe.
Concerning the career objectives of the fellow, the goal of holding a permanent academic position in France has been reached because the experienced researcher will be hired as an assistant professor at the University of Nantes (France) on September 2019.
This work was then extended periplasmic proteins from electroactive bacteria such as flavocytochrome c3, a tetraheme FAD-containing periplasmic flavoenzyme isolated from the bacterium Shewanella putrefaciens, taken as a model pH-dependent redox protein from electroactive bacteria. The production and purification of the flavocytochrome c3 enzyme from Shewanella putrefaciens DSM 9451 was carried out in the lab partner (ITQB, Lisbon) of Secondment. In summary, the electrochemical detection of adsorbed flavocytochrome c3 onto edge plane pyrolytic graphite electrode was achieved before and after surface grafting of a catechol pH probe. The periplasmic flavocytochrome c3 redox enzyme from Shewanella putrefaciens was successfully immobilized onto electrode with a high surface coverage, following a simple and efficient strategy consisting of consecutive cyclic voltammetry in a flavocytochrome c3 solution containing polymyxin as co-adsorbate until a steady state current is reached. The electrochemical detection of both grafted catechol and adsorbed flavocytochrome c3 redox systems was achieved while maintaining intact their native electrochemical properties such as the pH-dependent electroactivity of the redox enzyme and its fumarate reductase activity. The pH-dependent redox properties of the grafted catechol were evaluated in the presence of the immobilized enzyme onto electrode and demonstrated the effectiveness of the catechol pH sensor in the presence of the immobilized protein.
In a future work, the electrochemical platform of MELBA could be extended to probe ionophore activity by immobilizing appropriate ion sensor onto electrode surface.
In addition, covalent grafted molecules with quinone-like moieties as suitable probes to evaluate the pH changes at biofilm/electrode interface in microbial fuel cells as a promising biotechnology.
A personal impact of MELBA for the experienced researcher will be to submit a personal proposal for an ERC Starting grant on October 2019.