Serr spectroelectrochemistry as a guide for rational design of dyp-based bioelectronics devices

Funding Information: Acknowledgments: We acknowledge the support from Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE 2020-Programa Operacional Competitividade e Internacionalização (POCI), from FCT—Fundação para a Ciência e a Tecnologia (PTDC/BIA-BFS/31026/2017 and 2020.05017.BD) and from the European Union’s Horizon 2020 Research and Innovation Program, through TIMB3 and B-LigZymes projects (grant agreements numbers are 810856 and 824017, respectively). Molecular graphics and analyses were performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. Funding Information: This research was funded by FCT?Funda??o para a Ci?ncia e a Tecnologia (grant number: PTDC/BIA-BFS/31026/2017). The APC was funded by the TIMB3 project, European Union?s Horizon 2020 Research and Innovation Program (grant agreement No.: 810856). Funding Information: Funding: This research was funded by FCT—Fundação para a Ciência e a Tecnologia (grant number: PTDC/BIA-BFS/31026/2017). The APC was funded by the TIMB3 project, European Union’s Horizon 2020 Research and Innovation Program (grant agreement No.: 810856). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Immobilised dye-decolorizing peroxidases (DyPs) are promising biocatalysts for the development of biotechnological devices such as biosensors for the detection of H2O2 . To this end, these enzymes have to preserve native, solution properties upon immobilisation on the electrode surface. In this work, DyPs from Cellulomonas bogoriensis (CboDyP), Streptomyces coelicolor (ScoDyP) and Thermobifida fusca (TfuDyP) are immobilised on biocompatible silver electrodes functionalized with alkanethiols. Their structural, redox and catalytic properties upon immobilisation are evaluated by surface-enhanced resonance Raman (SERR) spectroelectrochemistry and cyclic voltammetry. Among the studied electrode/DyP constructs, only CboDyP shows preserved native structure upon attachment to the electrode. However, a comparison of the redox potentials of the enzyme in solution and immobilised states reveals a large discrepancy, and the enzyme shows no electrocatalytic activity in the presence of H2O2 . While some immobilised DyPs outperform existing peroxidase-based biosensors, others fail to fulfil the essential requirements that guarantee their applicability in the im-mobilised state. The capacity of SERR spectroelectrochemistry for fast screening of the performance of immobilised heme enzymes places it in the front-line of experimental approaches that can advance the search for promising DyP candidates. publishersversion published