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Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications
[Image: see text] Cellobiose dehydrogenase (CDH) is an attractive oxidoreductase for bioelectrochemical applications. Its two-domain structure allows the flavoheme enzyme to establish direct electron transfer to biosensor and biofuel cell electrodes. Yet, the application of CDH in these devices is i...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical Society
2021
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8296668/ https://www.ncbi.nlm.nih.gov/pubmed/34306835 http://dx.doi.org/10.1021/acssuschemeng.1c01165 |
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author | Geiss, Andreas F. Reichhart, Thomas M. B. Pejker, Barbara Plattner, Esther Herzog, Peter L. Schulz, Christopher Ludwig, Roland Felice, Alfons K. G. Haltrich, Dietmar |
author_facet | Geiss, Andreas F. Reichhart, Thomas M. B. Pejker, Barbara Plattner, Esther Herzog, Peter L. Schulz, Christopher Ludwig, Roland Felice, Alfons K. G. Haltrich, Dietmar |
author_sort | Geiss, Andreas F. |
collection | PubMed |
description | [Image: see text] Cellobiose dehydrogenase (CDH) is an attractive oxidoreductase for bioelectrochemical applications. Its two-domain structure allows the flavoheme enzyme to establish direct electron transfer to biosensor and biofuel cell electrodes. Yet, the application of CDH in these devices is impeded by its limited stability under turnover conditions. In this work, we aimed to improve the turnover stability of CDH by semirational, high-throughput enzyme engineering. We screened 13 736 colonies in a 96-well plate setup for improved turnover stability and selected 11 improved variants. Measures were taken to increase the reproducibility and robustness of the screening setup, and the statistical evaluation demonstrates the validity of the procedure. The selected CDH variants were expressed in shaking flasks and characterized in detail by biochemical and electrochemical methods. Two mechanisms contributing to turnover stability were found: (i) replacement of methionine side chains prone to oxidative damage and (ii) the reduction of oxygen reactivity achieved by an improved balance of the individual reaction rates in the two CDH domains. The engineered CDH variants hold promise for the application in continuous biosensors or biofuel cells, while the deduced mechanistic insights serve as a basis for future enzyme engineering approaches addressing the turnover stability of oxidoreductases in general. |
format | Online Article Text |
id | pubmed-8296668 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American
Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-82966682021-07-22 Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications Geiss, Andreas F. Reichhart, Thomas M. B. Pejker, Barbara Plattner, Esther Herzog, Peter L. Schulz, Christopher Ludwig, Roland Felice, Alfons K. G. Haltrich, Dietmar ACS Sustain Chem Eng [Image: see text] Cellobiose dehydrogenase (CDH) is an attractive oxidoreductase for bioelectrochemical applications. Its two-domain structure allows the flavoheme enzyme to establish direct electron transfer to biosensor and biofuel cell electrodes. Yet, the application of CDH in these devices is impeded by its limited stability under turnover conditions. In this work, we aimed to improve the turnover stability of CDH by semirational, high-throughput enzyme engineering. We screened 13 736 colonies in a 96-well plate setup for improved turnover stability and selected 11 improved variants. Measures were taken to increase the reproducibility and robustness of the screening setup, and the statistical evaluation demonstrates the validity of the procedure. The selected CDH variants were expressed in shaking flasks and characterized in detail by biochemical and electrochemical methods. Two mechanisms contributing to turnover stability were found: (i) replacement of methionine side chains prone to oxidative damage and (ii) the reduction of oxygen reactivity achieved by an improved balance of the individual reaction rates in the two CDH domains. The engineered CDH variants hold promise for the application in continuous biosensors or biofuel cells, while the deduced mechanistic insights serve as a basis for future enzyme engineering approaches addressing the turnover stability of oxidoreductases in general. American Chemical Society 2021-05-12 2021-05-24 /pmc/articles/PMC8296668/ /pubmed/34306835 http://dx.doi.org/10.1021/acssuschemeng.1c01165 Text en © 2021 The Authors. Published by American Chemical Society Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Geiss, Andreas F. Reichhart, Thomas M. B. Pejker, Barbara Plattner, Esther Herzog, Peter L. Schulz, Christopher Ludwig, Roland Felice, Alfons K. G. Haltrich, Dietmar Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications |
title | Engineering the Turnover Stability of Cellobiose Dehydrogenase
toward Long-Term Bioelectronic Applications |
title_full | Engineering the Turnover Stability of Cellobiose Dehydrogenase
toward Long-Term Bioelectronic Applications |
title_fullStr | Engineering the Turnover Stability of Cellobiose Dehydrogenase
toward Long-Term Bioelectronic Applications |
title_full_unstemmed | Engineering the Turnover Stability of Cellobiose Dehydrogenase
toward Long-Term Bioelectronic Applications |
title_short | Engineering the Turnover Stability of Cellobiose Dehydrogenase
toward Long-Term Bioelectronic Applications |
title_sort | engineering the turnover stability of cellobiose dehydrogenase
toward long-term bioelectronic applications |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8296668/ https://www.ncbi.nlm.nih.gov/pubmed/34306835 http://dx.doi.org/10.1021/acssuschemeng.1c01165 |
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