Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications.

Autor:	Geiss AF; Biocatalysis and Biosensing Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria., Reichhart TMB; Biocatalysis and Biosensing Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.; DirectSens Biosensors GmbH, Am Rosenbühel 38, 3400 Klosterneuburg, Austria., Pejker B; Biocatalysis and Biosensing Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria., Plattner E; DirectSens Biosensors GmbH, Am Rosenbühel 38, 3400 Klosterneuburg, Austria., Herzog PL; DirectSens Biosensors GmbH, Am Rosenbühel 38, 3400 Klosterneuburg, Austria., Schulz C; DirectSens Biosensors GmbH, Am Rosenbühel 38, 3400 Klosterneuburg, Austria., Ludwig R; Biocatalysis and Biosensing Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.; DirectSens Biosensors GmbH, Am Rosenbühel 38, 3400 Klosterneuburg, Austria., Felice AKG; DirectSens Biosensors GmbH, Am Rosenbühel 38, 3400 Klosterneuburg, Austria., Haltrich D; Biocatalysis and Biosensing Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
Jazyk:	angličtina
Zdroj:	ACS sustainable chemistry & engineering [ACS Sustain Chem Eng] 2021 May 24; Vol. 9 (20), pp. 7086-7100. Date of Electronic Publication: 2021 May 12.
DOI:	10.1021/acssuschemeng.1c01165
Abstrakt:	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. Competing Interests: The authors declare no competing financial interest. (© 2021 The Authors. Published by American Chemical Society.)
Databáze:	MEDLINE
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