posted on 2021-05-12, 19:03authored byAndreas
F. Geiss, Thomas M. B. Reichhart, Barbara Pejker, Esther Plattner, Peter L. Herzog, Christopher Schulz, Roland Ludwig, Alfons K. G. Felice, Dietmar Haltrich
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.