Abstract
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.
Highlights
Cellobiose dehydrogenase (CDH, EC 1.1.99.18) is an extracellular glycoenzyme of ∼90 kDa secreted by various fungi, both Ascomycota and Basidiomycota.[1]
Significant oxidation was measured for the peptide fragment 682−694, which contains one methionine (M690), a stability hotspot known for CDH from previous studies.[58]
DCIP is reduced directly at the DH of CDH, and its reduction does not involve the interdomain electron transfer, which is essential for the direct electron transfer ability of CDH on electrodes mediated by the cytochrome domain
Summary
Cellobiose dehydrogenase (CDH, EC 1.1.99.18) is an extracellular glycoenzyme of ∼90 kDa secreted by various fungi, both Ascomycota and Basidiomycota.[1]. CDH is the only currently known extracellular flavoheme enzyme.[3] The N-terminal cytochrome domain and the C-terminal DH span about 180 Å in longitudinal dimension[4−6] and are connected by a ∼20 amino-acid-long linker peptide. This linker is responsible for high domain mobility,[7] which plays a major role in the function of CDH. CDH oxidizes various sugars, including cellobiose or lactose and in some instances even glucose, at its DH while concurrently electrons are transferred to FAD. Reoxidation of FADH2 can occur directly by reduction of various quinones as electron acceptors, or by interdomain electron transfer (IET) to the heme group, from where they can be passed on to cytochrome c as an artificial electron acceptor or to lytic polysaccharide monooxygenases (LPMO), the presumed natural interaction partner.[8−10] CDH and LPMO are part of an extracellular electron transfer system efficiently fueling the breakdown of recalcitrant lignocellulose by sequential transfer of electrons from soluble sugars via FAD and heme b to the active site copper in LPMO.[11]
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