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

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.

An Oxygen Insensitive Amperometric Glucose Biosensor Based on An Engineered Cellobiose Dehydrogenase: Direct versus Mediated Electron Transfer Responses

“…The K M,app of 37.9±5.4 mM obtained for the optimized MET biosensor is similar to the value of 36±1 mM reported for a WT Ch CDH DET biosensor [21] . However, the j max value of 719±43 μA cm −2 achieved with the MET biosensor is 15‐fold higher than the 47 μA cm −2 obtained for that DET biosensor, [21] and more than 30‐fold higher than that obtained for the optimized WT Ch CDH DET biosensor reported on here. This higher j max value results in 17‐fold increased sensitivity, and a wider linear range, to glucose for the MET biosensor over the WT Ch CDH DET biosensor operating in PBS (Table 1).…”

“…While K M,app values for biosensors are influenced by the physicochemical properties of the films on the surface and the differences in enzyme immobilization methods, the K M,app value for the WT Ch CDH DET biosensor is nearly 3‐fold lower than the value of 36±1 mM reported for a WT Ch CDH DET biosensor prepared by drop‐coating 1 μL of a 15 mg mL −1 WT Ch CDH solution on electrodes [21] . As a consequence the linear range of 0–5 mM and j max of 21.8±0.3 μA cm −2 for the DET biosensor based on an enzyme volume of 18 μL (180 μg), Figure 3 and Table 1, are lower than the 0–10 mM and 47 μA cm −2 obtained at the WT Ch CDH DET biosensor from a previous report [21] . Signal sensitivity is, however, similar for both type of electrodes at ∼1 μA cm −2 mM −1 (Table 1).…”

“…The WT Ch CDH biosensors developed do not display a significant change in sensor response in the presence of oxygen (Figure 5). However, WT Ch CDH turnover stability in the presence of oxygen and excess glucose has been shown to be affected by the presence of oxygen, most likely related to the susceptibility of methionine residues to oxidation given that replacement of methionine residues results in improved turnover stability [21] …”

“…Purified enzymes were concentrated and rebuffered to 1 mM phosphate buffer, pH 7.4, with centrifugal filters (Amicon; 30 kDa mass cutoff) to a concentration of approximately 15 mg mL −1 and stored at 4 °C. The enzyme is referred to as wild‐type (WT Ch CDH) throughout this text to enable comparison to previously published data on the enzyme [21] …”

“…[13] CDH-based biosensors have been realized using carbon [18] and gold [19] as electrode material. Engineering of CDH has been performed to generate CDH variants for several purposes [10,20,21] . For instance, de-glycosylation of CDH can increase the faradaic efficiency [22][23][24] while amino acid substitutions in the active site can be used to change the substrate specificity of CDH.…”