5.2.1. How Far Can General Enzyme Stabilization Strategies Be Extended to Bioelectrocatalysis?

Table 1 reports bioelectrode stability for two main enzymatic reactions, i.e., O<sup>2</sup> reduction and H<sup>2</sup> oxidation. These two enzymatic reactions have been chosen as relevant for the development of biofuel cells, devices currently developed as alternatives to platinum-based fuel cells. As can be seen, many of the strategies detailed in the previous chapters of this review are involved in bioelectrode stabilization. It should be noted that in most cases stabilization refers to operational duration. Thus, the stability parameter is mainly evaluated through the percentage of preserved catalytic current. Although crucial for improving bioelectrocatalysis, only rare studies report enzyme conformational changes upon redox immobilization of enzymes on electrode.

From the survey of the literature reported in Table 1, general features of enzyme stabilization inducing bioelectrode stability can be recognized. This is the case for redox enzymes extracted from extremophiles yielding efficient bioelectrocatalysis while expected to be more stable than their mesophilic homologues even at room temperature. We already provided in Section 3 one relevant example showing that the NiFe HASE from the hyperthermophilic bacterium *A. aeolicus* was much more stable than its mesophilic homologue from *R. eutropha* [111]. This enhanced stability of the hyperthermophilic enzyme is fruitfully exploited to design an H2/O<sup>2</sup> fuel cell displaying at the same time high catalytic currents reported to the mass of enzymes (in the order 1 A/mg enzyme), delivering mWs of power in a range of temperature 20–60 ◦C, and a half-life for the bioanode of one month, a quite encouraging stability compared to other reported devices [198]. It should be also mentioned that at the cathodic side, the thermostable BOD from *B. pumilus* is used which also shows enhanced stability compared to the widely used *M. verrucaria* BOD. Other recent works proved the efficiency of extremophilic enzymes in bioelectrochemical systems, as illustrated in the two following examples. *Bacillus* sp. FNT thermophilic LAC showed 60–80% of remaining activity after two weeks of storage at room temperature when the activity of mesophilic *T. versicolor* LAC was zero in the same conditions [199]. The activity of cellobiose dehydrogenase from *Corynascus thermophilus* retained more than 50% activity after 5 days of multicycle voltammetry mode and about 30% after 9 days [200].

As for non-redox enzymatic catalysis, stability of enzymatic electrodes requires to avoid enzyme leaching from the electrode. Entrapment in porous matrices and covalent immobilization through suitable functionalization of the electrode are ways to improve redox enzyme-based bioelectrode stabilization. However, it must be considered that the redox enzyme will sense an electric field during a bioelectrocatalytic process and that some species can be generated through the electrochemical potential application, thus requiring stabilization strategies more specific to bioelectrochemistry. In addition, the electron exchange between the electrode and the enzyme which is the basis of bioelectrocatalysis imposes specific constraints that may prevent the use of the strategies developed above. Among these constraints, the host matrix must be conductive, cross-linking that yields heterogeneities in enzyme orientation can make a population of enzymes incapable of electron transferring; addition of polyols may electrically isolate the enzyme preventing any electron transfer, or use of high concentrations of salts in the electrolyte solution can favor the leaching of the enzymes from the electrode surface. Hence, any stabilization protocols described above must be evaluated in light of their adaptability to bioelectrochemistry with redox enzymes.


**Table 1.** Bioelectrode stability: cases of multicopper oxidases (MCOs) and hyperthermophilic hydrogenase (HASEs).


**Table 1.***Cont.*


**Table1.***Cont.*



**Table1.***Cont.*
