5.2.2. Engineering of Enzymes Seeking for Both Enhanced ET Efficiency and Stability

The general engineering procedures developed to enhance enzyme stability can be adapted for redox enzymes, including introduction of stronger bonds, removal of potential degradation sites, or oligomeric structure formation [238–240]. However, the most interesting strategies here are to obtain enhanced stability and ET efficiency at the same time. Then, the main targets for protein engineering will lie (i) in the vicinity of the active site or (ii) at specific points of the enzyme surface able to anchor it with the best orientation for the highest ET rate (Figure 11). For strategy (i), the vicinity of the heme cavity of peroxidase [241] or of the CuT1 site of LAC has been targeted [242,243]. Two recent reports illustrate this concept. Two alanine residues were mutated to one leucine and one valine. Not only did the introduction of hydrophobic residues increase the redox potential of the CuT1 site by 50 mV, but it also protected the CuT1 site from the solvent as well as enhanced temperature and pH stability of the variant. The mutant was stabilized at the electrode. It retained roughly 80% of its initial activity after 96 h incubation at pH 4.0 and presented a half-life of 60 min at 70 ◦C against 23 min for the wild type protein [203]. With the aim of electricity production directly from seawater recycling, recombinant forms of CotA LAC from *B. licheniformis* were produced [244]. A double mutation, one in a region close to the CuT1 and the other on the surface of the protein, was shown to induce a synergic effect in bioelectrocatalysis. Conformational changes in the double mutant were proved by CD and fluorescence that allow the variant to catalyze O<sup>2</sup> reduction in seawater, a very unusual property for classical LACs. Protein leaching under such high ionic strength conditions must however be solved.

**Figure 11.** Enzyme engineering strategies for enhanced stability. (**Left**) Mutations in the vicinity of the active site; computer-guided mutagenesis and directed evolution of a fungal LAC in CuT1 vicinity induce enhanced stability at pH 4 compared to the WT. Reproduced with permission from [243]. (**Right**) Covalent immobilization of BOD through site-directed mutagenesis and reaction with maleimide groups on the electrode surface induce enhanced stability of the mutant (black curve) compared to the WT (red curve). Adapted with permission from [227].

For strategy (ii), covalent immobilization of enzymes at electrodes was realized by producing site-directed variants with cysteine residues located at different sites of the enzymes able to react with maleimide groups immobilized on the electrode. This elegant method combines controlled orientation of the protein at the electrode for ET, sufficient flexibility

for activity, and expected enhanced stability. In the case of cellulose dehydrogenase, 40% of initial electroactivity was recovered after 2 months of storage while the wild type enzyme loses progressively its activity within 20 days [245,246]. In addition, controlled orientation allowed investigating mechanistic aspects of the ET. The immobilization method was extended to BOD from *Magnaporthe oryzae* showing an enhanced stability with almost no loss of electroactivity for 3 days [227] (Figure 11).
