**1. Introduction**

In the search for a more sustainable way of life, our society must adapt nowadays to reduce the use of harmful products or high carbon energy sources deleterious for our planet, hence for our lives. Many industrial sectors are concerned by this revolution, from fine product synthesis to environmental monitoring, clinical diagnosis or energy production. Catalysis of the involved chemical reactions is a key process towards sustainability by accelerating kinetics, or increasing selectivity. However, chemical catalysts themselves are very often not produced and used in eco-friendly manner, requiring organic solvents for their synthesis or being based on metals rare on earth. In that sense, enzymes can be regarded as sustainable alternatives with great advantages directly linked to their intrinsic properties required to sustain life [1] In particular, many different enzymes are involved in a variety of reactions in microorganisms where they operate in mild conditions, developing high catalytic activity and specificity for their substrate. Their amazing functional diversity directly originates from the chemistry and polarity of amino acids that fold in a diversity of structures. In addition, they are produced in mild aqueous conditions and are totally biodegradable. However, their weak stability and ability to maintain biological activity during storage and operation, freeze-thaw steps, and in non-physiological environments lower their attractiveness. Development of strategies to enhance enzyme shelf life while maintaining catalytic activity have been the subject of many researches during the last decades [2–4].

Redox enzymes belong to a class of enzymes containing redox cofactors that are necessary for their activity. These oxidoreductases transform their substrate by exchanging electrons with their physiological partners in the metabolic chain. Intra-enzyme redox components can be organic cofactors such as flavin adenine dinucleotide (FAD) for sugar oxidation [5]. They can be metal centers, including copper for oxygen reduction [6], iron and nickel for hydrogen evolution and uptake or CO<sup>2</sup> reduction [7–9], manganese for water oxidation in photosynthesis [10], etc. Some others are dependent on nicotinamide adenine dinucleotide phosphate (NADPH) to realize enzymatic transformations [11]. Such enzymes

**Citation:** Beaufils, C.; Man, H.-M.; de Poulpiquet, A.; Mazurenko, I.; Lojou, E. From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes. *Catalysts* **2021**, *11*, 497. https://doi.org/10.3390/catal11040497

Academic Editor: Giovanni Gadda

Received: 17 March 2021 Accepted: 10 April 2021 Published: 14 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

have been envisioned as biocatalysts in biosensors [12–14], biosynthesis reactors [15,16], or biofuel cells [17,18]. This domain is referred as Bioelectrochemistry. As for non-redox enzymes, one of the main limitations in the large-scale development of these biotechnologies is the weak stability of bioelectrodes based on redox enzymes. In particular, in the different biotechnological devices listed just before, enzymes may face high temperatures or high salt concentrations. They may have to operate at gas-liquid interfaces or even at the three phase boundaries. In addition, to exploit the catalytic properties of these redox enzymes in biotechnology, a further step is to ensure electron exchange between the protein and a conductive surface [19–21]. This prerequisite imposes the enzyme adopts certain configurations once immobilized, so that not only the structural conformation and dynamic remains for substrate accessibility and activity, but also enzyme positioning and orientation on the solid interface allows electron transfer.

The purpose of this review is to discuss whether general strategies available for all enzyme stabilization can be applied for the special case of redox enzymes used in bioelectrochemistry. To reach this objective, the fundamental bases for all enzyme stabilization by general reported strategies will be first detailed, with a focus on the immobilization procedure as an important way for stabilization. In a second step, we will emphasize the particular features of redox enzymes that must be considered before applying any available stabilization strategies. Ultimately, we will discuss which additional specific strategies must be set up for redox protein, and by extension for bioelectrode, stabilization. Because the topic is very large, this review will not provide an exhaustive list of references but will instead aim to highlight major issues related to enzyme and redox enzyme stability with illustrations taken from papers published during the last few years.
