*6.1. Extending the Lifetime of Membrane Enzymes*

A major drawback of enzymes in EBC is their limited active lifetime, which usually ranges from few hours to several days [191]. This applies particularly when membrane enzymes in detergent solutions are used, and functional reconstitution of membrane enzymes into an amphiphilic bilayer, such as liposome or polymersome vesicles, could represent a strategy to extend the enzyme lifetime. Liposomes offer great biocompatibility because they mimic the natural environment of membrane proteins, but they lack chemical and physical long-term stability [192]. Polymersomes offer a more robust amphiphilic polymer environment with increased chemical and physical stability [193,194]. However, this non-native polymeric environment might be limiting the functional incorporation of a wider range of membrane proteins [195]. Recently, hybrid vesicle systems composed of a mixture of lipids and block copolymers, have been developed with the rationale to provide a compromise between the biocompatibility of liposomes and the stability and robustness of polymersomes. In 2016 we showed that hybrid vesicles, composed of biocompatible lipids and stable PBd–PEO copolymer, supported higher activity of reconstituted cytochrome *bo*<sup>3</sup> than the proteopolymersomes and significantly extended the functional lifetime of the membrane enzyme when compared to standard proteoliposomes [196] (Figure 5). In 2018, we achieved increased stability of cytochrome *bo*<sup>3</sup> reconstituted in hybrid vesicles up to 500 days [197]. Similarly, recent work from Dimova group showed that functional integration of cytochrome *bo*<sup>3</sup> oxidase in synthetic membranes made of PDMS-*g*-PEO was capable of lumen

acidification and such reconstituted system showed to increase the active lifetime and resistance to free radicals [198]. In 2017, Otrin et al. [199] demonstrated a similar ability to store gradients by reconstituting cytochrome *bo*<sup>3</sup> together with an ATP synthase in hybrid vesicles constituted of the same copolymer, PDMS-*g*-PEO. In 2018, Smirnova et al. presented a method that allowed transfer of a functional membrane protein, cytochrome *c* oxidase (cytochrome *aa*<sup>3</sup> or yeast Complex IV), with a disc of its native lipids into pre-formed liposomes of well-defined lipid composition and size using amphipathic styrene maleic acid (SMA) copolymer [200]. This recent advance in using SMA copolymer for membrane enzymes isolation and reconstitution offers the advantage to maintain the native phospholipids environment surrounding the proteins and, moreover, could reduce time and cost for enzyme isolation and reconstitution processes by avoiding detergent mediated extraction [201]. Further research into affordable purification strategies and extending the stability of commercially-relevant membrane enzymes is required for membrane enzymes to find applications in bioelectrocatalysis. An alternative approach would be to omit purification altogether and exploit the regenerative capacity of micro-organisms in microbial electrosynthesis.

**Figure 5.** Stability of reconstituted cytochrome *bo*<sup>3</sup> in hybrid vesicles. (**a**) Ribbon diagram of cytochrome *bo*<sup>3</sup> . (**b**) Schematic representation of cytochrome *bo*<sup>3</sup> reactions (**c**) schematic representation of proteo-phospholipid/block copolymer hybrid vesicles. (**d**) Comparison of cytochrome *bo*<sup>3</sup> activity in reconstituted hybrid vesicles with increasing polymer content over a period of 41 days. Reprinted with the permission from ref [196]. Copyright (2016), The Royal Society of Chemistry.
