Escherichia coliscopper efflux oxidase (CueO) has rarely been employed in the cathodic compartment of enzymatic biofuel cells (EBFCs) due to its low redox potential (0.36 Vv s. Ag/AgCl, pH 5.5) towards O 2 reduction. Herein, directed evolution of CueO towards am ore positive onset potential was performed in an electrochemical screening system. An improved CueO variant (D439T/L502K) was obtained with asignificantly increased onset potential (0.54 V), comparable to that of high-redox-potential fungal laccases. Upon coupling with an anodic compartment, the EBFC exhibited an open-circuit voltage (V oc )o f0 .56 V. Directed enzyme evolution by tailoring enzymes to application conditions in EBFCs has been validated and might, in combination with molecular understanding,e nable future breakthroughs in EBFC performance

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Understanding interactions between polymers and enzymes to boost enzymatic activity is of high importance for application of enzymes in multicomponent systems, such as laundry, food, pharmaceuticals, or cosmetics. Proteases are widely used in industries and increased performance in the presence of polymers has been reported. Boosting of enzymes activity by polymers and understanding of the molecular principles is of high interest in biomedical and biotechnological applications. A molecular understanding of the boosting effect of poly­(acrylic acid) (PAA) and poly­(l-γ-glutamic acid) (γ-PGA) for a nonspecific subtilisin protease (Protein Database (PDB) ID: 1ST3) was generated through biophysical characterization (fluorescence correlation and circular dichroism spectroscopies, isothermal titration calorimetry), molecular dynamics simulations, and protease reengineering (site-saturation mutagenesis). Our study revealed that enthalpically driven interactions via key amino acid residues close to the protease Ca2+ binding sites cause the boosting effect in protease activity. On the molecular level electrostatic interactions results in the formation of protease-polyelectrolyte complexes. Site-saturation mutagenesis on positions S76, I77, A188, V238, N242, and K245 yielded an increased proteolytic performance against a complex protein mixture (trademark CO-3; up to ∼300% and ∼70%) in the presence of PAA and γ-PGA. Being able to fine-tune interactions between proteins and negatively charged polymers through integrative use of computational design, protein re-engineering and biophysical characterization proved to be an efficient workflow to improve protease performance.

The high interfacial activity of protein-polymer conjugates has inspired their use as stabilizers for Pickering emulsions, resulting in many interesting applications such as synthesis of templated micro-compartments and protocells or vehicles for drug and gene delivery. In this study we report, for the first time, the stabilization of Pickering emulsions with conjugates of a genetically modified transmembrane protein, ferric hydroxamate uptake protein component A (FhuA). The lysine residues of FhuA with open pore (FhuA ΔCVF) were modified to attach an initiator and consequently controlled radical polymerization (CRP) carried out via the grafting-from technique. The resulting conjugates of FhuA ΔCVF with poly(N-isopropylacrylamide) (PNIPAAm) and poly((2-dimethylamino)ethyl methacrylate) (PDMAEMA), the so-called building blocks based on transmembrane proteins (BBTP), have been shown to engender larger structures. The properties such as pH-responsivity, temperature-responsivity and interfacial activity of the BBTP were analyzed using UV-Vis spectrophotometry and pendant drop tensiometry. The BBTP were then utilized for the synthesis of highly stable Pickering emulsions, which could remain non-coalesced for well over a month. A new UV-crosslinkable monomer was synthesized and copolymerized with NIPAAm from the protein. The emulsion droplets, upon crosslinking of polymer chains, yielded micro-compartments. Fluorescence microscopy proved that these compartments are of micrometer scale, while cryo-scanning electron microscopy and scanning force microscopy analysis yielded a thickness in the range of 11.1 ± 0.6 to 38.0 ± 18.2 nm for the stabilizing layer of the conjugates. Such micro-compartments would prove to be beneficial in drug delivery applications, owing to the possibility of using the channel of the transmembrane protein as a gate and the smart polymer chains as trigger switches to tune the behavior of the capsules.