Directed Evolution of a Bacterial Laccase (CueO) for Enzymatic Biofuel Cells

Abstract

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

Rapid and Oriented Immobilization of Laccases on Electrodes via a Methionine-Rich Peptide

“…26 Recently, successful protein engineering of CueO aiming at improved electrocatalytic performance has been reported. 27,28 Attractively, CueO adsorbed in a superfast manner (20 s) is able to saturate the catalytic current, 27,28 much faster than the immobilization of other laccases, even most of the oxidoreductases. X-ray crystallographic structure determination shows that CueO, unlike other laccases, displays an additional 45-residue insert (residues 355−399) over the T1 Cu active site.…”

Water/Oxygen Circulation-Based Biophotoelectrochemical System for Solar Energy Storage and Release

“…Noticeably, when BOD was selected as cathodic catalyst in discharging process, the current has a slight decrease after 12 cycles. The main reason could be ascribed to the activity decline of enzyme biocatalyst, which may arise from the inherent drawbacks of enzyme proteins, such as short active lifetime, 35 fragility of protein structure, 35,36 and time-dependent degradation in biocatalytic performance 37 (Figure S13). Fundamentally, on the basis of the modular system design, the working performance of this integrated device can be further improved by optimizing the individual components.…”

“…Developing a photoanode material, 38 with narrower bandgap and higher photocatalytic activity, and/or introduction of a electrocatalytic overlayer to the surface of semiconductors, 39 may be an alternative way to improve photocharging performance. Additionally, directed evolution of multicopper oxidases, through iterative rounds of diversity generation and screening, 36,40 and/or optimizing substrate modified strategy is also a feasible method to enhance biodischarging performance. Hence, according to different requirements of operational conditions, solar energy storage and release processes can be optimized independently, which makes this BPECS better to satisfy the demands of practical application.…”

Multicopper oxidases: modular structure, sequence space, and evolutionary relationships

“…The application of standard numbering will allow researchers to compare MCOs with those that have already been subjected to mutagenesis studies and allow for a deeper insight into which type of approaches should be taken (eg, iterative saturation mutagenesis, random mutagenesis, or site-directed mutagenesis). In addition, insights can be gained from the various studies reported in literature about amino acid residues near or in the substrate binding site in order to change substrate specificity, [33][34][35][36] about residues located near the T1 copper for improved redox potential, 36,37 about residues located in the secondary coordination sphere of the T1 copper that may play a role in substrate binding and redox potential, 33,38,39 about residues located near the TNC for a greater understanding of the reduction of dioxygen to water, 40 about the role of the C-terminal tail in solvent access to the TNC, 41 as well as about the role of surface-located residues that either contribute towards improved stability or result in improved expression and secretion in a recombinant host. 41,42 The mutations at equivalent standard positions (Table S4)…”