Fungal high-redox-potential laccases (HRPLs) are multicopper oxidases with a relaxed substrate specificity that is highly dependent on their binding affinity and redox potential of the T1Cu site (E T1). In this study, we combined computational design with directed evolution to tailor an HRPL variant with increased E T1 and activity toward high-redox-potential mediators as well as enhanced stability. Laccase mutant libraries were screened in vitro using synthetic high-redox-potential mediators with different oxidation routes and chemical natures, while computer-aided evolution experiments were run in parallel to guide benchtop mutagenesis, without compromising protein stability. Through this strategy, the E T1 of the evolved HRPL increased from 740 to 790 mV, with a concomitant improvement in thermal and acidic pH stability. The kinetic constants for high-redox-potential mediators were markedly improved and were then successfully tested within laccase mediator systems (LMSs). Two hydrophobic substitutions surrounding the T1Cu site appeared to underlie these effects, and they were rationalized at the atomic level. Together, this study represents a proof-of-concept of the joint elevation of the E T1, redox mediator activity, and stability in an HRPL, making this versatile biocatalyst a promising candidate for future LMS applications and for the development of bioelectrochemical devices.

No abstract

Dedicated to the 60 th birthdayo fProfessor DoctorW olfgang Schuhmann 1Introduction Electroreduction and electrooxidation can be commonly referredt oa se lectrocatalysis.E lectroreduction processes occur in av arietyo fe lectrochemical devices,i ncluding sensors ande lectrolyzers,a sw ell as fuel cells and some supercapacitors.T hese devices are used for analytical and synthetic purposes,a sw ell as for energy conversion and storage,r espectively.Avariety of simple and more complex compounds can be useda so xidants,i ncludingh ydronium ion (H 3 O + ), organica nd inorganic peroxides (ROOR'), nitric oxide (NO), nitrite( NO 2 À ), and molecular oxygen (O 2 ).Among different reductive processes of different oxidants,r eductiono fO 2 is the key reactionn ot only in many naturala nd artificial systems,b ut also in many living organisms, i.e. aerobes. As the oxygen partial pressure in the atmospherei ncreased, aerobic organisms came to dominate the planet'ss pecies [1].T hey,i ncluding humans,u se the aerobic oxidation of different biofuels in order to extracta sm uch energy as possible.S imultaneously,p artial reduction of O 2 ,o ccurring during aerobic respiration, will generate harmful reactive oxygen species, such as hydrogenp eroxide (H 2 O 2 ), superoxide and hydroxylr adicals,w hich need to be safely disposed of by the organism. In many electrochemical devices similar problems exist, i.e. the complete direct four-electron O 2 reduction to the innocuous product H 2 Oi sn ot fully realized, and H 2 O 2 will be produced, whichs houldb ea ddi-

Here we report on an entirely new kind of bioelectronic devicea conventional biosupercapacitor, which is built from copper containing redox proteins. Prior to biodevice fabrication, detailed spectroelectrochemical studies of the protein, viz. Acidithiobacillus ferrooxidans rusticyanin, in solution and in adsorbed state, were performed, including estimation of the redox potential of the T1 site (0.62 V vs. NHE), protein midpoint potential when adsorbed on a self-assembled monolayer (0.34 V vs. NHE), as well as biocapacitance of rusticyanin modified gold electrodes (115 µF cm-2). The symmetrical biosupercapacitor based on two identical gold electrodes modified with rusticyanin is able to capacitively store electricity and deliver electric power accumulated mostly in the form of biopseudocapacitance, when charged and discharged externally. When charged during just 5 sec, the biosupercapacitor with a total capacitance of about 73 F cm-2 provided a maximum of 4 A cm-2 peak current at 0.40 V. The biodevice, which can be charged and discharged at least 50 times without a significant loss of ability to store electric energy, had a low leakage current below 50 nA cm-2 .

No abstract