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We present a transparent and flexible self-charging biosupercapacitor based on an optimised mediator- and membrane-free enzymatic glucose/oxygen biofuel cell. Indium tin oxide (ITO) nanoparticles were spray-coated on transparent conducting ITO supports resulting in a flocculent, porous and nanostructured electrode surface. By this, high capacitive currents caused by an increased electrochemical double layer as well as enhanced catalytic currents due to a higher number of immobilised enzyme molecules were obtained. After a chemical pre-treatment with a silane derivative, bilirubin oxidase from Myrothecium verrucaria was immobilized onto the ITO nanostructured electrode surface under formation of a biocathode, while bioanodes were obtained by either immobilisation of cellobiose dehydrogenase from Corynascus thermophilus or soluble PQQ-dependent glucose dehydrogenase from Acinetobacter calcoaceticus. The latter showed a lower apparent KM value for glucose conversion and higher catalytic currents at µM glucose concentrations. Applying the optimised device as a biosupercapacitor in a discontinuous charge/discharge mode led to a generated power output of 0.030mW/cm2 at 50µM glucose, simulating the glucose concentration in human tears. This represents an enhancement by a factor of 350 compared to the power density obtained from the continuously operating biofuel cell with a maximum power output of 0.086µW/cm2 under the same conditions. After 17h of charging/discharging cycles a remarkable current enhancement was still measured. The entire device was transferred to flexible materials and applied for powering a flexible display showing its potential applicability as an intermittent power source in smart contact lenses.

The interface between protein and material surface is of great research interest in applications varying from implants, tissue engineering to bioelectronics. Maintaining functionality of bioelements depends greatly on the immobilization process. In the present study direct electron transfer of cellobiose dehydrogenase from Humicola insolens ( Hi CDH), adsorbed on four different self-assembled monolayers (SAMs) formed by 5–6 chain length carbon thiols varying in terminal group structure was investigated. By using a combination of quartz crystal microbalance with dissipation, ellipsometry and electrochemistry the formation and function of the Hi CDH film was studied. It was found that the presence of charged pyridinium groups was needed to successfully establish direct electron contact between the enzyme and electrode. SAMs formed from hydrophilic charged thiols achieved nearly two times higher current densities compared to hydrophobic charged thiols. Additionally, the results also indicated proportionality between Hi CDH catalytic constant and water content of the enzyme film. Enzyme films on charged pyridine thiols had smaller variations in water content and viscoelastic properties than films adsorbed on the more hydrophobic thiols. This work highlights several perspectives on the underlying factors affecting performance of immobilized Hi CDH.

Cellobiose dehydrogenase (CDH) is a fungal extracellular flavocytochrome capable of direct electron transfer (DET). Unlike other CDHs, the pH optimum for CDHs from Corynascus thermophilus (CtCDH) and Humicola insolens (HiCDH) is close to the human physiological pH in blood (7.4). These are, therefore, interesting candidates for glucose measurements in human blood and the application in enzymatic fuel cells is, however, limited by their relatively low activity with this substrate. In this work, the substrate specificities of CtCDH and HiCDH have been altered by a single cysteine to tyrosine substitution in the active sites of CtCDH (position 291) and HiCDH (position 285), which resulted in improved kinetic constants with glucose while decreasing the activity with several disaccharides, including maltose. The DET properties of the generated CDH variants were tested in the absence and in the presence of substrates, on graphite electrodes and thiolic self-assembled monolayer (SAM)-modified Au electrodes. Seven different thiols with different spacer lengths were used, containing -COOH, -OH, and -NH2 end groups. The length and head functionality of the thiol govern the efficiency of the DET reaction and indicate different DET properties of CtCDH and HiCDH (Less)

A combination of thylakoid membranes (TMs) as photobiocatalysts with high-surface-area electroactive materials could hold great potential for sustainable “green” solar energy conversion. We have studied the orientated immobilization of TMs on high-surface-area graphene electrodes, which were fabricated by electroreduction of graphene oxide and simultaneous electrodeposition with further aminoaryl functionalization. We have achieved the highest performance to date under direct electron transfer conditions through a biocompatible “wiring” of TMs to graphene sheets. The photobiocurrent density generated by the optimized mediator-free TM-based bioanodes yielded up to 5.24 ± 0.50 μA cm–2. The photobioelectrochemical cell integrating the photobioanode in combination with an oxygen reducing enzymatic biocathode delivered a maximum power output of 1.79 ± 0.19 μW cm–2. Our approach ensures a simplified cell design, a greater load of photosynthetic units, a minimized overpotential loss, and an enhanced overall performance., The authors thank the following agencies for financial support: the European Commission (”Bioenergy” FP7-PEOPLE-2013-ITN-607793), the Ørsted-COFUND Postdoc fellowship at DTU (Agreement No. 2014-5908), and the Swedish Research Council (project 2014-5908), and Independent Research Fund Denmark-Nature Sciences (DFF-FNU, Project No. DFF-7014-00302).

Direct electron transfer (DET) between cellobiose dehydrogenase from Humicola insolens ascomycete (HiCDH) and gold nanoparticles (AuNPs) was achieved by modifying AuNPs with a novel, positively charged thiol N-(6-mercapto)hexylpyridinium (MHP). The DET enabled the use of the HiCDH enzyme as an anodic biocatalyst in the design of a mediatorless carbohydrate/oxygen enzymatic fuel cell (EFC). A biocathode of the EFC was based on bilirubin oxidase from Myrothecium verrucaria (MvBOx) directly immobilised on the surface of AuNPs. The following parameters of the EFC based on Au/AuNP/MHP/HiCDH bioanode and Au/AuNP/MvBOx biocathode were obtained in quiescent air saturated PBS, pH 7.4, containing: (i) 5 mM glucose-open-circuit voltage (OCV) of 0.65 ± 0.011 V and the maximal power density of 4.77 ± 1.34 μW cm−2 at operating voltage of 0.50 V; or (ii) 10 mM lactose-OCV of 0.67 ± 0.006 V and the maximal power density of 8.64 ± 1.91 μW cm−2 at operating voltage of 0.50 V. The half-life operation times of the EFC were estimated to be at least 13 and 44 h in air saturated PBS containing 5 mM glucose and 10 mM lactose, respectively. Among advantages of HiCDH/MvBOx FCs are (i) simplified construction, (ii) relatively high power output with glucose as biofuel, and (iii) the absence of the inhibition of the HiCDH based bioanode by lactose, when compared with the best previously reported CDH based bioanode.

A novel electrochemical enzyme biosensor was developed for real-time detection of cellulase activity when acting on their natural insoluble substrate, cellulose. The enzyme biosensor was constructed with pyranose dehydrongease (PDH) from Agaricus meleagris that was immobilized on the surface of a carbon paste electrode, which contained the mediator 2,6-dichlorophenolindophenol (DCIP). An oxidation current of the reduced form of DCIP, DCIPH2, produced by the PDH-catalyzed reaction with either glucose or cellobiose, was recorded under constant-potential amperometry at +0.25 V (vs. Ag/AgCl). The PDH-biosensor was shown to be anomer unspecific and it can therefore be used in kinetic studies over broad time-scales of both retaining- and inverting cellulases (in addition to enzyme cocktails). The biosensor was used for real-time measurements of the activity of the inverting cellobiohydrolase Cel6A from Hypocrea jecorina (HjCel6A) on cellulosic substrates with different morphology (bacterial microcrystalline cellulose (BMCC) and Avicel). The steady-state rate of hydrolysis increased towards a saturation plateau with increasing loads of substrate. The experimental results were rationalized using a steady-state rate equation for processive cellulases, and it was found that the turnover for HjCel6A at saturating substrate concentration (i.e. maximal apparent specific activity) was similar (0.39–0.40 s−1) for the two substrates. Conversely, the substrate load at half-saturation was much lower for BMCC compared to Avicel. Biosensors covered with a polycarbonate membrane showed high operational stability of several weeks with daily use.

Gold disk electrodes modified with gold nanoparticles have been used as a scaffold for the covalent immobilization of bilirubin oxidase. The nanostructured bioelectrodes were tested as mediator-less biosensors for oxygen in a buffer that mimics the content and the composition of human physiological fluids. Chronoamperometry measurements showed a detection limit towards oxygen of 6 ± 1 μM with a linear range of 6-300 μM, i.e. exceeding usual physiological ranges of oxygen in human tissues and fluids. The biosensor presented is the first ever-reported oxygen amperometric biosensor based on direct electron transfer of bilirubin oxidase., This work was funded by the FP7 project “3D-Nanobiodevice” (NMP4-SL-2009-229255). We thank Novozymes A/S for the preparation of Myrothecium verrucaria BOx. M.P. acknowledges the 2009 Ramon y Cajal program from the Spanish MINECO.

A microscale membrane-less biofuel cell, capable of generating electrical energy from human lachrymal liquid, was developed by utilizing the ascorbate and oxygen naturally present in tears as fuel and oxidant. The biodevice is based on three-dimensional nanostructured gold electrodes covered with abiotic (conductive organic complex) and biological (redox enzyme) materials functioning as efficient anodic and cathodic catalysts, respectively. Three-dimensional nanostructured electrodes were fabricated by modifying 100 μm gold wires with 17 nm gold nanoparticles, which were further modified with tetrathiafulvalene-tetracyanoquinodimethane conducting complex to create the anode and with Myrothecium verrucaria bilirubin oxidase to create the biocathode. When operated in human tears, the biodevice exhibited the following characteristics: an open circuit voltage of 0.54 V, a maximal power density of 3.1 μW cm(-2) at 0.25 V and 0.72 μW cm(-2) at 0.4 V, with a stable current density output of over 0.55 μA cm(-2) at 0.4 V for 6 h of continuous operation. These findings support our proposition that an ascorbate/oxygen biofuel cell could be a suitable power source for glucose-sensing contact lenses to be used for continuous health monitoring by diabetes patients.

After initial testing and optimization of anode biocatalysts, a membraneless glucose/oxygen enzymatic biofuel cell possessing high coulombic efficiency and power output was fabricated and characterized. Two sugar oxidizing enzymes, namely, pyranose dehydrogenase from Agaricus meleagris (AmPDH) and flavodehydrogenase domains of various cellobiose dehydrogenases (DH(CDH)) were tested during the pre-screening. The enzymes were mixed, "wired" and entrapped in a low-potential Os-complex-modified redox-polymer hydrogel immobilized on graphite. This anode was used in combination with a cathode based on bilirubin oxidase from Myrothecium verrucaria adsorbed on graphite. Optimization showed that the current density for the mixed enzyme electrode could be further improved by using a genetically engineered variant of the non-glycosylated flavodehydrogenase domain of cellobiose dehydrogenase from Corynascus thermophilus expressed in E. coli (ngDH(CtCDHC310Y)) with a high glucose-turnover rate in combination with an Os-complex-modified redox polymer with a high concentration of Os complexes as well as a low-density graphite electrode. The optimized biofuel cell with the AmPDH/ngDH(CtCDHC310Y) anode showed not only a similar maximum voltage as with the biofuel cell based only on the ngDH(CtCDHC310Y) anode (0.55 V) but also a substantially improved maximum power output (20 μW cm(-2)) at 300 mV cell voltage in air-saturated physiological buffer. Most importantly, the estimated half-life of the mixed biofuel cell can reach up to 12 h, which is apparently longer than that of a biofuel cell in which the bioanode is based on only one single enzyme.

We detail a mediator- and membrane-free enzymatic glucose/oxygen biofuel cell based on transparent and nanostructured conducting supports. Chemically modified indium tin oxide nanoparticle modified electrodes were used to substantially increase the active surface area without significantly compromising transparency. Two different procedures for surface nanostructuring were employed, viz. spray-coating and drop-coating. The spray-coated biodevice showed superior characteristics as compared to the drop-coated enzymatic fuel cell, as a result of the higher nanostructured surface area as confirmed by electrochemical characterisation, as well as scanning electron and atomic force microscopy. Subsequent chemical modification with silanes, followed by the immobilisation of either cellobiose dehydrogenase from Corynascus thermophiles or bilirubin oxidase from Myrothecium verrucaria, were performed to obtain the bioanodes and biocathodes, respectively. The optimised biodevice exhibited an OCV of 0.67 V and power output of up to 1.4 µW/cm2 at an operating voltage of 0.35 V. This is considered a significant step forward in the field of glucose/oxygen membrane- and mediator-free, transparent enzymatic fuel cells., This work was supported in part by the European Commission through the Marie-Curie Project “Bioenergy” (PITN-GA-2013-607793), by the Swedish Research Council (621-2013-6006), and by the Cluster of Excellence RESOLV (EXC 1069) funded by the Deutsche Forschungsgemeinschaft (DFG).

Threonine aldolases are versatile pyridoxal 5' phosphate (PLP) dependent enzymes key to glycine serine and threonine metabolism. Because they catalyze the reversible addition of glycine to an aldehyde to give ß hydroxy a amino acids they are also attractive as biotechnological catalysts for the diastereoselective synthesis of many pharmaceutically useful compounds. To study and evolve such enzymes we have developed a simple selection system based on the simultaneous inactivation of four genes involved in glycine biosynthesis in Escherichia coli. Glycine prototrophy in the deletion strain is restored by expression of a gene encoding an aldolase that converts ß hydroxy a amino acids provided in the medium to glycine and the corresponding aldehyde. Combinatorial mutagenesis and selection experiments with a previously uncharacterized l threonine aldolase from Caulobacter crescentus CB15 (Cc LTA) illustrate the power of this system. The codons for four active site residues His91 Asp95 Glu96 and Asp176 were simultaneously randomized and active variants selected. The results show that only His91 which p stacks against the PLP cofactor and probably serves as the catalytic base in the carbon carbon bond cleavage step is absolutely required for aldolase activity. In contrast Asp176 one of the most conserved residues in this enzyme superfamily can be replaced conservatively by glutamate albeit with a >5000 fold decrease in efficiency. Though neither Asp95 nor Glu96 is catalytically essential they appear to modulate substrate binding and His91 activity respectively. The broad dynamic range of this novel selection system should make it useful for mechanistic investigations and directed evolution of many natural and artificial aldolases. © 2012 Elsevier Ltd. All rights reserved.

Many enzymes catalyze reactions with multiple chemical steps, requiring the stabilization of multiple transition states during catalysis. Such enzymes must strike a balance between the conformational reorganization required to stabilize multiple transition states of a reaction and the confines of a preorganized active site in the polypeptide tertiary structure. Here we investigate the compromise between structural reorganization during the catalytic process and preorganization of the active site for a multistep enzyme-catalyzed reaction, the hydrolysis of esters by the Ser-His-Asp/Glu catalytic triad. Quantum mechanical transition states were used to generate ensembles of geometries that can catalyze each individual step in the mechanism. These geometries are compared to each other by superpositions of catalytic atoms to find "consensus" geometries that can catalyze all steps with minimal rearrangement. These consensus geometries are found to be excellent matches for the natural active site. Preorganization is therefore found to be the major defining characteristic of the active site, and reorganizational motions often proposed to promote catalysis have been minimized. The variability of enzyme active sites observed by X-ray crystallography was also investigated empirically. A catalog of geometrical parameters relating active site residues to each other and to bound inhibitors was collected from a set of crystal structures. The crystal-structure-derived values were then compared to the ranges found in quantum mechanically optimized structures along the entire reaction coordinate. The empirical ranges are found to encompass the theoretical ranges when thermal fluctuations are taken into account. Therefore, the active sites are preorganized to a geometry that can be objectively and quantitatively defined as minimizing conformational reorganization while maintaining optimal transition state stabilization for every step during catalysis. The results provide a useful guiding principle for de novo design of enzymes with multistep mechanisms.

Wahrend die Natur ihre Katalysatoren uber Millionen von Jahren entwickelt hat, versuchen Enzymingenieure, dies ein wenig schneller zu bewerkstelligen. Die aktive Tasche eines Enzyms bildet eine stark optimierte Mikroumgebung fur die Katalyse chemischer Transformationen in biologischen Systemen – folglich konnen Anderungen in diesen Zentren die Enzymaktivitat stark beeinflussen. Daher bietet die Voraussage und Kontrolle solcher Effekte einen vielversprechenden Weg, um zu neuen Funktionen zu gelangen. Der Ansatz, bei dem minimale Modifikationen in den aktiven Taschen von Enzymen (z. B. durch gezielte Mutagenese und Hinzufugen neuer reaktiver Funktionalitaten) deren katalytisches Repertoire erweitern sollen, ist der Schwerpunkt dieses Aufsatzes. Dabei kann eine neue Aktivitat oft schon durch eine einzige Mutation erreicht werden. Die vielen erfolgreichen Beispiele fur die Umgestaltung von aktiven Taschen durch minimale Mutationen geben wertvolle Einblicke in die Evolution von Enzymen und bahnen neue Wege in der Erforschung der Biokatalysatoren.

Although nature evolves its catalysts over millions of years, enzyme engineers try to do it a bit faster. Enzyme active sites provide highly optimized microenvironments for the catalysis of biologically useful chemical transformations. Consequently, changes at these centers can have large effects on enzyme activity. The prediction and control of these effects provides a promising way to access new functions. The development of methods and strategies to explore the untapped catalytic potential of natural enzyme scaffolds has been pushed by the increasing demand for industrial biocatalysts. This Review describes the use of minimal modifications at enzyme active sites to expand their catalytic repertoires, including targeted mutagenesis and the addition of new reactive functionalities. Often, a novel activity can be obtained with only a single point mutation. The many successful examples of active-site engineering through minimal mutations give useful insights into enzyme evolution and open new avenues in biocatalyst research.

Small but faster: A small laccase from Streptomyces coelicolor (SLAC) has been engineered by structure-based design and site-directed mutagenesis to improve the activity on commercially relevant substrates. The variants generated showed up to 40-fold increased efficiency on 2,6-dimethoxyphenol and the ability to use mediators with considerably higher redox potentials (methylsyringate and TEMPO).

We describe the rational design of a novel, highly potent inhibitor of type II dehydroquinase, the dicarboxylate 6. The incorporation of a carboxylate at the 3-position mimics the putative enolate intermediate in the reaction mechanism, and allows a potential electrostatic binding interaction with the arginine on the active site flap. This results in a 1000-fold increase in potency, making the dicarboxylate 6 the most potent inhibitor of type II dehydroquinase reported to date, with a high ligand efficiency of -0.68 kcal mol(-1) per nonhydrogen atom. The systematic dissection of 6 in compounds 7-12, all of which show a drop in potency, confirm the synergistic importance of the two carboxylates, the C3 and C4 hydroxyl groups, and the anhydroquinate ring structure for the potency of 6.

Selective inhibitors of type II dehydroquinase were rationally designed to explore a second binding-pocket in the active-site. The molecular modelling, synthesis, inhibition studies and crystal structure determination are described.

A series of 1-substituted and 4-substituted benzyl analogues of the known inhibitor (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid has been synthesized and tested as inhibitors of Streptomyces coelicolor type II dehydroquinase. The solid-phase syntheses of 18 new analogues are reported. The most potent inhibitor, 2-nitrobenzyloxy analogue 5i, has K(i) of 8 microM, more than 30 times lower than the K(M) of the substrate and approximately 4 times more potent than the original inhibitor. The binding modes of the synthesized analogues in the active site were studied by molecular docking with GOLD 2.0.

Inhibitors of type II dehydroquinase were designed to straddle the two distinct binding sites identified for the inhibitor (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid and a glycerol molecule in a crystallographic study of the Streptomyces coelicolor enzyme. A number of compounds were designed to incorporate characteristics of both ligands. These analogues were synthesized from quinic acid, and were assayed against type I (Salmonella typhi) and type II (S. coelicolor) dehydroquinases. None of the analogues showed inhibition for type I dehydroquinase. Six of the analogues were shown to have inhibition constants in the micromolar to low millimolar range against the S. coelicolor type II dehydroquinase, while two showed no inhibition. The binding modes of the analogues in the active site of the S. coelicolor enzyme were studied by molecular docking with GOLD1.2. These studies suggest a binding mode where the ring is in a similar position to (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid in the crystal structure and the side-chain occupies part of the glycerol binding-pocket.

Aromatic analogues of chorismate were synthesised as potential inhibitors of anthranilate synthase. Molecular modelling using GOLD2.1 showed that these analogues docked into the active site of Serratia marcescens anthranilate synthase in the same conformation as chorismate. Most compounds were found to be micromolar inhibitors of S. marcescens anthranilate synthase. The most potent analogue, 3-(1-carboxy-ethoxy)-4-hydroxybenzoate (K(I) 3 microM), included a lactyl ether side chain. This appears to be a good replacement for the enol-pyruvyl side chain of chorismate.