Search

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)

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.

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.

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.