Polyethyleneimine as a Promoter Layer for the Immobilization of Cellobiose Dehydrogenase from <i>Myriococcum thermophilum</i> on Graphite Electrodes

Cellobiose dehydrogenase (CDH) is a promising enzyme for the construction of biofuel cell anodes and biosensors capable of oxidizing aldoses as cellobiose as well as lactose and glucose and with the ability to connect to an electrode through a direct electron transfer mechanism. In the present study, we point out the beneficial effect of a premodification of spectrographic graphite electrodes with the polycation polyethyleneimine (PEI) prior to adsorption of CDH from <i>Myriococcum thermophilum</i> (<i>Mt</i>CDH). The application of PEI shifts the pH optimum of the response of the <i>Mt</i>CDH modified electrode from pH 5.5 to 8. The catalytic currents to lactose were increased up to 140 times, and the <i>K</i>_M^app values were increased up to 9 times. The previously investigated, beneficial effect of divalent cations on the activity of CDH was also present for graphite/PEI/<i>Mt</i>CDH electrodes but was less pronounced. Polarization curves revealed a second unexpected catalytic wave for graphite/PEI/<i>Mt</i>CDH electrodes especially pronounced at pH 8. Square wave voltammetric studies revealed the presence of an unknown redox functionality present at 192 mV vs Ag|AgCl (0.1 M KCl) at pH 8, probably originating from an oxidized adenosine derivative. Adenosine is a structural part of the flavin adenine dinucleotide (FAD) cofactor of the dehydrogenase domain of CDH. It is suggested that for some enzyme molecules FAD leaks out from the active site, adsorbs onto graphite, and is oxidized on the electrode surface into a product able to mediate the electron transfer between CDH and the electrode. PEI is suggested and discussed to act in several manners by (a) increasing the surface loading of the enzyme, (b) possibly increasing the electron transfer rate between CDH and the electrode, and (c) facilitating the creation or immobilization of redox active adenosine derivatives able to additionally mediate the electron transfer between CDH and the electrode

Direct Electron Transfer from the FAD Cofactor of Cellobiose Dehydrogenase to Electrodes

Cellobiose dehydrogenase (CDH) is employed in the construction of biosensors and biofuel cells. The flavin adenine dinucleotide (FAD) containing, catalytic dehydrogenase domain (DH) of the enzyme oxidizes carbohydrates, while the cytochrome <i>b</i> containing domain (CYT) acts as an electron mediator and shuttles the electrons to the electrode. Here we demonstrate for the first time in an unequivocal manner direct electron transfer (DET) between the FAD and electrodes by showing clear nonturnover voltammetric waves in the absence and turnover waves in the presence of substrate by using cyclic voltammetry and square wave voltammetry. Results were obtained by entrapping CDH under a dialysis membrane on alkanethiol-modified, polycrystalline gold electrodes. DET from the FAD cofactor occurs at potentials 130 mV more negative than those previously reported and established DET from the electron-mediating CYT domain. However, direct electrochemistry was only observed for two types of basidiomycete class I CDHs from Trametes villosa and Phanerochaete sordida at pH values below 5 and not for ascomycete class II CDHs investigated under the same experimental conditions. The present findings are of high interest for the development of biosensors and biofuel cells featuring a lower substrate oxidation potential, which decreases the occurrence of interfering reactions and increases the cell voltage in biofuel cells. Furthermore, these findings may also be transferrable to structurally related enzymes such as glucose oxidase and glucose dehydrogenase

Catalytically Active Silica Nanoparticle-Based Supramolecular Architectures of Two Proteins – Cellobiose Dehydrogenase and Cytochrome <i>c</i> on Electrodes

Artificial nanobiomolecular architectures that follow natural examples in protein assembly become more and more important from basic and applied points of view. Our study describes the investigation on cellobiose dehydrogenase (CDH), cytochrome <i>c</i> (cyt <i>c</i>), and silica nanoparticles (SiNP's) for the construction of fully catalytically active supramolecular architectures on electrodes. We report on intraprotein, interprotein, and direct electron-transfer reaction cascades of cellobiose dehydrogenase and cytochrome <i>c</i> immobilized in multiple supramolecular layers. Carboxy-modified SiNP's are used to provide an artificial matrix, which enables protein arrangement in an electroactive form. Direct and interprotein electron transfer has been established for a two-protein system with CDH and cyt <i>c</i> in a layered architecture for the first time. We also highlight that the glycosylation of CDH and the silica nanoparticle size play key roles in the mode of operation in such a complex system. The response of the specific substrate, here lactose, can be tuned by the number of immobilized nanobiomolecular layers

A symmetric supercapacitor/biofuel cell hybrid device based on enzyme-modified nanoporous gold: an autonomous pulse generator

The integration of supercapacitors with enzymatic biofuel cells (BFCs) can be used to prepare hybrid devices in order to harvest significantly higher power output. In this study, a supercapacitor/biofuel cell hybrid device was prepared by the immobilisation of redox enzymes with electrodeposited poly(3,4-ethylenedioxythiophene) (PEDOT) and the redox polymer [Os(2,2′-bipyridine)2(polyvinylimidazole)10Cl]+/2+(Os(bpy)2PVI) on dealloyed nanoporous gold. The thickness of the deposition layer can be easily controlled by tuning the deposition conditions. Once charged by the internal BFC, the device can be discharged as a supercapacitor at a current density of 2 mA cm−2 providing a maximum power density of 608.8 μW cm−2, an increase of a factor of 468 when compared to the power output from the BFC itself. The hybrid device exhibited good operational stability for 50 charge/discharge cycles and ca. 7 h at a discharge current density of 0.2 mA cm−2. The device could be used as a pulse generator, mimicking a cardiac pacemaker delivering pulses of 10 μA for 0.5 ms at a frequency of 0.2 Hz

Direct Electrochemistry of Phanerochaete chrysosporium Cellobiose Dehydrogenase Covalently Attached onto Gold Nanoparticle Modified Solid Gold Electrodes

Achieving efficient electrochemical communication between redox enzymes and various electrode materials is one of the main challenges in bioelectrochemistry and is of great importance for developing electronic applications. Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome composed of a catalytic FAD containing dehydrogenase domain (DH_CDH), a heme <i>b</i> containing cytochrome domain (CYT_CDH), and a flexible linker region connecting the two domains. Efficient direct electron transfer (DET) of CDH from the basidiomycete Phanerochaete chrysosporium (<i>Pc</i>CDH) covalently attached to mixed self-assembled monolayer (SAM) modified gold nanoparticle (AuNP) electrode is presented. The thiols used were as follows: 4-aminothiophenol (4-ATP), 4-mercaptobenzoic acid (4-MBA), 4-mercaptophenol (4-MP), 11-mercapto-1-undecanamine (MUNH₂), 11-mercapto-1-undecanoic acid (MUCOOH), and 11-mercapto-1-undecanol (MUOH). A covalent linkage between <i>Pc</i>CDH and 4-ATP or MUNH₂ in the mixed SAMs was formed using glutaraldehyde as cross-linker. The covalent immobilization and the surface coverage of <i>Pc</i>CDH were confirmed with surface plasmon resonance (SPR). To improve current density, AuNPs were cast on the top of polycrystalline gold electrodes. For all the immobilized <i>Pc</i>CDH modified AuNPs electrodes, cyclic voltammetry exhibited clear electrochemical responses of the CYT_CDH with fast electron transfer (ET) rates in the absence of substrate (lactose), and the formal potential was evaluated to be +162 mV vs NHE at pH 4.50. The standard ET rate constant (<i>k</i>_s) was estimated for the first time for CDH and was found to be 52.1, 59.8, 112, and 154 s^–1 for 4-ATP/4-MBA, 4-ATP/4-MP, MUNH₂/MUCOOH, and MUNH₂/MUOH modified electrodes, respectively. At all the mixed SAM modified AuNP electrodes, <i>Pc</i>CDH showed DET only via the CYT_CDH. No DET communication between the DH_CDH domain and the electrode was found. The current density for lactose oxidation was remarkably increased by introduction of the AuNPs. The 4-ATP/4-MBA modified AuNPs exhibited a current density up to 30 μA cm^–2, which is ∼70 times higher than that obtained for a 4-ATP/4-MBA modified polycrystalline gold electrode. The results provide insight into fundamental electrochemical properties of CDH covalently immobilized on gold electrodes and promote further applications of CDHs for biosensors, biofuel cells, and bioelectrocatalysis

Electron-Transfer Studies with a New Flavin Adenine Dinucleotide Dependent Glucose Dehydrogenase and Osmium Polymers of Different Redox Potentials

A new extracellular flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase from Glomerella cingulata (<i>Gc</i>GDH) was electrochemically studied as a recognition element in glucose biosensors. The redox enzyme was recombinantly produced in Pichia pastoris and homogeneously purified, and its glucose-oxidizing properties on spectrographic graphite electrodes were investigated. Six different Os polymers, the redox potentials of which ranged in a broad potential window between +15 and +489 mV versus the normal hydrogen electrode (NHE), were used to immobilize and “wire” <i>Gc</i>GDH to the spectrographic graphite electrode’s surface. The <i>Gc</i>GDH/Os polymer modified electrodes were evaluated by chronoamperometry using flow injection analysis. The current response was investigated using a stepwisely increased applied potential. It was observed that the ratio of <i>Gc</i>GDH/Os polymer and the overall loading of the enzyme electrode significantly affect the performance of the enzyme electrode for glucose oxidation. The best-suited Os polymer [Os(4,4′-dimethyl-2,2′-bipyridine)₂(PVI)Cl]⁺ had a potential of +309 mV versus NHE, and the optimum <i>Gc</i>GDH/Os polymer ratio was 1:2 yielding a maximum current density of 493 μA·cm^–2 at a 30 mM glucose concentration

Effect of Deglycosylation of Cellobiose Dehydrogenases on the Enhancement of Direct Electron Transfer with Electrodes

Cellobiose dehydrogenase (CDH) is a monomeric extracellular flavocytochrome composed of a catalytic dehydrogenase domain (DH_CDH) containing flavin adenine dinucleotide (FAD), a cytochrome domain (CYT_CDH) containing heme <i>b</i>, and a linker region connecting the two domains. In this work, the effect of deglycosylation on the electrochemical properties of CDH from Phanerochaete chrysosporium (<i>Pc</i>CDH) and Ceriporiopsis subvermispora (<i>Cs</i>CDH) is presented. All the glycosylated and deglycosylated enzymes show direct electron transfer (DET) between the CYT_CDH and the electrode. Graphite electrodes modified with deglycosylated <i>Pc</i>CDH (d<i>Pc</i>CDH) and <i>Cs</i>CDH (d<i>Cs</i>CDH) have a 40–65% higher <i>I</i>_max value in the presence of substrate than electrodes modified with their glycosylated counterparts. <i>Cs</i>CDH trapped under a permselective membrane showed similar changes on gold electrodes protected by a thiol-based self-assembled monolayer (SAM), in contrast to <i>Pc</i>CDH for which deglycosylation did not exhibit any different electrocatalytical response on SAM-modified gold electrodes. Glycosylated <i>Pc</i>CDH was found to have a 30% bigger hydrodynamic radius than d<i>Pc</i>CDH using dynamic light scattering. The basic bioelectrochemistry as well as the bioelectrocatalytic properties are presented

MOESM2 of A fast and sensitive activity assay for lytic polysaccharide monooxygenase

Additional file 2. Spectra of the oxidation of sinapic acid, gallic acid, and pyrocatechol by NcLPMO9C

Multipoint Precision Binding of Substrate Protects Lytic Polysaccharide Monooxygenases from Self-Destructive Off-Pathway Processes

Lytic polysaccharide monooxygenases (LPMOs) play a crucial role in the degradation of polysaccharides in biomass by catalyzing powerful oxidative chemistry using only a single copper ion as a cofactor. Despite the natural abundance and importance of these powerful monocopper enzymes, the structural determinants of their functionality have remained largely unknown. We have used site-directed mutagenesis to probe the roles of 13 conserved amino acids located on the flat substrate-binding surface of CBP21, a chitin-active family AA10 LPMO from <i>Serratia marcescens</i>, also known as <i>Sm</i>LPMO10A. Single mutations of residues that do not interact with the catalytic copper site, but rather are involved in substrate binding had remarkably strong effects on overall enzyme performance. Analysis of product formation over time showed that these mutations primarily affected enzyme stability. Investigation of protein integrity using proteomics technologies showed that loss of activity was caused by oxidation of essential residues in the enzyme active site. For most enzyme variants, reduced enzyme stability correlated with a reduced level of binding to chitin, suggesting that adhesion to the substrate prevents oxidative off-pathway processes that lead to enzyme inactivation. Thus, the extended and highly evolvable surfaces of LPMOs are tailored for precise multipoint substrate binding, which provides the confinement that is needed to harness and control the remarkable oxidative power of these enzymes. These findings are important for the optimized industrial use of LPMOs as well as the design of LPMO-inspired catalysts

MOESM4 of A fast and sensitive activity assay for lytic polysaccharide monooxygenase

Additional file 4. Increase of the 2,6-DMP absorbance at 469 nm in control experiments