The accurate determination of analyte concentrations with selective, fast, and robust methods is the key for process control, product analysis, environmental compliance, and medical applications. ...Enzyme-based biosensors meet these requirements to a high degree and can be operated with simple, cost efficient, and easy to use devices. This review focuses on enzymes capable of direct electron transfer (DET) to electrodes and also the electrode materials which can enable or enhance the DET type bioelectrocatalysis. It presents amperometric biosensors for the quantification of important medical, technical, and environmental analytes and it carves out the requirements for enzymes and electrode materials in DET-based third generation biosensors. This review critically surveys enzymes and biosensors for which DET has been reported. Single- or multi-cofactor enzymes featuring copper centers, hemes, FAD, FMN, or PQQ as prosthetic groups as well as fusion enzymes are presented. Nanomaterials, nanostructured electrodes, chemical surface modifications, and protein immobilization strategies are reviewed for their ability to support direct electrochemistry of enzymes. The combination of both biosensor elements—enzymes and electrodes—is evaluated by comparison of substrate specificity, current density, sensitivity, and the range of detection.
Efficient direct electron transfer (DET) between a cellobiose dehydrogenase mutant from Corynascus thermophilus (CtCDH C291Y) and a novel glassy carbon (GC)-modified electrode, obtained by direct ...electrodeposition of gold nanoparticles (AuNPs) was realized. The electrode was further modified with a mixed self-assembled monolayer of 4-aminothiophenol (4-APh) and 4-mercaptobenzoic acid (4-MBA), by using glutaraldehyde (GA) as cross-linking agent. The CtCDH C291Y/GA/4-APh,4-MBA/AuNPs/GC platform showed an apparent heterogeneous electron transfer rate constant (ks) of 19.4 ± 0.6 s-1, with an enhanced theoretical and real enzyme surface coverage (Γtheor and Γreal) of 5287 ± 152 pmol cm-2 and 27 ± 2 pmol cm-2, respectively. The modified electrode was successively used as glucose biosensor exhibiting a detection limit of 6.2 μM, an extended linear range from 0.02 to 30 mM, a sensitivity of 3.1 ± 0.1 μA mM-1 cm-2 (R2 = 0.995), excellent stability and good selectivity. These performances compared favourably with other glucose biosensors reported in the literature. Finally, the biosensor was tested to quantify the glucose content in human saliva samples with successful results in terms of both recovery and correlation with glucose blood levels, allowing further considerations on the development of non-invasive glucose monitoring devices.
Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears ...to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization.
Lytic polysaccharide monooxygenase (LPMO) has recently been discovered to depolymerize cellulose.
Dynamic imaging was applied to reveal the effects of LPMO and cellulase activity on solid cellulose surface.
Critical features of surface morphology for LPMO synergy with cellulases are recognized.
Direct insights into cellulose deconstruction by LPMO alone and in synergy with cellulases are obtained.
This review aims to present current knowledge of the fungi involved in lignocellulose degradation with an overview of the various classes of lignocellulose‐acting enzymes engaged in the pretreatment ...and saccharification step. Fungi have numerous applications and biotechnological potential for various industries including chemicals, fuel, pulp, and paper. The capability of fungi to degrade lignocellulose containing raw materials is due to their highly effective enzymatic system. Along with the hydrolytic enzymes consisting of cellulases and hemicellulases, responsible for polysaccharide degradation, they have a unique nonenzymatic oxidative system which together with ligninolytic enzymes is responsible for lignin modification and degradation. An overview of the enzymes classification is given by the Carbohydrate‐Active enZymes (CAZy) database as the major database for the identification of the lignocellulolytic enzymes by their amino acid sequence similarity. Finally, the recently discovered novel class of recalcitrant polysaccharide degraders‐lytic polysaccharide monooxygenases (LPMOs) are presented, because of these enzymes importance in the cellulose degradation process.
•The redox potential of fungal glucose dehydrogenase is reported for the first time.•A redox potential of −0.265 V vs SHE was determined by two independent methods.•2-Hydroxy-1,4-naphthoquinone works ...as an efficient redox mediator.•The red anionic semiquinone form of FAD was also observed.
The redox potential is important to rationally employ oxidoreductases as bioelectrocatalysts to minimize electroactive interferences and maximize current density or cell voltage. For many FAD-dependent enzymes redox potentials are published, but not for FAD-dependent glucose dehydrogenase (GDH) from the glucose-methanol-choline (GMC)-oxidoreductase family, which are widely used in glucose biosensors. GDH is contacted via redox mediators or redox polymers and features a reasonably high substrate specificity and oxygen insensitivity. Here we report the redox potential of Glomerella cingulata GDH that was determined by two methods. Spectroelectrochemically we obtained a reduction potential of −0.265 ± 0.003 V vs SHE and with the xanthine oxidase assay using Janus green B a potential of −0.267 ± 0.016 V vs SHE. The determined redox potential of GcGDH differs greatly from that of Aspergillus niger glucose oxidase (−0.080 V vs SHE) despite an almost similar protein fold as GcGDH. Taking this excitingly low redox potential of GcGDH to develop optimized redox mediators and redox polymers for the fabrication of glucose oxidizing bioanodes with a low operating potential can drastically reduce the susceptibility of glucose biosensors towards electroactive substances or increase the cell voltage in biofuel cells.
The direct electron transfer (DET) of enzymes has been utilized to develop biosensors and enzymatic biofuel cells on micro‐ and nanostructured electrodes. Whereas some enzymes exhibit direct electron ...transfer between their active‐site cofactor and an electrode, other oxidoreductases depend on acquired cytochrome domains or cytochrome subunits as built‐in redox mediators. The physiological function of these cytochromes is to transfer electrons between the active‐site cofactor and a redox partner protein. The exchange of the natural electron acceptor/donor by an electrode has been demonstrated for several cytochrome carrying oxidoreductases. These multi‐cofactor enzymes have been applied in third generation biosensors to detect glucose, lactate, and other analytes. This review investigates and classifies oxidoreductases with a cytochrome domain, enzyme complexes with a cytochrome subunit, and covers designed cytochrome fusion enzymes. The structurally and electrochemically best characterized proponents from each enzyme class carrying a cytochrome, that is, flavoenzymes, quinoenzymes, molybdenum‐cofactor enzymes, iron‐sulfur cluster enzymes, and multi‐haem enzymes, are featured, and their biochemical, kinetic, and electrochemical properties are compared. The cytochromes molecular and functional properties as well as their contribution to the interdomain electron transfer (IET, between active‐site and cytochrome) and DET (between cytochrome and electrode) with regard to the achieved current density is discussed. Protein design strategies for cytochrome‐fused enzymes are reviewed and the limiting factors as well as strategies to overcome them are outlined.
It′s classified: Multi‐cofactor enzymes with a cytochrome domain use it to transfer electrons to physiological redox partner proteins, but the cytochrome can also establish direct electron transfer (DET) to electrodes. The enzymes are classified, compared, and analyzed in terms of their redox and DET properties to obtain guidelines for selecting and designing suitable enzymes for third‐generation biosensors.
Cellobiose dehydrogenase catalyses the oxidation of aldoses—a simple reaction, a boring enzyme? No, neither for the envisaged bioelectrochemical applications nor mechanistically. The catalytic cycle ...of this flavocytochrome is complex and modulated by its flexible cytochrome domain, which acts as a built‐in redox mediator. This intramolecular electron transfer is modulated by the pH, an adaptation to the environmental conditions encountered or created by the enzyme‐producing fungi. The cytochrome domain forms the base from which electrons can jump to large terminal electron acceptors, such as redox proteins, and also enables by that path direct electron transfer from the catalytically active flavodehydrogenase domain to electrode surfaces. The application of electrochemical techniques to the elucidation of the molecular and catalytic properties of cellobiose dehydrogenase is discussed and compared to biochemical methods. The results lead to valuable insights into the function of this cellulose‐bound enzyme, but also form the basis of exciting applications in biosensors, biofuel cells and bioelectrocatalysis.
An enzyme with contacts: Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome consisting of flavodehydrogenase (DHCDH) and cytochrome (CYTCDH) domains with the ability to communicate with an electrode by direct (DET) or mediated electron transfer (MET; see picture). Bioelectrochemical techniques used to elucidate basic mechanistic and kinetic properties of CDH and pioneer potential applications in biosensors, biofuel cells and bioelectrosynthesis are presented.
Lytic polysaccharide monooxygenase (LPMO) supports biomass hydrolysis by increasing saccharification efficiency and rate. Recent studies demonstrate that H
O
rather than O
is the cosubstrate of the ...LPMO-catalyzed depolymerization of polysaccharides. Some studies have questioned the physiological relevance of the H
O
-based mechanism for plant cell wall degradation. This study reports the localized and time-resolved determination of LPMO activity on poplar wood cell walls by measuring the H
O
concentration in their vicinity with a piezo-controlled H
O
microsensor. The investigated Neurospora crassa LPMO binds to the inner cell wall layer and consumes enzymatically generated H
O
. The results point towards a high catalytic efficiency of LPMO at a low H
O
concentration that auxiliary oxidoreductases in fungal secretomes can easily generate. Measurements with a glucose microbiosensor additionally demonstrate that LPMO promotes cellobiohydrolase activity on wood cell walls and plays a synergistic role in the fungal extracellular catabolism and in industrial biomass degradation.
Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to ...fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61–3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end.
Background: Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that cleave polysaccharides.
Results: We describe a novel LPMO and use a range of analytical methods to characterize its activity.
Conclusion: Cellulose and cello-oligosaccharides are cleaved by oxidizing the sugar at the nonreducing end in the C4 position.
Significance: This study provides unequivocal evidence for C4 oxidation of the nonreducing end sugar and demonstrates a novel LPMO substrate specificity.
A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); ...however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.
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