Search

The function of cellobiose dehydrogenase (CDH) in biosensors, biofuel cells, and as a physiological redox partner of lytic polysaccharide monooxygenase (LPMO) is based on its role as an electron donor. Before donating electrons to LPMO or electrodes, an interdomain electron transfer from the catalytic FAD-containing dehydrogenase domain to the electron shuttling cytochrome domain of CDH is required. This study investigates the role of two crucial amino acids located at the dehydrogenase domain on domain interaction and interdomain electron transfer by structure-based engineering. The electron transfer kinetics of wild-type Myriococcum thermophilum CDH and its variants M309A, R698S, and M309A/R698S were analyzed by stopped-flow spectrophotometry and structural effects were studied by small-angle X-ray scattering. The data show that R698 is essential to pull the cytochrome domain close to the dehydrogenase domain and orient the heme propionate group towards the FAD, while M309 is an integral part of the electron transfer pathway - its mutation reducing the interdomain electron transfer 10-fold. Structural models and molecular dynamics simulations pinpoint the action of these two residues on the domain interaction and interdomain electron transfer.

The interdomain electron transfer (IET) between the catalytic flavodehydrogenase domain and the electron-transferring cytochrome domain of cellobiose dehydrogenase (CDH) plays an essential role in biocatalysis, biosensors and biofuel cells, as well as in its natural function as an auxiliary enzyme of lytic polysaccharide monooxygenase. We investigated the mobility of the cytochrome and dehydrogenase domains of CDH, which is hypothesised to limit IET in solution by small angle X-ray scattering (SAXS). CDH from Myriococcum thermophilum (syn. Crassicarpon hotsonii, syn. Thermothelomyces myriococcoides) was probed by SAXS to study the CDH mobility at different pH and in the presence of divalent cations. By comparison of the experimental SAXS data, using pair-distance distribution functions and Kratky plots, we show an increase in CDH mobility at higher pH, indicating alterations of domain mobility. To further visualise CDH movement in solution, we performed SAXS-based multistate modelling. Glycan structures present on CDH partially masked the resulting SAXS shapes, we diminished these effects by deglycosylation and studied the effect of glycoforms by modelling. The modelling shows that with increasing pH, the cytochrome domain adopts a more flexible state with significant separation from the dehydrogenase domain. On the contrary, the presence of calcium ions decreases the mobility of the cytochrome domain. Experimental SAXS data, multistate modelling and previously reported kinetic data show how pH and divalent ions impact the closed state necessary for the IET governed by the movement of the CDH cytochrome domain.

Abstract Background Cellobiose dehydrogenase (CDH) is an extracellular fungal oxidoreductase with multiple functions in plant biomass degradation. Its primary function as an auxiliary enzyme of lytic polysaccharide monooxygenase (LPMO) facilitates the efficient depolymerization of cellulose, hemicelluloses and other carbohydrate-based polymers. The synergistic action of CDH and LPMO that supports biomass-degrading hydrolases holds significant promise to harness renewable resources for the production of biofuels, chemicals, and modified materials in an environmentally sustainable manner. While previous phylogenetic analyses have identified four distinct classes of CDHs, only class I and II have been biochemically characterized so far. Results Following a comprehensive database search aimed at identifying CDH sequences belonging to the so far uncharacterized class III for subsequent expression and biochemical characterization, we have curated an extensive compilation of putative CDH amino acid sequences. A sequence similarity network analysis was used to cluster them into the four distinct CDH classes. A total of 1237 sequences encoding putative class III CDHs were extracted from the network and used for phylogenetic analyses. The obtained phylogenetic tree was used to guide the selection of 11 cdhIII genes for recombinant expression in Komagataella phaffii. A small-scale expression screening procedure identified a promising cdhIII gene originating from the plant pathogen Fusarium solani (FsCDH), which was selected for expression optimization by signal peptide shuffling and subsequent production in a 5-L bioreactor. The purified FsCDH exhibits a UV-Vis spectrum and enzymatic activity similar to other characterized CDH classes. Conclusion The successful production and functional characterization of FsCDH proved that class III CDHs are catalytical active enzymes resembling the key properties of class I and class II CDHs. A detailed biochemical characterization based on the established expression and purification strategy can provide new insights into the evolutionary process shaping CDHs and leading to their differentiation into the four distinct classes. The findings have the potential to broaden our understanding of the biocatalytic application of CDH and LPMO for the oxidative depolymerization of polysaccharides.

Background: Cellobiose dehydrogenase (CDH) is an extracellular fungal oxidoreductase with multiple functions in plant biomass degradation. Its primary function as an auxiliary enzyme of lytic polysaccharide monooxygenase (LPMO) facilitates the efficient depolymerization of cellulose, hemicelluloses and other carbohydrate-based polymers. The synergistic action of CDH and LPMO that supports biomass-degrading hydrolases holds significant promise to harness renewable resources for the production of biofuels, chemicals, and modified materials in an environmentally sustainable manner. While previous phylogenetic analyses have identified four distinct classes of CDHs, only class I and II have been biochemically characterized so far. Results: Following a comprehensive database search aimed at identifying CDH sequences belonging to the so far uncharacterized class III for subsequent expression and biochemical characterization, we have curated an extensive compilation of putative CDH amino acid sequences. A sequence similarity network analysis was used to cluster them into the four distinct CDH classes. A total of 1237 sequences encoding putative class III CDHs were extracted from the network and used for phylogenetic analyses. The obtained phylogenetic tree was used to guide the selection of 11 cdhIII genes for recombinant expression in Komagataella phaffii. A small-scale expression screening procedure identified a promising cdhIII gene originating from the plant pathogen Fusarium solani (FsCDH), which was selected for expression optimization by signal peptide shuffling and subsequent production in a 5-L bioreactor. The purified FsCDH exhibits a UV-Vis spectrum and enzymatic activity similar to other characterized CDH classes. Conclusion: The successful production and functional characterization of FsCDH proved that class III CDHs are catalytical active enzymes resembling the key properties of class I and class II CDHs. A detailed biochemical characterization based on the established expression and purification strategy can provide new insights into the evolutionary process shaping CDHs and leading to their differentiation into the four distinct classes. The findings have the potential to broaden our understanding of the biocatalytic application of CDH and LPMO for the oxidative depolymerization of polysaccharides. [ABSTRACT FROM AUTHOR]

Cellobiose dehydrogenase (CDH) is applied as a bioelectrocatalyst in biosensors because its mobile cytochrome domain is capable of direct electron transfer. This study investigates the electron transfer mechanism of CDH molecules embedded in the polycation polyethyleneimine (PEI), which has been reported as a current‐boosting component of CDH‐based biosensors. By immobilizing different concentrations of CDH and its isolated cytochrome domain in PEI films, we found that increasing concentrations of cytochrome enhanced the film conductivity (up to 251±8 mS cm−1) through improved electron transfer between the protein redox centers. The increased electrical conductivity of the film contacts CDH molecules at a greater distance from the electrode. The cross‐linker poly(ethylene glycol) diglycidyl ether improves the packing and contacting of the cytochrome domains, whereas glutaraldehyde reduces the current obtained. Deglycosylation of CDH enhances the conductivity of enzyme‐polymer films by up to 34 %, implying a higher number of productive electron‐hopping events between cytochrome domains due to enhanced mobility or reduced shielding. By balancing negative charges on the CDH surface at neutral and alkaline pH, PEI increases the interdomain electron transfer and the electrical film conductivity. The resulting increased current output is relevant for in vivo bioanalytical applications. [ABSTRACT FROM AUTHOR]

Cellobiose dehydrogenase (CDH) is one of the cellulase auxiliary proteins, which is widely used in the field of biomass degradation. However, how to efficiently and cheaply apply it in industrial production still needs further research. Aspergillus niger C112 is a significant producer of cellulase and has a relatively complete lignocellulose degradation system, but its CDH activity was only 3.92 U. To obtain a recombinant strain of A. niger C112 with high cellulases activity, the CDH from the readily available white-rot fungus Grifola frondose had been heterologously expressed in A. niger C112, under the control of the gpdA promoter. After cultivation in the medium with alkali-pretreated poplar fiber as substrate, the enzyme activity of recombinant CDH reached 36.63 U/L. Compared with the original A. niger C112, the recombinant A. niger transformed with Grifola frondosa CDH showed stronger lignocellulase activity, the activities of cellulases, ß-1, 4-glucosidase and manganese peroxidase increased by 28.57, 35.07 and 121.69%, respectively. The result showed that the expression of the gcdh gene in A. niger C112 could improve the activity of some lignocellulose degrading enzymes. This work provides a theoretical basis for the further application of gcdh gene in improving biomass conversion efficiency. [ABSTRACT FROM AUTHOR]

We report a novel production process for lactobionic acid (LBA) production using an engineered Neurospora crassa strain F5. The wild-type N. crassa strain produces cellobiose dehydrogenase (CDH) and uses lactose as a carbon source. N. crassa strain F5, which was constructed by deleting six out of the seven β-glucosidases in the wild type, showed a much slower lactose utilization rate and produced a much higher level of cellobiose dehydrogenase (CDH) than the wild type. Strain N. crassa F5 produced CDH and laccase simultaneously on the pretreated wheat straw with 3 µM of cycloheximide added as the laccase inducer. The deproteinized cheese whey was added directly to the shake flasks with the fungus present to achieve LBA production. Strain F5 produced about 37 g/L of LBA from 45 g/L of lactose in 27 h since deproteinized cheese whey addition. The yield of LBA from consumed lactose was about 85%, and the LBA productivity achieved was about 1.37 g/L/h. [ABSTRACT FROM AUTHOR]

Third-generation biosensors use enzymes capable of direct electron transfer (DET) to the sensor surface. They are of interest for continuous glucose monitoring in blood or interstitial fluid, but they are rarely investigated. One reason is the hindered DET of the enzymes to the metallic electrodes. In this publication, a novel method for the immobilization of cellobiose dehydrogenase (CDH) DET enzymes employing conductive poly(3,4-ethylenedioxythiophene)-poly (styrene sulfonate) (PEDOT:PSS) inks and a protective polyethylene glycol dimethacrylate (PEG-DMA) hydrogel layer on gold electrodes is reported. This layer stack showed a glucose-specific current response for voltages between −0.2 and 0.4 V in physiological PBS buffer, and enabled interference-less sensing in a solution of acetaminophen, ascorbic acid, dopamine, and uric acid at 0 V. A Michaelis–Menten fit led to a maximum current density (Imax) of 257 ± 7.9 nA/mm2 and a Michaelis–Menten constant (Km) of 28.4 ± 2.2 mM, with a dynamic range of 0.1–20 mM glucose and a limit of detection of 0.1 mM. After 16 h of continuous measurement of 20 mM glucose, the signal decreased to 60% of its initial value. Storage stability was successfully verified until up to 10 days. In summary, this paper shows a simplified approach for the fabrication of third-generation biosensors using CDH-PEDOT:PSS and PEG-DMA hydrogel inks. [ABSTRACT FROM AUTHOR]

In this work the green synthesis of gold nanoparticles (Au-NPs) using the oxidoreductive enzymes Myriococcum thermophilum cellobiose dehydrogenase (Mt CDH), Glomerella cingulata glucose dehydrogenase (Gc GDH), and Aspergillus niger glucose oxidase (An GOX)) as bioreductants was investigated. The influence of reaction conditions on the synthesis of Au-NPs was examined and optimised. The reaction kinetics and the influence of Au ions on the reaction rate were determined. Based on the kinetic study, the mechanism of Au-NP synthesis was proposed. The Au-NPs were characterized by UV–Vis spectroscopy and transmission electron microscopy (TEM). The surface plasmon resonance (SPR) absorption peaks of the Au-NPs synthesised with Mt CDH and Gc GDH were observed at 535 nm, indicating an average size of around 50 nm. According to the image analysis performed on a TEM micrograph, the Au-NPs synthesized with Gc GDH have a spherical shape with an average size of 2.83 and 6.63 nm after 24 and 48 h of the reaction, respectively. Key points: • The Au NPs were synthesised by the action of enzymes CDH and GDH. • The synthesis of Au-NPs by CDH is related to the oxidation of cellobiose. • The synthesis of Au-NPs by GDH was not driven by the reaction kinetic. [ABSTRACT FROM AUTHOR]

Cellobiose dehydrogenase (CDH) catalyzes the oxidation of cello‐oligosaccharides and transfers electrons to oxygen or other electron receptors, such as lytic polysaccharide oxygenases (LPMOs). Cellobiose dehydrogenase has the potential to be used as an electron donor for LPMOs. Therefore, it is important to obtain CDHs under reaction conditions that are compatible with those of cellulases and LPMOs. Here, a CDH gene from Myceliophthora thermophila (MtCDH‐A) was constitutively expressed in Trichoderma reesei, and its ability to donate electrons to the LPMO was validated. MtCDH‐A has an optimal pH of 6.0 and maintains relatively high activity over a wide pH range. The optimal temperature was 60°C, and it remained stable at 50°C for a long time. These enzymatic properties were compatible with the catalytic conditions of fungal LPMOs and cellulases. When MtCDH‐A was used as an electron donor, the synergistic effect of LPMO and cellulase improved the degradation efficiency of phosphoric acid‐swollen cellulose by 141%. This work provides a novel insight into finding a stable electron donor for LPMOs and a new direction for finding more efficient thermostable CDHs in newly identified fungal species. © 2023 Society of Industrial Chemistry and John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

This paper presents the development of an electrochemical sensor based on a modified ionic liquid, 3-Amine-N-[3-(N-pyrrole)propyl]imidazole bromide (APPIBr), for the detection of lactose content in dairy products. The sensor utilizes cellobiose dehydrogenase (CDH) as the catalytic enzyme for lactose oxidation. The APPIBr/CDH-modified electrode exhibits excellent electrocatalytic activity, enabling sensitive and selective lactose detection. The sensor offers a wide linear range of 50 µM to 3 mM and a low detection limit of 0.58 µM. The developed sensor eliminates the need for complex sample preparation steps, providing rapid and on-site analysis of lactose in dairy products. Compared to traditional lactose detection methods such as HPLC, the sensor demonstrates comparable accuracy and repeatability. The findings highlight the potential of the APPIBr/CDH-based electrochemical sensor for practical applications in the analysis of lactose content in dairy products. [ABSTRACT FROM AUTHOR]

Abstract Cellobiose dehydrogenase (CDH) is applied as a bioelectrocatalyst in biosensors because its mobile cytochrome domain is capable of direct electron transfer. This study investigates the electron transfer mechanism of CDH molecules embedded in the polycation polyethyleneimine (PEI), which has been reported as a current‐boosting component of CDH‐based biosensors. By immobilizing different concentrations of CDH and its isolated cytochrome domain in PEI films, we found that increasing concentrations of cytochrome enhanced the film conductivity (up to 251±8 mS cm−1) through improved electron transfer between the protein redox centers. The increased electrical conductivity of the film contacts CDH molecules at a greater distance from the electrode. The cross‐linker poly(ethylene glycol) diglycidyl ether improves the packing and contacting of the cytochrome domains, whereas glutaraldehyde reduces the current obtained. Deglycosylation of CDH enhances the conductivity of enzyme‐polymer films by up to 34 %, implying a higher number of productive electron‐hopping events between cytochrome domains due to enhanced mobility or reduced shielding. By balancing negative charges on the CDH surface at neutral and alkaline pH, PEI increases the interdomain electron transfer and the electrical film conductivity. The resulting increased current output is relevant for in vivo bioanalytical applications.

Third-generation biosensors use enzymes capable of direct electron transfer (DET) to the sensor surface. They are of interest for continuous glucose monitoring in blood or interstitial fluid, but they are rarely investigated. One reason is the hindered DET of the enzymes to the metallic electrodes. In this publication, a novel method for the immobilization of cellobiose dehydrogenase (CDH) DET enzymes employing conductive poly(3,4-ethylenedioxythiophene)-poly (styrene sulfonate) (PEDOT:PSS) inks and a protective polyethylene glycol dimethacrylate (PEG-DMA) hydrogel layer on gold electrodes is reported. This layer stack showed a glucose-specific current response for voltages between −0.2 and 0.4 V in physiological PBS buffer, and enabled interference-less sensing in a solution of acetaminophen, ascorbic acid, dopamine, and uric acid at 0 V. A Michaelis–Menten fit led to a maximum current density (Imax) of 257 ± 7.9 nA/mm2 and a Michaelis–Menten constant (Km) of 28.4 ± 2.2 mM, with a dynamic range of 0.1–20 mM glucose and a limit of detection of 0.1 mM. After 16 h of continuous measurement of 20 mM glucose, the signal decreased to 60% of its initial value. Storage stability was successfully verified until up to 10 days. In summary, this paper shows a simplified approach for the fabrication of third-generation biosensors using CDH-PEDOT:PSS and PEG-DMA hydrogel inks.

Lactobionic acid (LBA) is a bioactive compound that has become increasingly popular in medicine in recent years due to its unique properties. This chemical can be formed via the enzymatic oxidation of lactose using fungal oxidoreductive enzymes. This study aimed to intensify the synthesis of LBA using immobilised enzymes (cellobiose dehydrogenase from Phanerochaete chrysosporium (PchCDH) and laccase from Cerrena unicolor (CuLAC)) on chitosan microspheres. We used three different crosslinking agents: genipin, glutaraldehyde, and polyethyleneimine to activate the chitosan. The FTIR and CellDrop techniques were used to characterise the activated microspheres. Quantitative (HPLC) and qualitative (TLC) methods were used to determine the obtained LBA. The results show that the type of activator used influences the efficiency of the binding of the enzyme to the matrix. Furthermore, the amount of LBA formed depends on the type of system used. The use of a system in which one of the enzymes is immobilised on a PEI-activated carrier (PchCDH) and the other is free (CuLAC) proved to be the most optimal, as it yielded almost 100% conversion of lactose to lactobionic acid. Summarising the data obtained the following: lactobionic acid immobilised on chitosan microspheres has great potential for medical applications. [ABSTRACT FROM AUTHOR]

Abstract Background The Carbohydrate-Active enZymes (CAZy) auxiliary activity family 3 (AA3) comprises flavin adenine dinucleotide-dependent (FAD) oxidoreductases from the glucose–methanol–choline (GMC) family, which play auxiliary roles in lignocellulose conversion. The AA3 subfamily 1 predominantly consists of cellobiose dehydrogenases (CDHs) that typically comprise a dehydrogenase domain, a cytochrome domain, and a carbohydrate-binding module from family 1 (CBM1). Results In this work, an AA3_1 gene from T. myriococcoides CBS 398.93 encoding only a GMC dehydrogenase domain was expressed in Aspergillus niger. Like previously characterized CDHs, this enzyme (TmXdhA) predominantly accepts linear saccharides with β-(1 → 4) linkage and targets the hydroxyl on the reducing anomeric carbon. TmXdhA was distinguished, however, by its preferential activity towards xylooligosaccharides over cellooligosaccharides. Amino acid sequence analysis showed that TmXdhA possesses a glutamine at the substrate-binding site rather than a threonine or serine that occupies this position in previously characterized CDHs, and structural models suggest the glutamine in TmXdhA could facilitate binding to pentose sugars. Conclusions The biochemical analysis of TmXdhA revealed a catalytic preference for xylooligosaccharide substrates. The modeled structure of TmXdhA provides a reference for the screening of oxidoreductases targeting xylooligosaccharides. We anticipate TmXdhA to be a good candidate for the conversion of xylooligosaccharides to added-value chemicals by its exceptional catalytic ability.

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoprotein catalyzing the oxidation reaction of β-1,4-glycosidic-bonded sugars (lactose or cellobiose), which results in the formation of aldobionic acids and hydrogen peroxide as a byproduct. The biotechnological application of CDH requires the immobilization of the enzyme on a suitable support. As a carrier of natural origin used for CDH immobilization, chitosan seems to increase the catalytic potential of the enzyme, especially for applications as packaging in the food industry and as a dressing material in medical applications. The present study aimed to immobilize the enzyme on chitosan beads and determine the physicochemical and biological properties of immobilized CDHs obtained from different fungal sources. The chitosan beads with immobilized CDHs were characterized in terms of their FTIR spectra or SEM microstructure. The most effective method of immobilization in the proposed modification was the covalent bonding of enzyme molecules using glutaraldehyde, resulting in efficiencies ranging from 28 to 99%. Very promising results, compared to free CDH, were obtained in the case of antioxidant, antimicrobial, and cytotoxic properties. Summarizing the obtained data, chitosan seems to be a valuable material for the development of innovative and effective immobilization systems for biomedical applications or food packaging, preserving the unique properties of CDH. [ABSTRACT FROM AUTHOR]

Background: The Carbohydrate-Active enZymes (CAZy) auxiliary activity family 3 (AA3) comprises flavin adenine dinucleotide-dependent (FAD) oxidoreductases from the glucose–methanol–choline (GMC) family, which play auxiliary roles in lignocellulose conversion. The AA3 subfamily 1 predominantly consists of cellobiose dehydrogenases (CDHs) that typically comprise a dehydrogenase domain, a cytochrome domain, and a carbohydrate-binding module from family 1 (CBM1). Results: In this work, an AA3_1 gene from T. myriococcoides CBS 398.93 encoding only a GMC dehydrogenase domain was expressed in Aspergillus niger. Like previously characterized CDHs, this enzyme (TmXdhA) predominantly accepts linear saccharides with β-(1 → 4) linkage and targets the hydroxyl on the reducing anomeric carbon. TmXdhA was distinguished, however, by its preferential activity towards xylooligosaccharides over cellooligosaccharides. Amino acid sequence analysis showed that TmXdhA possesses a glutamine at the substrate-binding site rather than a threonine or serine that occupies this position in previously characterized CDHs, and structural models suggest the glutamine in TmXdhA could facilitate binding to pentose sugars. Conclusions: The biochemical analysis of TmXdhA revealed a catalytic preference for xylooligosaccharide substrates. The modeled structure of TmXdhA provides a reference for the screening of oxidoreductases targeting xylooligosaccharides. We anticipate TmXdhA to be a good candidate for the conversion of xylooligosaccharides to added-value chemicals by its exceptional catalytic ability. [ABSTRACT FROM AUTHOR]

Lactobionic acid (LBA) is a bioactive compound that has become increasingly popular in medicine in recent years due to its unique properties. This chemical can be formed via the enzymatic oxidation of lactose using fungal oxidoreductive enzymes. This study aimed to intensify the synthesis of LBA using immobilised enzymes (cellobiose dehydrogenase from Phanerochaete chrysosporium (PchCDH) and laccase from Cerrena unicolor (CuLAC)) on chitosan microspheres. We used three different crosslinking agents: genipin, glutaraldehyde, and polyethyleneimine to activate the chitosan. The FTIR and CellDrop techniques were used to characterise the activated microspheres. Quantitative (HPLC) and qualitative (TLC) methods were used to determine the obtained LBA. The results show that the type of activator used influences the efficiency of the binding of the enzyme to the matrix. Furthermore, the amount of LBA formed depends on the type of system used. The use of a system in which one of the enzymes is immobilised on a PEI-activated carrier (PchCDH) and the other is free (CuLAC) proved to be the most optimal, as it yielded almost 100% conversion of lactose to lactobionic acid. Summarising the data obtained the following: lactobionic acid immobilised on chitosan microspheres has great potential for medical applications.

Cellobiose dehydrogenase (CDH) is capable of oxidizing cellobiose and related carbohydrates and generating electrical current at carbon‐based electrodes through direct electron transfer (DET) or mediated electron transfer (MET) mechanisms. As a result, CDHs have been utilized as biocatalysts in biosensors and biofuel cell anodes. A novel engineered ascomycetous Class II CDH with enhanced glucose activity was tested as a bioelectrocatalyst for application to DET or MET‐based glucose biosensors with the electrode component amount selection optimized for maximum current in 5 mM glucose solutions. The optimised DET biosensor showed a similar sensitivity and 3‐fold lower KM,app when compared to non‐optimised DET sensor based on the same engineered CDH. The optimized MET biosensor had a similar KM,app to non‐optimized MET biosensor. However, it showed 15‐fold improvement in jmax and 17‐fold improvement in sensitivity over the DET biosensor. The sensor signals are not affected by the presence of oxygen, although operation in artificial serum results in 43 % and 28 % lower sensitivity for the DET and MET sensors, respectively. While no individually tested potential interferent breaches a mean absolute relative difference of 20 % of the current, the cumulative co‐operative effect in complex media, such as artificial serum, decreases the glucose oxidation current signal. [ABSTRACT FROM AUTHOR]

Cellobiose dehydrogenase (CDH) plays a crucial role in lignocellulose degradation and bioelectrochemical industries, making it highly in demand. However, the production and purification of CDH through fungal heterologous expression methods is time-consuming, costly, and challenging. In this study, we successfully displayed Pycnoporus sanguineus CDH (ps CDH) on the surface of Bacillus subtilis spores for the first time. Enzymatic characterization revealed that spore surface display enhanced the tolerance of ps CDH to high temperature (80 °C) and low pH levels (3.5) compared to free ps CDH. Furthermore, we found that glycerol, lactic acid, and malic acid promoted the activity of immobilized spore-displayed ps CDH; glycerol has a more significant stimulating effect, increasing the activity from 16.86 ± 1.27 U/mL to 46.26 ± 3.25 U/mL. After four reuse cycles, the ps CDH immobilized with spores retained 48% of its initial activity, demonstrating a substantial recovery rate. In conclusion, the spore display system, relying on cotG, enables the expression and immobilization of CDH while enhancing its resistance to adverse conditions. This system demonstrates efficient enzyme recovery and reuse. This approach provides a novel method and strategy for the immobilization and stability enhancement of CDH. • Integration of CDH fixation and stability enhancement through spore surface display. • Glycerol, lactic acid, and malic acid promote the activity of spore-displayed CDH. • CDH immobilized with spores demonstrating a substantial recovery rate. [ABSTRACT FROM AUTHOR]

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoprotein catalyzing the oxidation reaction of β-1,4-glycosidic-bonded sugars (lactose or cellobiose), which results in the formation of aldobionic acids and hydrogen peroxide as a byproduct. The biotechnological application of CDH requires the immobilization of the enzyme on a suitable support. As a carrier of natural origin used for CDH immobilization, chitosan seems to increase the catalytic potential of the enzyme, especially for applications as packaging in the food industry and as a dressing material in medical applications. The present study aimed to immobilize the enzyme on chitosan beads and determine the physicochemical and biological properties of immobilized CDHs obtained from different fungal sources. The chitosan beads with immobilized CDHs were characterized in terms of their FTIR spectra or SEM microstructure. The most effective method of immobilization in the proposed modification was the covalent bonding of enzyme molecules using glutaraldehyde, resulting in efficiencies ranging from 28 to 99%. Very promising results, compared to free CDH, were obtained in the case of antioxidant, antimicrobial, and cytotoxic properties. Summarizing the obtained data, chitosan seems to be a valuable material for the development of innovative and effective immobilization systems for biomedical applications or food packaging, preserving the unique properties of CDH.

Abstract Background Cellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDH Tr ) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDH Pp ). Results PcCDH Tr was recombinantly produced with a yield of 600 U L−1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDH Tr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDH Tr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDH Tr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-d-glucosamine additions at the six potential N-glycosylation sites of PcCDH Tr , which indicates the presence of an endo-N-acetyl-β-d-glucosaminidase in the supernatant. Conclusions Heterologous production of PcCDH Tr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDH Tr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDH Tr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent redox enzymes that cleave recalcitrant biopolymers such as cellulose, chitin, starch and hemicelluloses. Although LPMOs receive ample interest in industry and academia, their reaction mechanism is not yet fully understood. Recent studies showed that H2O2 is a more efficient cosubstrate for the enzyme than O2, which could greatly affect the utilization of LPMOs in industrial settings. Results We probe the reactivity of LPMO9C from the cellulose-degrading fungus Neurospora crassa with a turbidimetric assay using phosphoric acid-swollen cellulose (PASC) as substrate and H2O2 as a cosubstrate. The measurements were also followed by continuous electrochemical H2O2 detection and LPMO reaction products were analysed by mass spectrometry. Different systems for the in situ generation of H2O2 and for the reduction of LPMO’s active-site copper were employed, including glucose oxidase, cellobiose dehydrogenase, and the routinely used reductant ascorbate. Conclusions We found for all systems that the supply of H2O2 limited LPMO’s cellulose depolymerization activity, which supports the function of H2O2 as the relevant cosubstrate. The turbidimetric assay allowed rapid determination of LPMO activity on a cellulosic substrate without the need for time-consuming and instrumentally elaborate analysis methods.

From the biotechnological viewpoint, the enzymatic disintegration of plant lignocellulosic biomass is a promising goal since it would deliver fermentable sugars for the chemical sector. Cellobiose dehydrogenase (CDH) is a vital component of the extracellular lignocellulose-degrading enzyme system of fungi and has a great potential to improve catalyst efficiency for biomass processing. In the present study, a CDH from a newly isolated strain of the agaricomycete Coprinellus aureogranulatus (CauCDH) was successfully purified with a specific activity of 28.9 U mg−1. This pure enzyme (MW = 109 kDa, pI = 5.4) displayed the high oxidative activity towards β-1–4-linked oligosaccharides. Not least, CauCDH was used for the enzymatic degradation of rice straw without chemical pretreatment. As main metabolites, glucose (up to 165.18 ± 3.19 mg g−1), xylose (64.21 ± 1.22 mg g−1), and gluconic acid (5.17 ± 0.13 mg g−1) could be identified during the synergistic conversion of this raw material with the fungal hydrolases (e.g., esterase, cellulase, and xylanase) and further optimization by using an RSM statistical approach. [ABSTRACT FROM AUTHOR]

Polysaccharides are biopolymers composed of simple sugars like glucose, galactose, mannose, fructose, etc. The major natural sources for the production of polysaccharides include plants and microorganisms. In the present work, four bacterial and two fungal polysaccharides (PS or EPS) were used for the modification and preservation of Pycnoporus sanguineus cellobiose dehydrogenase (CDH) activity. It was found that the presence of polysaccharide preparations clearly enhanced the stability of cellobiose dehydrogenase compared to the control value (4 °C). The highest stabilization effect was observed for CDH modified with Rh110EPS. Changes in the optimum pH in the samples of CDH incubated with the chosen polysaccharide modifiers were evidenced as well. The most significant effect was observed for Rh24EPS and Cu139PS (pH 3.5). Cyclic voltammetry used for the analysis of electrochemical parameters of modified CDH showed the highest peak values after 30 days of incubation with polysaccharides at 4 °C. In summary, natural polysaccharides seem to be an effective biotechnological tool for the modification of CDH activity to increase the possibilities of its practical applications in many fields of industry. [ABSTRACT FROM AUTHOR]

Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H2O2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H2O2 is therefore of importance for their use in biotechnological settings. Here, we have employed a variety of spectroscopic and biochemical approaches to probe the reaction of the fungal LPMO9C from N. crassa using H2O2 as a cosubstrate and xyloglucan as a polysaccharide substrate. We show that a single 'priming' electron transfer reaction from the cellobiose dehydrogenase partner protein supports up to 20 H2O2‐driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped‐flow spectroscopy, alongside electron paramagnetic resonance and UV‐Vis spectroscopy, we reveal how H2O2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H2O2 cosubstrate supports fungal LPMO catalysis and leaves the enzyme in the reduced Cu+ state following a single enzyme turnover, thus preventing the need for external protons and electrons from reducing agents or cellobiose dehydrogenase and supporting the binding of H2O2 for further catalytic steps. We observe that the presence of the substrate xyloglucan stabilises the Cu+ state of LPMOs, which may prevent the formation of uncoupled side reactions. [ABSTRACT FROM AUTHOR]

Termitomyces sp. OE 147 is one of the active cellulose degraders in the ecosphere and produces large amount of cellobiose dehydrogenase (CDH) and β-glucosidases when cultivated on cellulose. In order to investigate its effect on cellulose, a highly purified preparation of CDH was obtained from the culture supernatant of the fungus cultivated on cellulose. A combination of ultrafiltration, ion-exchange and gel-filtration chromatography was used to purify CDH by ∼172-fold to a high specific activity of ∼324 U/mg protein on lactose which was used for routine measurement of enzyme activity. The enzyme displayed a pH optimum of 5.0 and stability between pH 5.0 and 8.0 with maximum catalytic efficiency (kcat/Km) of 397 mM−1 s−1 on cellobiose. Incubation of microcrystalline cellulose with the purified CDH led to production of reducing sugars which was accelerated by the addition of FeCl3 during the early stages of incubation. A mass spectrometric analysis revealed fragmentation products of cellulose which were concluded to be cellodextrins, sugars, and corresponding aldonic acids suggesting that CDH can release reducing sugars in the absence of externally added lytic polysaccharide monooxygenases. Polymerized products of glucose were also detected at low intensity. [ABSTRACT FROM AUTHOR]

Abstract Objective Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. Results The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489–2780 mg L−1), and were secreted at moderate to high percentages of the total extracellular protein (51–94%). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5–6-fold, reaching enzyme activity levels (1020–202 U L−1) that could allow their direct application.

Long‐range electron transfer (ET) in metalloenzymes is a general and fundamental process governing O2 activation and reduction. Lytic polysaccharide monooxygenases (LPMOs) are key enzymes for the oxidative cleavage of insoluble polysaccharides, but their reduction mechanism by cellobiose dehydrogenase (CDH), one of the most commonly used enzymatic electron donors, via long‐range ET is still an enigma. Using multiscale simulations, we reveal that interprotein ET between CDH and LPMO is mediated by the heme propionates of CDH and solvent waters. We also show that oxygen binding to the copper center of LPMO is coupled with the long‐range interprotein ET. This process, which is spin‐regulated and enhanced by the presence of O2, directly leads to LPMO−CuII−O2−, bypassing the formation of the generally assumed LPMO−CuI species. The uncovered ET mechanism rationalizes experimental observations and might have far‐reaching implications for LPMO catalysis as well as the O2‐ or CO‐binding‐enhanced long‐range ET processes in other metalloenzymes. [ABSTRACT FROM AUTHOR]

Background: Cellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp). Results: PcCDHTr was recombinantly produced with a yield of 600 U L−1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-d-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-β-d-glucosaminidase in the supernatant. Conclusions: Heterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells. [ABSTRACT FROM AUTHOR]

Production of lactobionic acid using an immobilized cellobiose dehydrogenase/laccase system on magnetic chitosan spheres • A new cellobiose dehydrogenase was identified from Aspergillus fumigatus. • Immobilized Af CDH showed better thermal stability than free enzyme. • Immobilized Af CDH/ Laccase system was efficient for oxidizing lactose into lactobionic acid. • Immobilized bi-enzyme system has good reusability retaining 70 % activity after 10 cycles. Lactobionic acid (LBA) has numerous promising applications in food, pharmaceutical, cosmetics and chemical fields. LBA can be produced by cellobiose dehydrogenase (CDH)/laccase (Lac) bi‐enzymatic system with redox mediator, in which CDH catalyzes the oxidation of lactose into LBA, while LAC serves as a regenerative enzyme to simultaneously regenerate the redox mediator as electron acceptor for CDH. Despite this CDH/LAC system is effective in catalyzing the oxidation of lactose into LBA, easy deactivation of free enzymes throughout the biocatalysis process limited its industrial implementation. In this study, a new cellobiose dehydrogenase from Aspergillus fumigatus (Af CDH) was characterized. Af CDH and LAC were separately immobilized on the glutaraldehyde-modified magnetic chitosan spheres (MCS) with the activity yields of immobilization 61.54 % and 14.1 %, respectively. Immobilized Af CDH displayed better thermal stability and retained 60 % of initial activity after incubation at 50 °C for 4 h, in contrast, the residual activity of free Af CDH was hardly detected under the same condition. Our study demonstrated that the immobilized Af CDH/LAC system was efficient for bioproduction of LBA. 100 g/L of lactose was completely converted to LBA with a space-time yield of 7.14 g/L h This immobilized Af CDH/LAC system had good reusability and retained 70 % activity after 10 cycles of reuse. [ABSTRACT FROM AUTHOR]

Cellobiose dehydrogenase (CDH) is one of the cellulase auxiliary proteins, which is widely used in the field of biomass degradation. However, how to efficiently and cheaply apply it in industrial production still needs further research. Aspergillus niger C112 is a significant producer of cellulase and has a relatively complete lignocellulose degradation system, but its CDH activity was only 3.92 U. To obtain a recombinant strain of A. niger C112 with high cellulases activity, the CDH from the readily available white-rot fungus Grifola frondose had been heterologously expressed in A. niger C112, under the control of the gpdA promoter. After cultivation in the medium with alkali-pretreated poplar fiber as substrate, the enzyme activity of recombinant CDH reached 36.63 U/L. Compared with the original A. niger C112, the recombinant A. niger transformed with Grifola frondosa CDH showed stronger lignocellulase activity, the activities of cellulases, β -1, 4-glucosidase and manganese peroxidase increased by 28.57, 35.07 and 121.69%, respectively. The result showed that the expression of the gcdh gene in A. niger C112 could improve the activity of some lignocellulose degrading enzymes. This work provides a theoretical basis for the further application of gcdh gene in improving biomass conversion efficiency., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhong, Guo, Li, Jia and Zeng.)

Cellobiose dehydrogenases (CDHs) are a group of enzymes belonging to the hemoflavoenzyme group, which are mostly found in fungi. They play an important role in the production of acid sugar. In this research, CDH annotated from the actinobacterium Cellulomonas palmilytica EW123 ( Cp CDH) was cloned and characterized. The Cp CDH exhibited a domain architecture resembling class-I CDH found in Basidiomycota. The cytochrome c and flavin-containing dehydrogenase domains in Cp CDH showed an extra-long evolutionary distance compared to fungal CDH. The amino acid sequence of Cp CDH revealed conservative catalytic amino acids and a distinct flavin adenine dinucleotide region specific to CDH, setting it apart from closely related sequences. The physicochemical properties of Cp CDH displayed optimal pH conditions similar to those of CDHs but differed in terms of optimal temperature. The Cp CDH displayed excellent enzymatic activity at low temperatures (below 30°C), unlike other CDHs. Moreover, Cp CDH showed the highest substrate specificity for disaccharides such as cellobiose and lactose, which contain a glucose molecule at the non-reducing end. The catalytic efficiency of Cp CDH for cellobiose and lactose were 2.05 x 10 5 and 9.06 x 10 4 (M -1 s -1 ), respectively. The result from the Fourier-transform infrared spectroscopy (FT-IR) spectra confirmed the presence of cellobionic and lactobionic acids as the oxidative products of Cp CDH. This study establishes Cp CDH as a novel and attractive bacterial CDH, representing the first report of its kind in the Cellulomonas genus.

• The Co-hemin MOF/chitosan was developed as a mediator for biosensor. • The CDH was immobilized on the modified electrode for lactose detection. • The biosensor was shown broad range detection of lactose with high sensitivity. An electrochemical biosensor containing Co-hemin metal organic frameworks (Co-hemin MOF)/chitosan composite and cellobiose dehydrogenase (CDH) was developed for lactose dectection. The formation of the Co-hemin MOF was verified by scanning electron microscopy, Fourier transform infrared spectroscopy, UV–vis absorption spectroscopy and electrochemical techniques. The Co-hemin MOF/chitosan composite exhibited outstanding electrochemical performance on lactose detection with high sensitivity (102.3 μA mM−1 cm−2) and fast response (5 s). More importantly, the lower determination limit of 4 mM and the broad linear ranges of 10 to 100 mM were obtained to detect lactose. With broad linear ranges, the feasibility of dairy application of the biosensor based on Co-hemin MOF electrode was demonstrated in this study. [ABSTRACT FROM AUTHOR]

Second-generation bioethanol is a sustainable energy source that can be produced from different renewable materials. However, there is a challenge we must overcome to significantly enhance bioethanol production: the hydrolysis of lignocellulosic biomass to fermentable sugars. Synergistic enzymes, such as endoglucanases, β-glucosidases, cellobiohydrolases, and, more recently, lytic polysaccharide monooxygenases and cellobiose dehydrogenases have been used with great success to hydrolyze pretreated biomass. Further advances in the field of second-generation bioethanol production will likely depend on an increased understanding of the interactions between enzymes and lignocellulosic substrates, the development of enzyme engineering, and the optimization of enzyme mixtures to enhance cellulose hydrolysis. [ABSTRACT FROM AUTHOR]

Microbial degradation of lignocellulose and soil organic matter is an important process controlling CO2 flow into the atmosphere, which is mostly carried out by fungi. The alkali-soluble fraction, which is represented mostly by the so-called humic acids (HA), dark-colored compounds of phenolic nature, constitute ~30‒70% of soil Corg. This is the first report on efficient degradation of soil HA under cellulolytic conditions; cellobiose dehydrogenase (CDH), an oxidative enzyme of the cellulolytic complex, was shown to play the key role in this process. Growth of a wood-decomposing fungus Cerrena unicolor on cellulose (CDH and laccase production) and glucose (laccase production) resulted in HA decolorization by 60 and 20%, respectively. HA depolymerization in the presence of CDH and its polymerization in the presence of laccase were shown by gel filtration experiments with the culture liquid and pure enzyme preparations. HA degradation in the presence of CDH was inhibited by radical scavengers, indicating the radical nature of this process (probably the Fenton's reaction). HA depolymerization by CDH decreased in the presence of laccase, while HA polymerization by laccase decreased in the presence of CDH. Thus, the interaction of these enzymes affects HA transformation under cellulolytic conditions. These results improve our understanding of the mechanisms of microbial degradation of the aromatic components of soil humus and of the ecological functions of wood-decomposing fungi grown under cellulolytic conditions. [ABSTRACT FROM AUTHOR]

Background: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent redox enzymes that cleave recalcitrant biopolymers such as cellulose, chitin, starch and hemicelluloses. Although LPMOs receive ample interest in industry and academia, their reaction mechanism is not yet fully understood. Recent studies showed that H2O2 is a more efficient cosubstrate for the enzyme than O2, which could greatly affect the utilization of LPMOs in industrial settings. Results: We probe the reactivity of LPMO9C from the cellulose-degrading fungus Neurospora crassa with a turbidimetric assay using phosphoric acid-swollen cellulose (PASC) as substrate and H2O2 as a cosubstrate. The measurements were also followed by continuous electrochemical H2O2 detection and LPMO reaction products were analysed by mass spectrometry. Different systems for the in situ generation of H2O2 and for the reduction of LPMO's active-site copper were employed, including glucose oxidase, cellobiose dehydrogenase, and the routinely used reductant ascorbate. Conclusions: We found for all systems that the supply of H2O2 limited LPMO's cellulose depolymerization activity, which supports the function of H2O2 as the relevant cosubstrate. The turbidimetric assay allowed rapid determination of LPMO activity on a cellulosic substrate without the need for time-consuming and instrumentally elaborate analysis methods. [ABSTRACT FROM AUTHOR]

The catalytic function of lytic polysaccharide monooxygenases (LPMOs) to cleave and decrystallize recalcitrant polysaccharides put these enzymes in the spotlight of fundamental and applied research. Here we demonstrate that the demand of LPMO for an electron donor and an oxygen species as cosubstrate can be fulfilled by a single auxiliary enzyme: an engineered fungal cellobiose dehydrogenase (CDH) with increased oxidase activity. The engineered CDH was about 30 times more efficient in driving the LPMO reaction due to its 27 time increased production of H2O2 acting as a cosubstrate for LPMO. Transient kinetic measurements confirmed that intra‐ and intermolecular electron transfer rates of the engineered CDH were similar to the wild‐type CDH, meaning that the mutations had not compromised CDH's role as an electron donor. These results support the notion of H2O2‐driven LPMO activity and shed new light on the role of CDH in activating LPMOs. Importantly, the results also demonstrate that the use of the engineered CDH results in fast and steady LPMO reactions with CDH‐generated H2O2 as a cosubstrate, which may provide new opportunities to employ LPMOs in biomass hydrolysis to generate fuels and chemicals. [ABSTRACT FROM AUTHOR]

Copyright of Advancements of Microbiology is the property of Sciendo and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

• CDH of Termitomyces clypeatus possesses distinct carbohydrate binding module. • First report of three domains- cytochrome, dehydrogenase, CBM in basidiomycete CDH. • Dehydrogenase domain is devoid of cellulose binding residues. • Phylogenetic analysis clusters it with the CDH of ascomycetes group of fungi. • Possible phylogenetic status of this fungus have been discussed. Filamentous fungi secrete various oxidative enzymes to degrade the glycosidic bonds of polysaccharides. Cellobiose dehydrogenase (CDH) (E.C.1.1.99.18) is one of the important lignocellulose degrading enzymes produced by various filamentous fungi. It contains two stereo specific ligand binding domains, cytochrome and dehydrogenase - one for heme and the other for flavin adenine dinucleotide (FAD) respectively. The enzyme is of commercial importance for its use in amperometric biosensor, biofuel production, lactose determination in food, bioremediation etc. Termitomyces clypeatus , an edible fungus belonging to the basidiomycetes group, is a good producer of CDH. In this paper we have analyzed the structural properties of this enzyme from T. clypeatus and identified a distinct carbohydrate binding module (CBM) which is not present in most fungi belonging to the basidiomycetes group. In addition, the dehydrogenase domain of T. clypeatus CDH exhibited the absence of cellulose binding residues which is in contrast to the dehydrogenase domains of CDH of other basidiomycetes. Sequence analysis of cytochrome domain showed that the important residues of this domain were conserved like in other fungal CDHs. Phylogenetic tree, constructed using basidiomycetes and ascomycetes CDH sequences, has shown that very surprisingly the CDH from T. clypeatus, which is classified as a basidiomycetes fungus, is clustered with the ascomycetes group. A homology model of this protein has been constructed using the CDH enzyme of ascomycetes fungus Myricoccum thermophilum as a template since it has been found to be the best match sequence with T. clypeatus CDH. We also have modelled the protein with its substrate, cellobiose, which has helped us to identify the substrate interacting residues (L354, P606, T629, R631, Y649, N732, H733 and N781) localized within its dehydrogenase domain. Our computational investigation revealed for the first time the presence of all three domains - cytochrome, dehydrogenase and CBM - in the CDH of T. clypeatus, a basidiomycetes fungus. In addition to discovering the unique structural attributes of this enzyme from T. clypeatus , our study also discusses the possible phylogenetic status of this fungus. [ABSTRACT FROM AUTHOR]

Protein‐nanoparticle (NP) interactions offer a powerful tool to modulate enzymatic activity and activate biocatalysis. However, strong scattering effects may complicate conventional spectrophotometric studies of NP‐assisted biocatalysis. Here, bioelectrocatalytic oxidation of cellobiose by the Humicola insolens cellobiose dehydrogenase (CDH) was electrochemically interrogated with two redox mediators, ferricyanide and cytochrome c, and two types of carbon NPs (CNPs), unmodified (CNP) and negatively charged 4‐sulfophenyl‐modified (CNP‐) ones. We show that both CNPs activate the bioelectrocatalytic oxidation of cellobiose and improve the enzymatic specificity reflected in the kf/KM relationship of 6680 (no CNPs), 15500 (CNP‐) and 10444 M−1 s−1 (CNP) shown for ferricyanide‐mediated and 1262 (no CNPs), 3630 (CNP‐), and 6375 M−1 s−1 (CNP) for cytochrome c‐mediated reactions. The bioelectrocatalysis activation by CNPs apparently results from the interfacial/conformational regulation of the CDH activity and the CDH‐electron acceptor reactivity, which may also be the case of other nanomaterials such as frequently used in bioelectrochemistry carbon nanotubes. [ABSTRACT FROM AUTHOR]

Highlights • Phanerochaete chrysosporium cellobiose dehydrogenase was cloned and expressed in S.cerevisiae. • Enzymatic assay in microtiter plates based on 2,6-dichloroindophenol was optimized. • Several mutants of cellobiose dehydrogenase with increased activity were found. • Recombinant cellobiose dehydrogenases were purified and characterized. Abstract Cellobiose dehydrogenase (CDH) can be used in industry for lactobionic acid production, as a part of biosensors for disaccharides and in wound healing. In fungi it is involved in lignocellulose degradation. CDH gene from Phanerochaete chrysosporium has been cloned in pYES2 plasmid for extracellular expression and protein engineering in yeast Saccharomyces cerevisiae InvSC1 for the first time. A CDH gene library was generated using error-prone PCR and screened by spectrophotometric enzymatic assay based on 2,6-dichloroindophenol reduction detection in microtiter plates. Several mutants with increased activity and specificity towards lactose and cellobiose were found, purified and characterized in detail. Recombinant CDH enzymes showed a broad molecular weight between 120 and 150 KDa due to hyper-glycosylation and the best S137 N mutant showed 2.2 times increased k cat and 1.5 and 2 times increased specificity constant for lactose and cellobiose compared to the wild type enzyme. pH optimum of mutants was not changed while thermostability of selected mutants improved and S137 N mutant retained 30% of it's original activity after 15 min at 70 °C compared to 10% of activity that the wild type enzyme retained. Mutants M65S and S137 N showed also 1.6 and 1.5 times increased productivity of hydrogen peroxide in the presence of 30 mM lactose compared to the wild type. [ABSTRACT FROM AUTHOR]

Featured Application: Developed fluorescent assay and expression system can be used for obtaining improved cellobiose dehydrogenase whole cell biocatalysts for lactobionic acid production and building of biosensors and biofuel cells. Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium can be used in lactobionic acid production, biosensor for lactose, biofuel cells, lignocellulose degradation, and wound-healing applications. To make it a better biocatalyst, CDH with higher activity in an immobilized form is desirable. For this purpose, CDH was expressed for the first time on the surface of S. cerevisiae EBY100 cells in an active form as a triple mutant tmCDH (D20N, A64T, V592M) and evolved further for higher activity using resazurin-based fluorescent assay. In order to decrease blank reaction of resazurin with yeast cells and to have linear correlation between enzyme activity on the cell surface and fluorescence signal, the assay was optimized with respect to resazurin concentration (0.1 mM), substrate concentration (10 mM lactose and 0.08 mM cellobiose), and pH (6.0). Using optimized assay an error prone PCR gene library of tmCDH was screened. Two mutants with 5 (H5) and 7 mutations (H9) were found having two times higher activity than the parent tmCDH enzyme that already had improved activity compared to wild type CDH whose activity could not be detected on the surface of yeast cells. [ABSTRACT FROM AUTHOR]

Abstract Lipidic cubic phase systems (LCPs) are excellent carriers for immobilized enzymes due to their biocompatibility and well-defined nanoporous structure. Lipidic cubic phases act as a convenient matrix to incorporate enzymes and hold them in the vicinity of electrode surfaces in their fully active forms. Corynascus thermophilus cellobiose dehydrogenase (Ct CDH) was trapped in a monoolein cubic phase, which increased not only its stability, but also its catalytic performance with both enhanced mediated and direct electron transfer with electrodes. For studies of mediated electron transfer, three mediators with different formal potentials (E°′) were employed: horse-heart cytochrome c (cyt c), electron acceptor active with the cytochrome domain of Ct CDH, and 2,6-dichlorophenolindophenol (DCPIP) as well as hexaammineruthenium(II) chloride [Ru(NH 3)Cl 2 ] both electron acceptors with the dehydrogenase domain. Ru(NH 3)Cl 2 , having the most negative E°′ of − 0.138 V vs. Ag | AgCl at pH 7.5, gave a catalytic current for lactose oxidation of 32.10 μA cm− 2 in MOPS buffer at pH 7.5. The process carried out in the same solution but under direct electron conditions transfer resulted in a catalytic current of 9.22 μA cm− 2. Electrodes covered with Ct CDH in a LCP film retained their catalytic activity after 28 days showing a slightly increased current density after 6 days. Graphical abstract Image 2 Highlights • Lipidic cubic phase enhances enzyme catalytic activity. • Electrodes with CtCDH in cubic phase are more stable than with adsorbed enzyme. • Both DET and MET mechanism of Ct CDH can be observed in cubic phase. [ABSTRACT FROM AUTHOR]

Abstract In the present study, the effect of carbon nanotubes (CNTs) either single walled (SWCNT) or multi walled (MWCNT) on sugar oxidation by Corynascus thermophilus cellobiose dehydrogenase (Ct CDH) is investigated. The current observed for the direct electron transfer (DET) communication between the enzyme and the electrode is compared to that of the mediated electron transfer (MET) obtained from Ct CDH/CNT/Os-polymer modified electrodes. The CNTs provide a high surface area for the immobilization of Ct CDH and the Os-polymer and increases the amount of both on the electrode surface. The amperometric current density measured for 50 mM glucose at pH 7.4 and an applied potential of 290 mV vs. SHE in DET mode is 6.90 μA cm−2, which is further improved to about 8.73 μA cm−2 when a cross-linker is applied, whereas a 14-folds increase to 97.6 μA cm−2 is observed for MET when Ct CDH is immobilized in the Os-polymer and cross-linked. The current densities extracted from CVs at 150 mV vs. SHE using 20 mM glucose for Ct CDH immobilized with the Os-polymer, MWCNT/Os-polymer and SWCNT/Os-polymer are 139.7, 180.3 and 328.2 μA cm−2, respectively so a remarkable increase is observed when using SWCNT for electrode modification. The bioanode also exhibits a good stability and it retains more than 50% of its activity in multicycle CV scan after five days. Thus the bioanode based on SWCNT/ Ct CDH/Os-polymer/cross-linker could be a promising electrode for application as anode in enzymatic biofuel cells. Graphical abstract Image 1 Highlights • Bioanode produced high current density of 0.55 mA cm−2 at pH 7.4 and 150 mV vs. SHE. • A fast MET between Ct CDH/Os-polymer and electrode was observed. • A novel and versatile bioanode for sugars oxidation was developed. • Good stability with more than 50% activity after five days of multicycle CVs. [ABSTRACT FROM AUTHOR]

Covalent coupling between a surface‐exposed cysteine residue and maleimide groups was used to immobilize variants of Myriococcum thermophilum cellobiose dehydrogenase (MtCDH) at multiwall carbon nanotube electrodes. By introducing individual cysteine residues at particular places on the surface of the flavodehydrogenase domain of the flavocytochrome, we are able to immobilize the different variants in different orientations. Our results show that direct electron transfer (DET) occurs exclusively through the haem b cofactor and that the redox potential of the haem is unaffected by the orientation of the enzyme. Electron transfer between the haem and the electrode is fast in all cases and, at high glucose concentrations, the catalytic currents are limited by the rate of inter‐domain electron transfer (IET) between the FAD and the haem. Using ferrocene carboxylic acid as a mediator, we find that the total amount of immobilized enzyme is 4 to 5 times greater than the amount of enzyme that participates in DET. The role of IET in the overall DET catalysed oxidation was also demonstrated by the effects of changing Ca2+ concentration and by proteolytic cleavage of the cytochrome domain on the DET and MET currents. IET can′t keep up: Covalent coupling between a cysteine residue and maleimide is used to immobilize four variants of cellobiose dehydrogenase at electrodes. Introducing the cysteine residues at different places gives different orientations for each variant. Direct electron transfer (DET) occurs exclusively through the haem b cofactor and is fast in all cases. At high substrate concentrations, the rate of inter‐domain electron transfer (IET) cannot keep up and it limits the catalytic current. [ABSTRACT FROM AUTHOR]

Abstract Fungi-derived flavin adenine dinucleotide glucose dehydrogenases (FADGDHs) are currently the most popular and advanced enzymes for self-monitoring of blood glucose sensors; however, the achievement of direct electron transfer (DET) with FADGDHs is difficult. In this study, a designer FADGDH was constructed by fusing Aspergillus flavus derived FADGDH (AfGDH) and a Phanerochaete chrisosporium CDH (PcCDH)-derived heme b -binding cytochrome domain to develop a novel FADGDH that is capable of direct electron transfer with an electrode. A structural prediction suggested that the heme in the CDH may exist in proximity to the FAD of AfGDH if the heme b -binding cytochrome domain is fused to the AfGDH N-terminal region. Spectroscopic observations of recombinantly produced designer FADGDH confirmed the intramolecular electron transfer between FAD and the heme. A decrease in pH and the presence of a divalent cation improved the intramolecular electron transfer. An enzyme electrode with the immobilized designer FADGDH showed an increase in current immediately after the addition of glucose in a glucose concentration-dependent manner, whereas those with wild-type AfGDH did not show an increase in current. Therefore, the designer FADGDH was confirmed to be a novel GDH that possesses electrode DET ability. The difference in the surface electrostatic potentials of AfGDH and the catalytic domain of PcCDH might be why their intramolecular electron transfer ability is inferior to that of CDH. These relevant and consistent findings provide us with a novel strategic approach for the improvement of the DET properties of designer FADGDH. (241 words) Highlights • The structural prediction suggested that the GDH N-terminal fusion of cytochrome domain of CDH may provide DET enzyme. • A designer FADGDH was constructed by fusing a fungal FADGDH and CDH derived a heme b -binding cytochrome domain. • The spectroscopic observation confirmed that the intra-molecular electron transfer between FAD and heme occurred. • The decrease in pH and  the addition of divalent cation improved the intra-molecular electron transfer of designer FADGDH. • The designer FADGDH is confirmed as a novel GDH possessing DET ability to electrode. [ABSTRACT FROM AUTHOR]