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Roland Ludwig - One of the best experts on this subject based on the ideXlab platform.
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Cellobiose Dehydrogenase: An extracellular flavocytochrome from the phytopathogenic basidiomycete Sclerotium (Athelia) rolfsii
2020Co-Authors: Dietmar Haltrich, Roland Ludwig, Marcel ZamockyAbstract:Cellobiose Dehydrogenase (CDH) is an extracellular flavocytochrome that is produced by wood-degrading and plant pathogenic fungi, both basidiomycetes and ascomycetes. Typically, CDH is a monomeric protein with a bipartitedomain organization, consisting of an N-terminal heme domain containing a cytochrome b type heme, and a C-terminal flavin domain with a noncovalently bound FAD. These are linked by a protease-sensitive linker region. Based on the currently known sequences CDH can be divided in two distinct classes, class-1 CDH comprising five known sequences from basidiomycetes, and class-2 CDH represented by three sequences from ascomycetes. CDH oxidizes Cellobiose and higher cellodextrins efficiently to their corresponding lactones. Concurrently a wide spectrum of different electron acceptors including various quinones, organic radical species and metal ion complexes are reduced. This report reviews recent progress made in understanding the physiology, structure and function of CDH.
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polysaccharide oxidation by lytic polysaccharide monooxygenase is enhanced by engineered Cellobiose Dehydrogenase
FEBS Journal, 2020Co-Authors: Christoph Sygmund, Daniel Kracher, Zarah Forsberg, Bastien Bissaro, Sonja Gangl, Marita Preims, Vincent G H Eijsink, Roland LudwigAbstract:: 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 H2 O2 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 H2 O2 -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 H2 O2 as a cosubstrate, which may provide new opportunities to employ LPMOs in biomass hydrolysis to generate fuels and chemicals.
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Cellobiose Dehydrogenase: Bioelectrochemical insights and applications.
Bioelectrochemistry, 2019Co-Authors: Stefan Scheiblbrandner, Roland LudwigAbstract:Abstract Cellobiose Dehydrogenase (CDH) is a flavocytochrome with a history of bioelectrochemical research dating back to 1992. During the years, it has been shown to be capable of mediated electron transfer (MET) and direct electron transfer (DET) to a variety of electrodes. This versatility of CDH originates from the separation of the catalytic flavoDehydrogenase domain and the electron transferring cytochrome domain. This uncoupling of the catalytic reaction from the electron transfer process allows the application of CDH on many different electrode materials and surfaces, where it shows robust DET. Recent X-ray diffraction and small angle scattering studies provided insights into the structure of CDH and its domain mobility, which can change between a closed-state and an open-state conformation. This structural information verifies the electron transfer mechanism of CDH that was initially established by bioelectrochemical methods. A combination of DET and MET experiments has been used to investigate the catalytic mechanism and the electron transfer process of CDH and to deduce a protein structure comprising of mobile domains. Even more, electrochemical methods have been used to study the redox potentials of the FAD and the haem b cofactors of CDH or the electron transfer rates. These electrochemical experiments, their results and the application of the characterised CDHs in biosensors, biofuel cells and biosupercapacitors are combined with biochemical and structural data to provide a thorough overview on CDH as versatile bioelectrocatalyst.
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direct electron transfer anisotropy of a site specifically immobilized Cellobiose Dehydrogenase
ACS Catalysis, 2019Co-Authors: Christophe V F P Laurent, Lo Gorton, Chris Oostenbrink, Marta Meneghello, Jani Tuoriniemi, Philip N Bartlett, Roland LudwigAbstract:To study the direct electron transfer (DET) of the multicofactor enzyme Cellobiose Dehydrogenase (CDH) in regard to its orientation on an electrode surface, a recently published, maleimide-based immobilization method was used in combination with site-directed mutagenesis to establish different orientations on an electrode surface. CDH from Myriococcum thermophilum was chosen for this study because its protein structure is resolved and the factors influencing the movement of its mobile cytochrome domain (CYT) are established. Seven CDH variants with a surface-exposed cysteine residue in different spatial positions were generated for site-specific maleimide coupling. Surface plasmon resonance and cyclic voltammetry showed that all CDH variants, but not the wild-type CDH, bound covalently to gold electrodes or glassy carbon electrodes and were catalytically active. For DET, the CYT domain needs to move from the closed-state conformation, where it obtains an electron from the catalytic flavin adenine dinucleo...
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interaction between Cellobiose Dehydrogenase and lytic polysaccharide monooxygenase
Biochemistry, 2019Co-Authors: Christophe V F P Laurent, Roland Ludwig, Erik Breslmayr, Daniel Tunega, Chris OostenbrinkAbstract:Lytic polysaccharide monooxygenases (LPMOs) are ubiquitous oxidoreductases, facilitating the degradation of polymeric carbohydrates in biomass. Cellobiose Dehydrogenase (CDH) is a biologically relevant electron donor in this process, with the electrons resulting from Cellobiose oxidation being shuttled from the CDH Dehydrogenase domain to its cytochrome domain and then to the LPMO catalytic site. In this work, we investigate the interaction of four Neurospora crassa LPMOs and five CDH cytochrome domains from different species using computational methods. We used HADDOCK to perform protein–protein docking experiments on all 20 combinations and subsequently to select four complexes for extensive molecular dynamics simulations. The potential of mean force is computed for a rotation of the cytochrome domain relative to LPMO. We find that the LPMO loops are largely responsible for the preferred orientations of the cytochrome domains. This leads us to postulate a hybrid version of NcLPMO9F, with exchanged loops...
Dietmar Haltrich - One of the best experts on this subject based on the ideXlab platform.
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Cellobiose Dehydrogenase: An extracellular flavocytochrome from the phytopathogenic basidiomycete Sclerotium (Athelia) rolfsii
2020Co-Authors: Dietmar Haltrich, Roland Ludwig, Marcel ZamockyAbstract:Cellobiose Dehydrogenase (CDH) is an extracellular flavocytochrome that is produced by wood-degrading and plant pathogenic fungi, both basidiomycetes and ascomycetes. Typically, CDH is a monomeric protein with a bipartitedomain organization, consisting of an N-terminal heme domain containing a cytochrome b type heme, and a C-terminal flavin domain with a noncovalently bound FAD. These are linked by a protease-sensitive linker region. Based on the currently known sequences CDH can be divided in two distinct classes, class-1 CDH comprising five known sequences from basidiomycetes, and class-2 CDH represented by three sequences from ascomycetes. CDH oxidizes Cellobiose and higher cellodextrins efficiently to their corresponding lactones. Concurrently a wide spectrum of different electron acceptors including various quinones, organic radical species and metal ion complexes are reduced. This report reviews recent progress made in understanding the physiology, structure and function of CDH.
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Cellobiose Dehydrogenase from the ligninolytic basidiomycete ceriporiopsis subvermispora
Applied and Environmental Microbiology, 2009Co-Authors: Wolfgang Harreither, Dietmar Haltrich, Christoph Sygmund, Evelyn Dunhofen, Rafael Vicuna, Roland LudwigAbstract:Cellobiose Dehydrogenase (CDH), an extracellular flavocytochrome produced by several wood-degrading fungi, was detected in cultures of the selective delignifier Ceriporiopsis subvermispora when grown on a cellulose- and yeast extract-based liquid medium. CDH amounted to up to 2.5% of total extracellular protein during latter phases of the cultivation and thus suggested an important function for the fungus under the given conditions. The enzyme was purified 44-fold to apparent homogeneity. It was found to be present in two glycoforms of 98 kDa and 87 kDa with carbohydrate contents of 16 and 4%, respectively. The isoelectric point of both glycoforms is around 3.0, differing by 0.1 units, which is the most acidic value so far reported for a CDH. By using degenerated primers of known CDH sequences, one cdh gene was found in the genomic DNA, cloned, and sequenced. Alignment of the 774-amino-acid protein sequence revealed a high similarity to CDH from other white rot fungi. One notable difference was found in the longer interdomain peptide linker, which might affect the interdomain electron transfer at higher temperatures. The preferred substrate of C. subvermispora CDH is Cellobiose, while glucose conversion is strongly discriminated by a 155,000-fold-lower catalytic efficiency. This is a typical feature of a basidiomycete CDH, as are the acidic pH optima for all tested electron acceptors in the range from 2.5 to 4.5.
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highly efficient and versatile anodes for biofuel cells based on Cellobiose Dehydrogenase from myriococcum thermophilum
Journal of Physical Chemistry C, 2008Co-Authors: Federico Tasca, Roland Ludwig, Dietmar Haltrich, Wolfgang Harreither, Lo Gorton, Gilbert NollAbstract:A powerful alternative to glucose oxidase as anode material in implantable biofuel cells is presented: Cellobiose Dehydrogenase (CDH) from the ascomycete Myriococcum thermophilum (MtCDH) catalyzes the electrochemical oxidation of glucose, lactose, and Cellobiose over a broad pH range. Current densities of more than 1 mA·cm−2 can be reached when MtCDH is wired to an Os redox polymer in the presence of single-walled carbon nanotubes and when lactose is used as a substrate at pH 8. In contrast to CDHs from basidiomycete fungi, which oxidize only β-1,4-linked di- and oligosaccharides efficiently, MtCDH is also able to oxidize glucose and other monosaccharides at relatively high turnover rates. The current density toward oxidation of 5 mM glucose under physiological conditions was about 100 μA·cm−2. Outstanding properties of MtCDH are high-temperature stability; a strong discrimination of oxygen turnover (and therefore no H2O2 production) in the presence of alternative electron acceptors; an ability to oxidize...
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investigation of graphite electrodes modified with Cellobiose Dehydrogenase from the ascomycete myriococcum thermophilum
Electroanalysis, 2007Co-Authors: Wolfgang Harreither, Roland Ludwig, Dietmar Haltrich, Vasile Coman, Lo GortonAbstract:The catalytic properties of Cellobiose Dehydrogenase (CDH) from the ascomycete fungus Myriococcum thermophilum adsorbed on a graphite electrode were investigated for a large variety of carbohydrate substrates. The effects of applied potential, pH and buffer composition were tested and optimized, and the most suitable conditions were used to evaluate the detection limit, linear range, and sensitivity of the sensor for different carbohydrates in the flow injection mode. Subsequently, the long term stability of the modified electrodes was determined. Additionally, the direct and mediated electron transfer between the active site of the enzyme and the electrode has been investigated by amperometric flow injection measurements in the absence and presence of the mediator 1,4-benzoquinone in the presence of Cellobiose or lactose.
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investigation of electron transfer between Cellobiose Dehydrogenase from myriococcum thermophilum and gold electrodes
Chemia Analityczna, 2007Co-Authors: Vasile Coman, Roland Ludwig, Dietmar Haltrich, Wolfgang Harreither, Lo GortonAbstract:Cellobiose Dehydrogenase (CDH) is a monomeric protein consisting of two subdomains: a larger flavin-associated domain (DHcdh) and a smaller heme-binding domain (CYTcdh), connected via a protease cleavable linker region. In this study, the inter-domain electron transfer, using the CDH from the ascomycete fungus Myriococcum thermophilum and thiol (SAM) modified gold electrodes, was investigated with cyclic voltammetry and UV-VIS spectroelectrochemistry. The effect of the SAM and pH on the formal potential of the heme domain of CDH and on the current generated by the electrocatalytic oxidation of Cellobiose and lactose was evaluated with voltammetric techniques. The oxidation-reduction midpoint potentials of the DHcdh, CYTcdh, and whole CDH unit were estimated at different pH values using a long-optical-pathway thin capillary-type spectroelectrochemical cell. (Less)
Georg M Guebitz - One of the best experts on this subject based on the ideXlab platform.
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Cellobiose Dehydrogenase based biomedical applications
Process Biochemistry, 2017Co-Authors: Gibson S Nyanhongo, Barbara Thallinger, Georg M GuebitzAbstract:Abstract Cellobiose Dehydrogenase (CDH) is an emerging enzyme that is being investigated for applications in many fields including biosensors, biofuels, bioremediation, and textile processing and recently for several biomedical applications. This review summarizes the recent advances in the application of CDH in the biomedical field, particularly as a novel antimicrobial and antibiofilm agent, as biosesensor, and for the production of many biomedically important biomolecules. In fact, studies showed that the past decade has witnessed great strides in developing CDH-based systems for biomedical applications that opens new strategies for solving some of the perennial problems encountered in the biomedical field.
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Cellobiose Dehydrogenase and chitosan based lysozyme responsive materials for antimicrobial wound treatment
Biotechnology and Bioengineering, 2017Co-Authors: Christoph Ohlknecht, Roland Ludwig, Gregor Tegl, Bianca Beer, Christoph Sygmund, Georg M GuebitzAbstract:: The treatment of wound infection still constitutes a major threat in health care due to the increasing number of bacterial resistances and the difficulty of timely infection detection. Here, we present a smart antimicrobial system that is activated in case of infection based on elevated lysozyme activities. N-acetyl chitosan (degree of N-acetylation: 40%) was synthesized and hydrolysis by lysozyme in artificial wound fluid (AWF) was demonstrated. This resulted in the formation of N-acetylated chito oligosaccharides (COS) with a degree of polymerization of 2-5 units. The COS were shown to serve as substrate for Cellobiose Dehydrogenase (CDH) leading to the production of 1 mM antimicrobial hydrogen peroxide (H2 O2 ) after 24 h incubation at 37°C in AWF. Growth inhibition was seen upon incubation of Escherichia coli and Staphylococcus aureus with this chitosan-CDH system over 8 h. This approach represents the first self-regulating system for the infection responsive inhibition of bacterial growth in response to lysozyme as infection biomarker. Biotechnol. Bioeng. 2017;114: 416-422. © 2016 Wiley Periodicals, Inc.
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antimicrobial Cellobiose Dehydrogenase chitosan particles
ACS Applied Materials & Interfaces, 2016Co-Authors: Gregor Tegl, Roland Ludwig, Gibson S Nyanhongo, Barbara Thallinger, Bianca Beer, Christoph Sygmund, Alexandra Rollett, Georg M GuebitzAbstract:Increasing prevalence of chronic wounds and microbial infection constitute a severe health challenge. The situation is further complicated by emerging multidrug resistance making the treatment of infections increasingly difficult. Here, a novel antimicrobial system based on in situ release of hydrogen peroxide (H2O2) by Cellobiose Dehydrogenase (CDH) immobilized on chitosan (CTS) particles is described. Covalent immobilization using carbodiimide coupling lead to a higher amount of protein immobilized on CTS (104 μg CDH/mg CTS) when compared to noncovalent immobilization, which, however, showed highest recovery of CDH activity (0.01 U/mg CTS). The CDH-CTS in situ generated H2O2 completely inhibited growth of Escherichia coli and Staphylococcus aureus over a period of 24 h. This resilient antimicrobial system represents a novel strategy for preventing infection with potential application in counteracting microbial colonization of chronic wounds.
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preventing microbial colonisation of catheters antimicrobial and antibiofilm activities of Cellobiose Dehydrogenase
International Journal of Antimicrobial Agents, 2014Co-Authors: Barbara Thallinger, Roland Ludwig, Gibson S Nyanhongo, Christoph Sygmund, Maya Argirova, Magdalena Lesseva, Angelika Schlick, Georg M GuebitzAbstract:Abstract The ability of Cellobiose Dehydrogenase (CDH) to produce hydrogen peroxide (H 2 O 2 ) for antimicrobial and antibiofilm functionalisation of urinary catheters was investigated. A recombinantly produced CDH from Myriococcum thermophilum was shown to completely inhibit the growth of Escherichia coli and Staphylococcus aureus both in liquid and solid media when supplemented with either 0.8mM or 2mM Cellobiose as substrate. Biofilm formation on silicone films was prevented by CDH when supplemented with 1mM Cellobiose. The CDH/Cellobiose system also successfully inhibited many common urinary catheter-colonising micro-organisms, including multidrug-resistant S. aureus , Staphylococcus epidermidis , Proteus mirabilis , Stenotrophomonas maltophilia , Acinetobacter baumannii and Pseudomonas aeruginosa. Interestingly, CDH was also able to produce H 2 O 2 during oxidation of extracellular polysaccharides (exPS) formed by micro-organisms in the absence of Cellobiose. The H 2 O 2 production and consequently antimicrobial and antibiofilm activities on these exPS were enhanced by incorporation of glycoside hydrolases such as amylases. Hydrolysis of polysaccharides by these enzymes increases the number of terminal reducing sugars as substrates for CDH as well as destabilises the biofilm. Furthermore, CDH suspended in catheter lubricants killed bacteria in biofilms colonising catheters. Incorporation of the CDH/Cellobiose system in the lubricant therefore makes it an easy strategy for preventing microbial colonisation of catheters.
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cellulose oxidation and bleaching processes based on recombinant myriococcum thermophilum Cellobiose Dehydrogenase
Enzyme and Microbial Technology, 2013Co-Authors: Annemarie Flitsch, Roland Ludwig, Gibson S Nyanhongo, Christoph Sygmund, Endry Nugroho Prasetyo, Georg M GuebitzAbstract:Abstract Myriococcum thermophilum Cellobiose Dehydrogenase ( Mt CDH) was expressed in Pichia pastoris using the pPICZαA expression vector under the control of methanol inducible AOX promoter. The purified recombinant Mt CDH with a specific activity of 3.1 U mg −1 was characterized to obtain kinetic constants for various carbohydrate substrates. Additionally, the C1 oxidation of the reducing ends of Cellobiose, cellotetraose and maltotriose by MtCDH was verified by HPLC-MS. Mt CDH was employed to oxidize several different cellulose-based materials by production of hydrogen peroxide. Based on the obtained results a one-pot enzymatic scouring/bleaching process for cotton fabrics was developed using pectinases as scouring agent and Mt CDH to produce H 2 O 2 for bleaching. An average increase in whiteness (Berger) Δ E of 26 and an average 95% increase in wettability were observed in all Mt CDH treated fabrics. In addition, Mt CDH oxidized typical colored cotton flavonoids (morin, rutin, isoquercitrin).
Lo Gorton - One of the best experts on this subject based on the ideXlab platform.
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direct electron transfer anisotropy of a site specifically immobilized Cellobiose Dehydrogenase
ACS Catalysis, 2019Co-Authors: Christophe V F P Laurent, Lo Gorton, Chris Oostenbrink, Marta Meneghello, Jani Tuoriniemi, Philip N Bartlett, Roland LudwigAbstract:To study the direct electron transfer (DET) of the multicofactor enzyme Cellobiose Dehydrogenase (CDH) in regard to its orientation on an electrode surface, a recently published, maleimide-based immobilization method was used in combination with site-directed mutagenesis to establish different orientations on an electrode surface. CDH from Myriococcum thermophilum was chosen for this study because its protein structure is resolved and the factors influencing the movement of its mobile cytochrome domain (CYT) are established. Seven CDH variants with a surface-exposed cysteine residue in different spatial positions were generated for site-specific maleimide coupling. Surface plasmon resonance and cyclic voltammetry showed that all CDH variants, but not the wild-type CDH, bound covalently to gold electrodes or glassy carbon electrodes and were catalytically active. For DET, the CYT domain needs to move from the closed-state conformation, where it obtains an electron from the catalytic flavin adenine dinucleo...
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direct electron transfer of Cellobiose Dehydrogenase on positively charged polyethyleneimine gold nanoparticles
ChemPlusChem, 2017Co-Authors: Mojtaba Tavahodi, Roland Ludwig, Christopher Schulz, Roberto Ortiz, Ali Ekhtiari, Behzad Haghighi, Lo GortonAbstract:Efficient conjugation between biomolecules and electrode materials is one of the main challenges in the field of biosensors. Cellobiose Dehydrogenase (CDH) is a monomeric enzyme, which consists of two separate domains: one catalytic Dehydrogenase domain (DHCDH) carrying strongly bound flavin adenine dinucleotide (FAD) in the active site and a cytochrome domain (CYTCDH) carrying a b-type heme connected by a flexible linker region. Herein, we report on the development of a lactose biosensor, based on direct electron transfer (DET) from CDH from Phanerochaete sordida (PsCDH) electrostatically attached onto polyethyleneimine-stabilized gold nanoparticles (PEI@AuNPs) used to cover a conventional polycrystalline solid gold disk electrode. PEI@AuNPs were synthesized in aqueous solution using PEI as reducing agent for AuIII and as stabilizer for the nanoparticles. The heterogeneous electron-transfer (ET) rate (ks) for the redox reaction of immobilized PsCDH at the modified electrodes was calculated based on the Laviron theory and was found to be (39.6±2.5)s-1. The proposed lactose biosensor exhibits good long term stability as well as high and reproducible sensitivity to lactose with a response time less than 5s and a linear range from 1 to 100μm. (Less)
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direct electron transfer from the fad cofactor of Cellobiose Dehydrogenase to electrodes
ACS Catalysis, 2016Co-Authors: Christopher Schulz, Roland Ludwig, Roman Kittl, Lo GortonAbstract: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 b 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 Tr...
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Cellobiose Dehydrogenase modified electrodes advances by materials science and biochemical engineering
Analytical and Bioanalytical Chemistry, 2013Co-Authors: Roland Ludwig, Wolfgang Harreither, Christoph Sygmund, Christopher Schulz, Roberto Ortiz, Lo GortonAbstract:The flavocytochrome Cellobiose Dehydrogenase (CDH) is a versatile biorecognition element capable of detecting carbohydrates as well as quinones and catecholamines. In addition, it can be used as an anode biocatalyst for enzymatic biofuel cells to power miniaturised sensor–transmitter systems. Various electrode materials and designs have been tested in the past decade to utilize and enhance the direct electron transfer (DET) from the enzyme to the electrode. Additionally, mediated electron transfer (MET) approaches via soluble redox mediators and redox polymers have been pursued. Biosensors for Cellobiose, lactose and glucose determination are based on CDH from different fungal producers, which show differences with respect to substrate specificity, pH optima, DET efficiency and surface binding affinity. Biosensors for the detection of quinones and catecholamines can use carbohydrates for analyte regeneration and signal amplification. This review discusses different approaches to enhance the sensitivity and selectivity of CDH-based biosensors, which focus on (1) more efficient DET on chemically modified or nanostructured electrodes, (2) the synthesis of custom-made redox polymers for higher MET currents and (3) the engineering of enzymes and reaction pathways. Combination of these strategies will enable the design of sensitive and selective CDH-based biosensors with reduced electrode size for the detection of analytes in continuous on-site and point-of-care applications.
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direct electrochemistry of phanerochaete chrysosporium Cellobiose Dehydrogenase covalently attached onto gold nanoparticle modified solid gold electrodes
Langmuir, 2012Co-Authors: Hirotoshi Matsumura, Roland Ludwig, Kiyohiko Igarashi, Masahiro Samejima, Roberto Ortiz, Lo GortonAbstract: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 (DHCDH), a heme b containing cytochrome domain (CYTCDH), and a flexible linker region connecting the two domains. Efficient direct electron transfer (DET) of CDH from the basidiomycete Phanerochaete chrysosporium (PcCDH) 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 PcCDH and 4-ATP or MUNH2 in the mixed SAMs was formed using glutaraldehyde...
Christoph Sygmund - One of the best experts on this subject based on the ideXlab platform.
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polysaccharide oxidation by lytic polysaccharide monooxygenase is enhanced by engineered Cellobiose Dehydrogenase
FEBS Journal, 2020Co-Authors: Christoph Sygmund, Daniel Kracher, Zarah Forsberg, Bastien Bissaro, Sonja Gangl, Marita Preims, Vincent G H Eijsink, Roland LudwigAbstract:: 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 H2 O2 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 H2 O2 -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 H2 O2 as a cosubstrate, which may provide new opportunities to employ LPMOs in biomass hydrolysis to generate fuels and chemicals.
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Cellobiose Dehydrogenase and chitosan based lysozyme responsive materials for antimicrobial wound treatment
Biotechnology and Bioengineering, 2017Co-Authors: Christoph Ohlknecht, Roland Ludwig, Gregor Tegl, Bianca Beer, Christoph Sygmund, Georg M GuebitzAbstract:: The treatment of wound infection still constitutes a major threat in health care due to the increasing number of bacterial resistances and the difficulty of timely infection detection. Here, we present a smart antimicrobial system that is activated in case of infection based on elevated lysozyme activities. N-acetyl chitosan (degree of N-acetylation: 40%) was synthesized and hydrolysis by lysozyme in artificial wound fluid (AWF) was demonstrated. This resulted in the formation of N-acetylated chito oligosaccharides (COS) with a degree of polymerization of 2-5 units. The COS were shown to serve as substrate for Cellobiose Dehydrogenase (CDH) leading to the production of 1 mM antimicrobial hydrogen peroxide (H2 O2 ) after 24 h incubation at 37°C in AWF. Growth inhibition was seen upon incubation of Escherichia coli and Staphylococcus aureus with this chitosan-CDH system over 8 h. This approach represents the first self-regulating system for the infection responsive inhibition of bacterial growth in response to lysozyme as infection biomarker. Biotechnol. Bioeng. 2017;114: 416-422. © 2016 Wiley Periodicals, Inc.
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Cellobiose Dehydrogenase functionalized urinary catheter as novel antibiofilm system
Journal of Biomedical Materials Research Part B, 2016Co-Authors: Barbara Thallinger, Roland Ludwig, Christoph Sygmund, Martin Brandauer, Peter Burger, Kristina Ivanova, Denis Scaini, Michael Burnet, Tzanko Tzanov, Gibson S NyanhongoAbstract:: Urinary catheters expose patients to a high risk of acquiring nosocomial infections. To prevent this risk of infection, Cellobiose Dehydrogenase (CDH), an antimicrobial enzyme able to use various oligosaccharides as electron donors to produce hydrogen peroxide using oxygen as an electron acceptor, was covalently grafted onto plasma-activated urinary polydimethylsiloxane (PDMS) catheter surfaces. Successful immobilization of CDH on PDMS was confirmed by Fourier transformed-infrared spectrometry and production of H2 O2 . The CDH functionalized PDMS surfaces reduced the amount of viable Staphylococcus aureus by 60%, total biomass deposited on the surface by 30% and 70% of biofilm formation. The immobilized CDH was relatively stable in artificial urine over 16 days, retaining 20% of its initial activity. The CDH coated PDMS surface did not affect the growth and physiology of HEK 239 and RAW 264,7 mammalian cells. Therefore this new CDH functionalized catheter system shows great potential for solving the current problems associated with urinary catheters. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1448-1456, 2016.
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antimicrobial Cellobiose Dehydrogenase chitosan particles
ACS Applied Materials & Interfaces, 2016Co-Authors: Gregor Tegl, Roland Ludwig, Gibson S Nyanhongo, Barbara Thallinger, Bianca Beer, Christoph Sygmund, Alexandra Rollett, Georg M GuebitzAbstract:Increasing prevalence of chronic wounds and microbial infection constitute a severe health challenge. The situation is further complicated by emerging multidrug resistance making the treatment of infections increasingly difficult. Here, a novel antimicrobial system based on in situ release of hydrogen peroxide (H2O2) by Cellobiose Dehydrogenase (CDH) immobilized on chitosan (CTS) particles is described. Covalent immobilization using carbodiimide coupling lead to a higher amount of protein immobilized on CTS (104 μg CDH/mg CTS) when compared to noncovalent immobilization, which, however, showed highest recovery of CDH activity (0.01 U/mg CTS). The CDH-CTS in situ generated H2O2 completely inhibited growth of Escherichia coli and Staphylococcus aureus over a period of 24 h. This resilient antimicrobial system represents a novel strategy for preventing infection with potential application in counteracting microbial colonization of chronic wounds.
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preventing microbial colonisation of catheters antimicrobial and antibiofilm activities of Cellobiose Dehydrogenase
International Journal of Antimicrobial Agents, 2014Co-Authors: Barbara Thallinger, Roland Ludwig, Gibson S Nyanhongo, Christoph Sygmund, Maya Argirova, Magdalena Lesseva, Angelika Schlick, Georg M GuebitzAbstract:Abstract The ability of Cellobiose Dehydrogenase (CDH) to produce hydrogen peroxide (H 2 O 2 ) for antimicrobial and antibiofilm functionalisation of urinary catheters was investigated. A recombinantly produced CDH from Myriococcum thermophilum was shown to completely inhibit the growth of Escherichia coli and Staphylococcus aureus both in liquid and solid media when supplemented with either 0.8mM or 2mM Cellobiose as substrate. Biofilm formation on silicone films was prevented by CDH when supplemented with 1mM Cellobiose. The CDH/Cellobiose system also successfully inhibited many common urinary catheter-colonising micro-organisms, including multidrug-resistant S. aureus , Staphylococcus epidermidis , Proteus mirabilis , Stenotrophomonas maltophilia , Acinetobacter baumannii and Pseudomonas aeruginosa. Interestingly, CDH was also able to produce H 2 O 2 during oxidation of extracellular polysaccharides (exPS) formed by micro-organisms in the absence of Cellobiose. The H 2 O 2 production and consequently antimicrobial and antibiofilm activities on these exPS were enhanced by incorporation of glycoside hydrolases such as amylases. Hydrolysis of polysaccharides by these enzymes increases the number of terminal reducing sugars as substrates for CDH as well as destabilises the biofilm. Furthermore, CDH suspended in catheter lubricants killed bacteria in biofilms colonising catheters. Incorporation of the CDH/Cellobiose system in the lubricant therefore makes it an easy strategy for preventing microbial colonisation of catheters.