Showing papers on "Cellobiose dehydrogenase published in 2015"

Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation.

Abstract: A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

A survey of genes encoding H2O2-producing GMC oxidoreductases in 10 Polyporales genomes

Abstract: The genomes of three representative Polyporales (Bjerkandera adusta, Phlebia brevispora and a member of the Ganoderma lucidum complex) recently were sequenced to expand our knowledge on the diversity and distribution of genes involved in degradation of plant polymers in this Basidiomycota order, which includes most wood-rotting fungi. Oxidases, including members of the glucose-methanol-choline (GMC) oxidoreductase superfamily, play a central role in the above degradative process because they generate extracellular H2O2 acting as the ultimate oxidizer in both white-rot and brown-rot decay. The survey was completed by analyzing the GMC genes in the available genomes of seven more species to cover the four Polyporales clades. First, an in silico search for sequences encoding members of the aryl-alcohol oxidase, glucose oxidase, methanol oxidase, pyranose oxidase, cellobiose dehydrogenase and pyranose dehydrogenase families was performed. The curated sequences were subjected to an analysis of their evolutionary relationships, followed by estimation of gene duplication/reduction history during fungal evolution. Second, the molecular structures of the near one hundred GMC oxidoreductases identified were modeled to gain insight into their structural variation and expected catalytic properties. In contrast to ligninolytic peroxidases, whose genes are present in all white-rot Polyporales genomes and absent from those of brown-rot species, the H2O2-generating oxidases are widely distributed in both fungal types. This indicates that the GMC oxidases provide H2O2 for both ligninolytic peroxidase activity (in white-rot decay) and Fenton attack on cellulose (in brown-rot decay), after the transition between both decay patterns in Polyporales occurred.

Inter-domain electron transfer in cellobiose dehydrogenase: modulation by pH and divalent cations

Abstract: The flavocytochrome cellobiose dehydrogenase (CDH) is secreted by wood-decomposing fungi, and is the only known extracellular enzyme with the characteristics of an electron transfer protein. Its proposed function is reduction of lytic polysaccharide mono-oxygenase for subsequent cellulose depolymerization. Electrons are transferred from FADH2 in the catalytic flavodehydrogenase domain of CDH to haem b in a mobile cytochrome domain, which acts as a mediator and transfers electrons towards the active site of lytic polysaccharide mono-oxygenase to activate oxygen. This vital role of the cytochrome domain is little understood, e.g. why do CDHs exhibit different pH optima and rates for inter-domain electron transfer (IET)? This study uses kinetic techniques and docking to assess the interaction of both domains and the resulting IET with regard to pH and ions. The results show that the reported elimination of IET at neutral or alkaline pH is caused by electrostatic repulsion, which prevents adoption of the closed conformation of CDH. Divalent alkali earth metal cations are shown to exert a bridging effect between the domains at concentrations of > 3 mm, thereby neutralizing electrostatic repulsion and increasing IET rates. The necessary high ion concentration, together with the docking results, show that this effect is not caused by specific cation binding sites, but by various clusters of Asp, Glu, Asn, Gln and the haem b propionate group at the domain interface. The results show that a closed conformation of both CDH domains is necessary for IET, but the closed conformation also increases the FAD reduction rate by an electron pulling effect.

Engineering Neurospora crassa for improved cellobiose and cellobionate production

Abstract: We report engineering Neurospora crassa to improve the yield of cellobiose and cellobionate from cellulose. A previously engineered strain of N. crassa (F5) with six of seven β-glucosidase (bgl) genes knocked out was shown to produce cellobiose and cellobionate directly from cellulose without the addition of exogenous cellulases. In this study, the F5 strain was further modified to improve the yield of cellobiose and cellobionate from cellulose by increasing cellulase production and decreasing product consumption. The effects of two catabolite repression genes, cre-1 and ace-1, on cellulase production were investigated. The F5 Δace-1 mutant showed no improvement over the wild type. The F5 Δcre-1 and F5 Δace-1 Δcre-1 strains showed improved cellobiose dehydrogenase and exoglucanase expression. However, this improvement in cellulase expression did not lead to an improvement in cellobiose or cellobionate production. The cellobionate phosphorylase gene (ndvB) was deleted from the genome of F5 Δace-1 Δcre-1 to prevent the consumption of cellobiose and cellobionate. Despite a slightly reduced hydrolysis rate, the F5 Δace-1 Δcre-1 ΔndvB strain converted 75% of the cellulose consumed to the desired products, cellobiose and cellobionate, compared to 18% converted by the strain F5 Δace-1 Δcre-1.

Characterization of a Novel PQQ-Dependent Quinohemoprotein Pyranose Dehydrogenase from Coprinopsis cinerea Classified into Auxiliary Activities Family 12 in Carbohydrate-Active Enzymes

Abstract: The basidiomycete Coprinopsis cinerea contains a quinohemoprotein (CcPDH named as CcSDH in our previous paper), which is a new type of pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase and is the first found among all eukaryotes. This enzyme has a three-domain structure consisting of an N-terminal heme b containing a cytochrome domain that is homologous to the cytochrome domain of cellobiose dehydrogenase (CDH; EC 1.1.99.18) from the wood-rotting basidiomycete Phanerochaete chrysosporium, a C-terminal family 1-type carbohydrate-binding module, and a novel central catalytic domain containing PQQ as a cofactor. Here, we describe the biochemical and electrochemical characterization of recombinant CcPDH. UV-vis and resonance Raman spectroscopic studies clearly reveal characteristics of a 6-coordinated low-spin heme b in both the ferric and ferrous states, as well as intramolecular electron transfer from the PQQ to heme b. Moreover, the formal potential of the heme was evaluated to be 130 mV vs. NHE by cyclic voltammetry. These results indicate that the cytochrome domain of CcPDH possesses similar biophysical properties to that in CDH. A comparison of the conformations of monosaccharides as substrates and the associated catalytic efficiency (kcat/Km) of CcPDH indicates that the enzyme prefers monosaccharides with equatorial C-2, C-3 hydroxyl groups and an axial C-4 hydroxyl group in the 1C4 chair conformation. Furthermore, a binding study shows a high binding affinity of CcPDH for cellulose, suggesting that CcPDH function is related to the enzymatic degradation of plant cell wall.

Spectroscopic Observation of Calcium‐Induced Reorientation of Cellobiose Dehydrogenase Immobilized on Electrodes and its Effect on Electrocatalytic Activity

Abstract: Cellobiose dehydrogenase catalyzes the oxidation of various carbohydrates and is considered as a possible anode catalyst in biofuel cells. It has been shown that the catalytic performance of this enzyme immobilized on electrodes can be increased by presence of calcium ions. To get insight into the Ca2+-induced changes in the immobilized enzyme we employ surface-enhanced vibrational (SERR and SEIRA) spectroscopy together with electrochemistry. Upon addition of Ca2+ ions electrochemical measurements show a shift of the catalytic turnover signal to more negative potentials while SERR measurements reveal an offset between the potential of heme reduction and catalytic current. Comparing SERR and SEIRA data we propose that binding of Ca2+ to the heme induces protein reorientation in a way that the electron transfer pathway of the catalytic FAD center to the electrode can bypass the heme cofactor, resulting in catalytic activity at more negative potentials.

Ultrasound coating of polydimethylsiloxanes with antimicrobial enzymes

Abstract: There is an urgent need for antimicrobial functionalization of urinary catheters to prevent microbial colonization and biofilm formation on them. Here, the antimicrobial hydrogen peroxide (H2O2) producing enzyme cellobiose dehydrogenase (CDH) was for the first time grafted onto polydimethylsiloxanes (PDMS) using an ultrasound assisted coating method. This resulted in the development of an effective in situ continous H2O2 producing system able to continuously prevent microbial colonization and biofilm formation on catheters. This enzyme has an added advantage that it uses various oligosaccharides including expolysaccharides (an important part of the bioflim produced by the microbes while colonizing biomaterials) as electron donors to produce H2O2. Successful immobilization of active CDH nanoparticles on PDMS was confirmed by ESEM and AFM analysis as well as quantification of H2O2. Depending on the initial enzyme concentration, CDH-nanoparticles of varying sizes from 65 ± 17 nm to 93 ± 17 nm were created by the ultrasonic waves and subsequently deposited on the PDMS surface. PDMS sheets treated for 3 min produced 18 μM of H2O2 within 2 hours which was sufficient to significantly reduce the amount of viable S. aureus cells as well as the total amount of biomass deposited on the surface. The ultrasound assisted coating of antimicrobial enzymes therefore provides an easy approach to immobilize enzymes and create a surface with antimicrobial properties.

Characterization of Cellobiose Dehydrogenase from a Biotechnologically Important Cerrena unicolor Strain

Abstract: Cellobiose dehydrogenase (CDH), a secreted flavocytochrome produced by a number of wood-degrading fungi, was detected in the culture supernatant of a biotechnologically important strain of Cerrena unicolor grown in a modified cellulose-based liquid medium. The enzyme was purified as two active fractions: CuCDH-FAD (flavin domain) (1.51-fold) with recovery of 8.35 % and CuCDH (flavo-heme enzyme) (21.21-fold) with recovery of 73.41 %. As CDH from other wood-rotting fungi, the intact form of cellobiose dehydrogenase of C. unicolor is a monomeric protein containing one flavin and one heme b with molecular mass 97 kDa and pI = 4.55. The enzyme is glycosylated (8.2 %) mainly with mannose and glucosamine residues. Moreover, the cellobiose dehydrogenase gene cdh1 and its corresponding cDNA from the fungus C. unicolor were isolated, cloned, and characterized. The 2316-bp full-length cDNA of cdh1 encoded a mature CDH protein containing 771 amino acids preceded by a signal peptide consisting of 18 amino acids. Moreover, both active fractions were characterized in terms of kinetics, temperature and pH optima, and antioxidant properties.

Production of cellobionate from cellulose using an engineered Neurospora crassa strain with laccase and redox mediator addition.

Abstract: We report a novel production process for cellobionic acid from cellulose using an engineered fungal strain with the exogenous addition of laccase and a redox mediator. A previously engineered strain of Neurospora crassa (F5∆ace-1∆cre-1∆ndvB) was shown to produce cellobionate directly from cellulose without the addition of exogenous cellulases. Specifically, N. crassa produces cellulases, which hydrolyze cellulose to cellobiose, and cellobiose dehydrogenase (CDH), which oxidizes cellobiose to cellobionate. However, the conversion of cellobiose to cellobionate is limited by the slow re-oxidation of CDH by molecular oxygen. By adding low concentrations of laccase and a redox mediator to the fermentation, CDH can be efficiently oxidized by the redox mediator, with in-situ re-oxidation of the redox mediator by laccase. The conversion of cellulose to cellobionate was optimized by evaluating pH, buffer, and laccase and redox mediator addition time on the yield of cellobionate. Mass and material balances were performed, and the use of the native N. crassa laccase in such a conversion system was evaluated against the exogenous Pleurotus ostreatus laccase. This paper describes a working concept of cellobionate production from cellulose using the CDH-ATBS-laccase system in a fermentation system.

Direct electron transfer of Phanerochaete chrysosporium cellobiose dehydrogenase at platinum and palladium nanoparticles decorated carbon nanotubes modified electrodes

Abstract: In the present work, platinum and palladium nanoparticles (PtNPs and PdNPs) were decorated on the surface of multi-walled carbon nanotubes (MWCNTs) by a simple thermal decomposition method. The prepared nanohybrids, PtNPs–MWCNTs and PdNPs–MWCNTs, were cast on the surface of spectrographic graphite electrodes and then Phanerochaete chrysosporium cellobiose dehydrogenase (PcCDH) was adsorbed on the modified layer. Direct electron transfer between PcCDH and the nanostructured modified electrodes was studied using flow injection amperometry and cyclic voltammetry. The maximum current responses (Imax) and the apparent Michaelis–Menten constants (KappM) for the different PcCDH modified electrodes were calculated by fitting the data to the Michaelis–Menten equation and compared. The sensitivity towards lactose was 3.07 and 3.28 μA mM−1 at the PcCDH/PtNPs–MWCNTs/SPGE and PcCDH/PdNPs–MWCNTs/SPGE electrodes, respectively, which were higher than those measured at the PcCDH/MWCNTs/SPGE (2.60 μA mM−1) and PcCDH/SPGE (0.92 μA mM−1). The modified electrodes were additionally tested as bioanodes for biofuel cell applications.

Native Macrofungi that Produce Lignin-Modifying Enzymes, Cellulases, and Xylanases with Potential Biotechnological Applications

Abstract: With the aim of identifying and exploiting the mycological resources available in the Mexican Sierra Madre Oriental, the lignocellulolytic and pectinolytic potential of autochthonous fungi were evaluated in the present work. A solid media selection system was established in which 74 isolated strains were tested and compared to six international reference strains. The macrofungi Xylaria sp CS121, Inonotus sp CU7, Basidiomycete CH32, Basidiomycete CH23, Xylaria poitei, and Trametes maxima CU1 showed the highest cellulolytic and pectinolytic potential. The greatest lignolytic capability was exhibited by T. maxima CU1 and Pycnoporus sanguineus CS43. Under stirred submerged culture, T. maxima CU1 (cellulases, cellobiose dehydrogenase, manganese peroxidase (MnP), and laccase, with 200, 359, 51, and 267 U/L, respectively) and Xylaria sp CS121 (198 U/L of xylanases) were the highest enzymatic producers. Under stationary conditions, the best producers were Inonotus sp CU7 for cellulases, P. sanguineus CS43 for cellobiose dehydrogenase and laccase, and T. maxima CU1 for xylanases and MnP (242, 467, 35, 165, and 31 U/L, respectively). These results demonstrate the efficiency of enzymatic profiling as a tool for enzyme discovery with Mexican native fungi.

Lignocellulose depolymerization occurs via an environmentally adapted metabolic cascades in the wood-rotting basidiomycete Phanerochaete chrysosporium.

Abstract: Plant biomass can be utilized by a lignocellulose-degrading fungus, Phanerochaete chrysosporium, but the metabolic and regulatory mechanisms involved are not well understood A polyomics-based analysis (metabolomics, proteomics, and transcriptomics) of P chrysosporium has been carried out using statistically optimized conditions for lignocellulolytic reaction Thirty-nine metabolites and 123 genes (14 encoded proteins) that consistently exhibited altered regulation patterns were identified These factors were then integrated into a comprehensive map that fully depicts all signaling cascades involved in P chrysosporium Despite the diversity of these cascades, they showed complementary interconnection among themselves, ensuring the efficiency of passive biosystem and thereby yielding energy expenditure for the cells Particularly, many factors related to intracellular regulatory networks showed compensating activity in homeostatic lignocellulolysis In the main platform of proactive biosystem, although several deconstruction-related targets (eg, glycoside hydrolase, ureidoglycolate hydrolase, transporters, and peroxidases) were systematically utilized, well-known supporters (eg, cellobiose dehydrogenase and ferroxidase) were rarely generated

Laccase–cellobiose dehydrogenase-catalyzed detoxification of phenolic-rich olive processing residues

Abstract: The combination of a laccase–hydroxybenzotriazole (HBT) mediator system with/without cellobiose dehydrogenase (CDH) or an additional Fenton reaction step for the elimination and/or detoxification of phenolic compounds in dry olive mill residues (DOR) and liquid olive mill wastewaters (OMW) was evaluated. The laccase–HBT–CDH and laccase–HBT–CDH–Fenton system were the most effective, removing at least 69 and 72 % of phenolic compounds from a total of 698 and 683 mg in OMW and DOR, respectively, in 12 h. The efficient removal of phenolic compounds was also accompanied by >80 % reduction in biochemical oxygen demand and chemical oxygen demand in both DOR and OMW. Microbial community analysis using single-strand conformation polymorphism (SSCP) gels showed that biogas reactors supplemented with untreated and laccase–HBT–CDH–Fenton-treated DOR and OMW strongly inhibited growth of microorganisms. In contrast, the laccase–HBT- and laccase–HBT–CDH-pretreated OMW and DOR were detoxified as evidenced by SSCP analysis, which also indicated a distinct sensitivity of the individual members of the anaerobic population toward the toxicants. Further, although the laccase–HBT–CDH–Fenton system was effective in bleaching and removing phenolic compounds in both OMW and DOR, it was not able to support methane production. However, laccase–HBT and laccase–HBT–CDH indeed supported biogas production. This study therefore shows that the laccase–HBT–CDH system has a potential for the detoxification of olive mill residues, which can be potentially used as substrates for downstream processes.

Factors Affecting the Induction of Lignin Peroxidase in Manganese-Deficient Cultures of the White Rot Fungus Phanerochaete chrysosporium

Abstract: The lignin peroxidase (LIP) production and regulation, in manganese ions (Mn2+) deficient cultures of the white rot fungus Phanerochaete chrysosporium, is still not clearly understood. Mn2+ deficiency is correlated to low levels of manganese containing superoxide dismutase (MnSOD). In this work, we show that despite the low activity level of MnSOD in Mn2+-deficient cultures, the presence of H2O2 is essential for the expression of the lip-H2 gene, which encodes for the major LIP isoenzyme produced (LIP-H2). Thus, the H2O2 present in Mn2+-deficient cultures is probably produced by other mechanisms rather than dismutation of superoxide ions by MnSOD. Glyoxal oxidase gene (glox) expression was significantly higher than MnSOD (MnSOD1) and cellobiose dehydrogenase (cdh1) expression in Mn2+-deficient cultures, indicating its clear involvement in H2O2 production in those cultures. Glyoxal oxidase may compensate the absence of MnSOD activity in Mn2+-deficient cultures. The high levels of reactive oxygen species (ROS) needed for the enhancement of LIP expression in Mn2+-deficient cultures were not directly correlated to the protein kinase C (PKC) activity involved in signal transduction pathway. High level of oxidative stress was observed in MnSOD silenced mutants, grown in the presence of Mn2+, indicating that oxidative stress in Mn2+-deficient cultures was caused by low levels of MnSOD rather than the deficiency in Mn2+. The results of this work can further contribute to the understanding of LIP regulation in Mn2+-deficient cultures.

Phenolic antioxidants and their role in quenching of reactive molecular species in the human skin injury

Abstract: Summary A large percentage of the population suffers from impaired wound healing related problems that are partly attributed to the continuous production and exposure of wounds to reactive species. The skin is prone to many adverse effects when e.g. damaged, exposed to ionizing UV radiation and/or environmental reactive pollutants. The reactive molecular species attacking the skin in excessive quantities quickly overwhelm tissue antioxidants and other oxidant-degrading pathways. The uncontrolled release of these reactive species is involved in the oxidation of biomolecules including lipids which in turn results in a number of human skin disorders. Phenolic antioxidants are emerging as contenders removing reactive species produced in these chronic wounds, thus accelerating the healing process. Building on this knowledge, a novel physiologically active wound dressing containing both phenolic antioxidants and an enzyme (cellobiose dehydrogenase) for the continuous recycling of oxidized phenolic antioxidants has been developed recently. This novel approach should protect the wound from damage and will help to create ideal conditions for the successful healing of chronic wounds.

Bioelectrochemical Behavior of the Composite PVP-Os/chitosan as a Mediator with Different Types of Enzymes at Graphite Electrode

Abstract: Chitosan was cross-linked to an osmium redox polymer, poly(4-vinylpyridine) osmium bipyridyl [PVP-Os-(bpy)2-Cl], to form PVP-Os-(bpy)2-Cl/chitosan composite known to make a porous and hydrophilic film with an enzyme. In this work we demonstrate such a composite is a useful trestle, for hosting various sugars oxidizing enzymes to construct biosensors. Glucose sensing ability has been proven with the following glucose oxidizing redox enzymes; Aspergillus niger glucose oxidase (AnGOX), Myriococcum thermophilum cellobiose dehydrogenase, (MtCDH), glycosylated Agaricus meleagris pyranose dehydrogenase (gAmPDH), fragmented deglycosylated Agaricus meleagris pyranose dehydrogenase (fdgAmPDH), and Aspergillus sp. glucose dehydrogenase (AspGDH), as well as recombinant Glomerella cingulata glucose dehydrogenase (rGcGDH).

Cellobiose dehydrogenase on electrodes - an electrochemical biosensor for various analytes tunable by positive charges

Abstract: Cellobiose dehydrogenase (CDH) is a sugar oxidizing enzyme secreted by various species of wood degrading fungi to assist the process of wood degradation. It can oxidise analytically relevant sugars as cellobiose, lactose or glucose leading to a gain of two electrons per sugar molecule. CDH consists of a flavin containing catalytic domain (DH) and a haem containing electron mediating domain (CYT). CDH is able to directly communicate with electrode surfaces via the CYT delivering its gained electrons making it a suitable candidate for the construction of mediatorless biosensors and biofuel cell anodes being applicable either for the detection of sugars or for the generation of electricity out of sugar containing solutions. In the present thesis the ability of CDH to electrically communicate with silver, gold and graphite electrode surfaces was investigated and employed mainly by electrochemical techniques as cyclic voltammetry and square wave voltammetry and was complemented by spectroscopic techniques. The central finding is the ability of cations to enhance the electro-catalytic activity of CDH. Especially divalent cations as Ca2+ were found to increase the internal electron transfer (IET) from the DH to the electron mediating CYT leading to higher current outputs. The effect was ascribed to a yet unknown, transient, electrostatic interaction of Ca2+ with negative charges present on the DH and CYT decreasing their repulsion leading to a faster IET. Similar effects were observed for CDH electrodes premodified with immobilised polycations as polyethylenimine (PEI) or polydiallyldimethylammonium chloride (PDADMAC) or premodified with PEI covered gold nanoparticles. The polycations were found to enhance the enzyme load onto electrode surfaces by electrostatic interactions but were also suggested to increase the IET comparable to Ca2+. The beneficial effect of cations and polycations on the electro-catalytic activity of CDH was employed to construct various biosensors to detect lactose, glucose, adenosine triphosphate and Ca2+ in various sensing schemes and analytes. A further observation regarding the electrochemistry of CDH could be obtained only recently. We could finally prove unequivocally, after more than a decade of efforts, that a direct electronic communication is also possible between electrodes and the DH domain occurring at lower voltages than the DET with CYT. This potentially increases the voltage of biofuel cells and lowers the problematic oxidation of common interferents of biosensors pushing the commercial exploitation of CDH as a bioelectrocatalyst to a new level.