Proteomic Characterization of Lignocellulolytic Enzymes Secreted by the Insect-Associated Fungus Daldinia decipiens oita, Isolated from a Forest in Northern Japan.

Wood-devastating insects utilize their symbiotic microbes with lignocellulose-degrading abilities to extract energy from recalcitrant woods. It is well known that free-living lignocellulose-degrading fungi secrete various carbohydrate-active enzymes (CAZymes) to degrade plant cell wall components, mainly cellulose, hemicellulose, and lignin. However, CAZymes from insect-symbiotic fungi have not been well documented except for a few examples. In this study, an insect-associated fungus, Daldinia decipiens oita, was isolated as a potential symbiotic fungus of female Xiphydria albopicta captured from Hokkaido forest. This fungus was grown in seven different media containing a single carbon source, glucose, cellulose, xylan, mannan, pectin, poplar, or larch, and the secreted proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 128 CAZymes, including domains of 92 glycoside hydrolases, 15 carbohydrate esterases, 5 polysaccharide lyases, 17 auxiliary activities, and 11 carbohydrate-binding modules, were identified, and these are involved in degradation of cellulose and hemicellulose but not lignin. Together with the results of polysaccharide-degrading activity measurements, we concluded that D. decipiens oita tightly regulates the expression of these CAZymes in response to the tested plant cell wall materials. Overall, this study described the detailed proteomic approach of a woodwasp-associated fungus and revealed that the new isolate, D. decipiens oita, secretes diverse CAZymes to efficiently degrade lignocellulose in the symbiotic environment.IMPORTANCE Recent studies show the potential impacts of insect symbiont microbes on biofuel application with regard to their degradation capability of a recalcitrant plant cell wall. In this study, we describe a novel fungal isolate, D. decipiens oita, as a single symbiotic fungus from the Xiphydria woodwasp found in the northern forests of Japan. Our detailed secretome analyses of D. decipiens oita, together with activity measurements, reveal that this insect-associated fungus exhibits high and broad activities for plant cell wall material degradation, suggesting potential applications within the biomass conversion industry for plant mass degradation.

Investigating the Function of Cryptic Cytochalasan Cytochrome P450 Monooxygenases Using Combinatorial Biosynthesis.

Tailoring enzymes in cytochalasan biosynthesis are relatively promiscuous. Exploiting this property, we deduced the function of four cryptic cytochrome P450 monooxygenases via heterologous expression of six cytochrome P450-encoding genes, originating from Hypoxylon fragiforme and Pyricularia oryzae, in pyrichalasin H ΔP450 strains. Three cryptic cytochrome P450 enzymes (HffD, HffG, and CYP1) restored pyrichalasin H production in mutant strains, while CYP3 catalyzed a site-selective epoxidation leading to the isolation of three novel cytochalasans.

Biosynthetic pathway for furanosteroid demethoxyviridin and identification of an unusual pregnane side-chain cleavage.

Furanosteroids, represented by wortmannin, viridin, and demethoxyviridin, are a special group of fungal-derived, highly oxygenated steroids featured by an extra furan ring. They are well-known nanomolar-potency inhibitors of phosphatidylinositol 3-kinase and widely used in biological studies. Despite their importance, the biosyntheses of these molecules are poorly understood. Here, we report the identification of the biosynthetic gene cluster for demethoxyviridin, consisting of 19 genes, and among them 15 biosynthetic genes, including six cytochrome P450 monooxygenase genes, are deleted. As a result, 14 biosynthetic intermediates are isolated, and the biosynthetic pathway for demethoxyviridin is elucidated. Notably, the pregnane side-chain cleavage requires three enzymes: flavin-dependent Baeyer-Villiger monooxygenase, esterase, and dehydrogenase, in sharp contrast to the single cytochrome P450-mediated process in mammalian cells. Structure-activity analyses of these obtained biosynthetic intermediates reveal that the 3-keto group, the C1ß-OH, and the aromatic ring C are important for the inhibition of phosphatidylinositol 3-kinase.

Roles of phospholipid methyltransferases in pycnidia development, stress tolerance and secondary metabolism in the taxol-producing fungus Pestalotiopsis microspore.

Phosphatidylcholine (PC) is an important membrane component of the eukaryotic cell. In yeast fungi, two phospholipid methyltransferases catalyze consecutive steps of methylation in the formation of phosphatidylcholine from phosphatidylethanolamine. However, roles of phospholipid methyltransferases in filamentous fungi remains less investigated. We report here the characterization of two genes, choA and choC, that putatively encoded phospholipid methyltransferases in the taxol-producing fungus Pestalotiopsis microspora. Deletion of choC resulted in defects in PC production, vegetative growth and development of asexual structure. The mutant strains exhibited multiple morphological abnormalities, e.g. swollen hyphal tips and enhanced hyphal branching, and even mycelial autolysis. Some novel roles for the genes were also revealed, for instance, the deletion of either choC or choA impaired the development of pycnidia and conidia, the cell wall integrity. The mutant strains displayed a hypersensitivity to stress conditions, e.g. osmotic stress, cold and metal ions. The osmotic hypersensitivity indicates a crosstalk of PC pathways to other signaling pathways, such as the HOG pathway. Still more, choA, but not choC, was required for the production of secondary metabolites, e.g. pestalotiollide B, suggesting distinct roles of the two genes. This work would contribute to better understanding the function of phospholipid methyltransferases in fungi.

Characterization of a Prenyltransferase for Iso-A82775C Biosynthesis and Generation of New Congeners of Chloropestolides.

Chloropupukeananin and chloropestolides are novel metabolites of the plant endophyte Pestalotiopsis fici, showing antimicrobial, antitumor, and anti-HIV activities. Their highly complex and unique skeletons were generated from the coisolated pestheic acid (1) and iso-A82775C (10) based on our previous studies. Here, we identified the biosynthetic gene cluster iac of 10 and characterized an iacE encoded prenyltransferase. Deletion of iacE abolished iso-A82775C production, accumulated the prenyl group-lacking siccayne (2), and generated four new chloropestolides (3-6). Compounds 5 and 6 showed antibacterial effects against Staphylococcus aureus and Bacillus subtilis, and 5 was also cytotoxic to human tumor cell lines HeLa, MCF-7, and SW480. These results provided the first genetic and biochemical insights into the biosynthesis of natural prenylepoxycyclohexanes and demonstrated the feasibility for generation of diversified congeners by manipulating the biosynthetic genes of 10.

Three new active members of the I-OnuI family of homing endonucleases.

In vitro characterization of 3 LAGLIDADG-type homing endonucleases (HEs) (I-CcaI, I-CcaII, and I-AstI) that belong to the I-OnuI family showed that they are functional HEs that cleave their respective cognate target sites. These endonucleases are encoded within group ID introns and appear to be orthologues that have inserted into 3 different mitochondrial genes: rns, rnl, and cox3. The endonuclease activity of I-CcaI was tested using various substrates, and its minimum DNA recognition sequence was estimated to be 26 nt. This set of HEs may provide some insight into how these types of mobile elements can migrate into new locations. This study provides additional endonucleases that can be added to the catalog of currently available HEs that may have various biotechnology applications.

Characterization of four endophytic fungi as potential consolidated bioprocessing hosts for conversion of lignocellulose into advanced biofuels.

Recently, several endophytic fungi have been demonstrated to produce volatile organic compounds (VOCs) with properties similar to fossil fuels, called "mycodiesel," while growing on lignocellulosic plant and agricultural residues. The fact that endophytes are plant symbionts suggests that some may be able to produce lignocellulolytic enzymes, making them capable of both deconstructing lignocellulose and converting it into mycodiesel, two properties that indicate that these strains may be useful consolidated bioprocessing (CBP) hosts for the biofuel production. In this study, four endophytes Hypoxylon sp. CI4A, Hypoxylon sp. EC38, Hypoxylon sp. CO27, and Daldinia eschscholzii EC12 were selected and evaluated for their CBP potential. Analysis of their genomes indicates that these endophytes have a rich reservoir of biomass-deconstructing carbohydrate-active enzymes (CAZys), which includes enzymes active on both polysaccharides and lignin, as well as terpene synthases (TPSs), enzymes that may produce fuel-like molecules, suggesting that they do indeed have CBP potential. GC-MS analyses of their VOCs when grown on four representative lignocellulosic feedstocks revealed that these endophytes produce a wide spectrum of hydrocarbons, the majority of which are monoterpenes and sesquiterpenes, including some known biofuel candidates. Analysis of their cellulase activity when grown under the same conditions revealed that these endophytes actively produce endoglucanases, exoglucanases, and ß-glucosidases. The richness of CAZymes as well as terpene synthases identified in these four endophytic fungi suggests that they are great candidates to pursue for development into platform CBP organisms.

An Insight into the Secondary Metabolism of Muscodor yucatanensis: Small-Molecule Epigenetic Modifiers Induce Expression of Secondary Metabolism-Related Genes and Production of New Metabolites in the Endophyte.

Muscodor spp. are proficient producers of bioactive volatile organic compounds (VOCs) with many potential applications. However, all members of this genus produce varying amounts and types of VOCs which suggests the involvement of epigenetics as a possible explanation. The members of this genus are poorly explored for the production of soluble compounds (extrolites). In this study, the polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes from an endophyte, Muscodor yucatanensis Ni30, were cloned and sequenced. The PKS genes belonged to reduced, partially reduced, non-reduced, and highly reduced subtypes. Strains over-expressing PKS genes were developed through the use of small-molecule epigenetic modifiers (suberoylanilide hydroxamic acid (SAHA) and 5-azacytidine). The putative epigenetic variants of this organism differed considerably from the wild type in morphological features and cultural characteristics as well as metabolites that were produced. Each variant produced a different set of VOCs distinct from the wild type, and several VOCs including methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)hexane-2,4-diol and 2-carboxymethyl-3-n-hexylmaleic appeared in the variant strains, the production of which could be attributed to the activity of otherwise silent PKS genes. The bioactive extrolite brefeldin A was isolated and characterized from the wild type. However, this metabolite was not detected in EV-1, but instead, two other products were isolated and characterized as ergosterol and xylaguaianol C. Hence, M. yucatanensis has the genetic potential to produce several previously undetectable VOCs and organic solvent soluble products. It is also the case that small-molecule epigenetic modifiers can be used to produce stable variant strains of fungi with the potential to produce new molecules. Finally, this work hints to the prospect that the epigenetics of an endophytic microorganism can be influenced by any number of environmental and chemical factors associated with its host plant which may help to explain the enormous chemical diversity of secondary metabolic products found in Muscodor spp.

Xylarinase: a novel clot busting enzyme from an endophytic fungus Xylaria curta.

Xylarinase is a bi-functional fibrinolytic metalloprotease isolated from the culture filtrate of endophytic fungus Xylaria curta which is monomeric with a molecular mass of ∼33.76 kDa. The enzyme displayed both plasmin and tissue plasminogen activator like activity under in vitro conditions. It hydrolyses Aα and Bß chains of the fibrinogen. Optimal fibrinolytic activity of xylarinase is observed at 35 °C, pH 8. Ca(2+) stimulated the fibrinolytic activity of xylarinase while Fe(2+) and Zn(2+) inhibited suggesting it to be a metalloprotease. The Km and Vmax values of xylarinase were 240.9 µM and 1.10 U/ml for fibrinogen and 246 µM and 1.22 U/ml for fibrin, respectively. Xylarinase was found to prolong the activated partial thromboplastin time and prothrombin time. The N-terminal sequence of xylarinase (SNGPLPGGVVWAG) did not show any homology with previously known fibrinolytic enzymes. Thus xylarinase is a novel fibrinolytic metalloprotease which could be possibly used as a new clot busting enzyme.

Rapid Discovery and Functional Characterization of Terpene Synthases from Four Endophytic Xylariaceae.

Endophytic fungi are ubiquitous plant endosymbionts that establish complex and poorly understood relationships with their host organisms. Many endophytic fungi are known to produce a wide spectrum of volatile organic compounds (VOCs) with potential energy applications, which have been described as "mycodiesel". Many of these mycodiesel hydrocarbons are terpenes, a chemically diverse class of compounds produced by many plants, fungi, and bacteria. Due to their high energy densities, terpenes, such as pinene and bisabolene, are actively being investigated as potential "drop-in" biofuels for replacing diesel and aviation fuel. In this study, we rapidly discovered and characterized 26 terpene synthases (TPSs) derived from four endophytic fungi known to produce mycodiesel hydrocarbons. The TPS genes were expressed in an E. coli strain harboring a heterologous mevalonate pathway designed to enhance terpene production, and their product profiles were determined using Solid Phase Micro-Extraction (SPME) and GC-MS. Out of the 26 TPS's profiled, 12 TPS's were functional, with the majority of them exhibiting both monoterpene and sesquiterpene synthase activity.

Activity of carbohydrate oxidases as influenced by wheat flour dough components.

The carbohydrate oxidase (COXMn) from Microdochium nivale may well have desired functionalities as a dough and bread improver, similarly to Aspergillus niger glucose oxidase (GOX). COXMn catalyses the oxidation of various monosaccharides as well as maltooligosaccharides for which the best activity is obtained towards the maltooligosaccharides of polymerisation degrees 3 and 4. For the same activity towards glucose under air saturation, we show that COXMn exhibits a similar efficiency towards maltose as GOX towards glucose whatever the oxygen supply. Assays with COXMn show that no competition exists between carbohydrates naturally present in the wheat flour. We show that reaction products (d-glucono-δ-lactone and hydrogen peroxide) and the wheat flour dough component, ferulic acid, have no noticeable specific effect on the COXMn activity. The demonstrated differences in kinetics between COXMn and GOX allow predicting of differences in the functional behaviours of those enzymes during wheat flour dough formation.

A Putative Histone Deacetylase Modulates the Biosynthesis of Pestalotiollide B and Conidiation in Pestalotiopsis microspora.

Fungi of the genus Pestalotiopsis have drawn attention for their capability to produce an array of bioactive secondary metabolites that have potential for drug development. Here, we report the determination of a polyketide derivative compound, pestalotiollide B, in the culture of the saprophytic fungus Pestalotiopsis microspora NK17. Structural information acquired by analyses with a set of spectroscopic and chromatographic techniques suggests that pestalotiollide B has the same skeleton as the penicillide derivatives, dibenzodioxocinones, which are inhibitors of cholesterol ester transfer protein (CETP), and as purpactins A and C', inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). Strain NK17 can make a fairly high yield of pestalotiollide B (i.e., up to 7.22 mg/l) in a constitutive manner in liquid culture. Moreover, we found that a putative histone deacetylase gene, designated as hid1, played a role in the biosynthesis of pestalotiollide B. In the hid1 null mutant, the yield of pestalotiollide B increased approximately 2-fold to 15.90 mg/l. In contrast, deletion of gene hid1 led to a dramatic decrease of conidia production of the fungus. These results suggest that hid1 is a modulator, concerting secondary metabolism and development such as conidiation in P. microspora. Our work may help with the investigation into the biosynthesis of pestalotiollide B and the development for new CETP and ACAT inhibitors.

Identification of the first diphenyl ether gene cluster for pestheic acid biosynthesis in plant endophyte Pestalotiopsis fici.

The diphenyl ether pestheic acid was isolated from the endophytic fungus Pestalotiopsis fici, which is proposed to be the biosynthetic precursor of the unique chloropupukeananes. The pestheic acid biosynthetic gene (pta) cluster was identified in the fungus through genome scanning. Sequence analysis revealed that this gene cluster encodes a nonreducing polyketide synthase, a number of modification enzymes, and three regulators. Gene disruption and intermediate analysis demonstrated that the biosynthesis proceeded through formation of the polyketide backbone, cyclization of a polyketo acid to a benzophenone, chlorination, and formation of the diphenyl ether skeleton through oxidation and hydrolyzation. A dihydrogeodin oxidase gene, ptaE, was essential for diphenyl ether formation, and ptaM encoded a flavin-dependent halogenase catalyzing chlorination in the biosynthesis. Identification of the pta cluster laid the foundation to decipher the genetic and biochemical mechanisms involved in the pathway.

Selective oxidation and N-coupling by purified laccase of xylaria polymorpha MTCC-1100.

The chemical route of oxidation of methyl group to its aldehyde is inconvenient because once a methyl group is attacked, it is likely to be oxidized to the carboxylic acid and it is very difficult to stop the reaction at the aldehyde stage. Fungal laccases can be used for such oxidation reaction and the reaction can be completed sharply within 1-2 hrs. Coupling of amines are another important reactions known forfungal laccases; coupling reactions generally take 3-7 hrs. We have used the purified laccase of molecular weight 63 kDa obtained from the fungal strainXylaria polymorpha MTCC-100 with activity of 1.95 IU/mL for selective oxidation of 2-fluorotoluene, 4-fluorotoluene, and 2-chlorotoluene to 2-fluorobenzaldehyde, 4-fluorobenzaldehyde, and 2-chlorobenzaldehyde, respectively, and syntheses of 3-(3,4-dihydroxyphenyl)-propionic acid derivatives by N-coupling of amines. In each oxidation reactions, ABTS was used as mediator molecule. All the syntheses are ecofriendly and were performed at room temperature.

Potential role of oxidative exoenzymes of the extremophilic fungus Pestalotiopsis palmarum BM-04 in biotransformation of extra-heavy crude oil.

Large amount of drilling waste associated with the expansion of the Orinoco Oil Belt (OOB), the biggest proven reserve of extra-heavy crude oil (EHCO) worldwide, is usually impregnated with EHCO and highly salinized water-based drilling fluids. Oxidative exoenzymes (OE) of the lignin-degrading enzyme system (LDS) of fungi catalyse the oxidation of a wide range of toxic pollutants. However, very little evidences on fungal degradation or biotransformation of EHCO have been reported, which contain high amounts of asphaltenes and its biodegradation rate is very limited. The aims of this work were to study the ability of Pestalotiopsis palmarum BM-04 to synthesize OE, its potential to biotransform EHCO and to survive in extreme environmental conditions. Enzymatic studies of the LDS showed the ability of this fungus to overproduce high amounts of laccase (LACp) in presence of wheat bran or lignin peroxidase (LIPp) with EHCO as sole carbon and energy source (1300 U mgP(-1) in both cases). FT-IR spectroscopy with Attenuated Total Reflectance (ATR) analysis showed the enzymatic oxidation of carbon and sulfur atoms in both maltenes and asphaltenes fractions of biotreated EHCO catalysed by cell-free laccase-enriched OE using wheat bran as inducer. UV-visible spectrophotometry analysis revealed the oxidation of the petroporphyrins in the asphaltenes fraction of biotreated EHCO. Tolerance assays showed the ability of this fungus to grow up to 50,000 p.p.m. of EHCO and 2000 mM of NaCl. These results suggest that P. palmarum BM-04 is a hopeful alternative to be used in remediation processes in extreme environmental conditions of salinity and EHCO contamination, such as the drilling waste from the OOB.

Characterization of salt-adapted secreted lignocellulolytic enzymes from the mangrove fungus Pestalotiopsis sp.

Fungi are important for biomass degradation processes in mangrove forests. Given the presence of sea water in these ecosystems, mangrove fungi are adapted to high salinity. Here we isolate Pestalotiopsis sp. NCi6, a halotolerant and lignocellulolytic mangrove fungus of the order Xylariales. We study its lignocellulolytic enzymes and analyse the effects of salinity on its secretomes. De novo transcriptome sequencing and assembly indicate that this fungus possesses of over 400 putative lignocellulolytic enzymes, including a large fraction involved in lignin degradation. Proteomic analyses of the secretomes suggest that the presence of salt modifies lignocellulolytic enzyme composition, with an increase in the secretion of xylanases and cellulases and a decrease in the production of oxidases. As a result, cellulose and hemicellulose hydrolysis is enhanced but lignin breakdown is reduced. This study highlights the adaptation to salt of mangrove fungi and their potential for biotechnological applications.

Functional expression of a foreign gene in Aspergillus oryzae producing new pyrone compounds.

Fungi from the genus Xylaria produce a wide range of polyketides with diverse structures, which provide important sources for pharmaceutical agents. At least seven polyketide synthase (PKS) genes, including pksmt, were found in Xylaria sp. BCC 1067. The multifunctional enzyme pksmt contains the following catalytic motifs: ß-ketosynthase (KS), acyltransferase (AT), dehydratase (DH), methyltransferase (MT), enoylreductase (ER), ketoreductase (KR), and acyl carrier region (ACP). The presence of multiple domains indicated that pksmt was an iterative type I highly-reduced-type PKS gene. To identify the gene function, pksmt was fused with a gene encoding green fluorescent protein (GFP) and introduced into a surrogate host, Aspergillus oryzae, and expressed under the control of a constitutive gpdA promoter. In the transformant, the pksmt gene was functionally expressed and translated as detected by a green fluorescence signal. This transformant produced two new 2-pyrone compounds, 4-(hydroxymethyl)-5,6-dihydro-pyran-2-one and 5-hydroxy-4-methyl-5,6-dihydro-pyran-2-one, as well as a previously identified 4-methyl-5,6-dihydro-pyran-2-one. Our results suggested that pksmt from Xylaria sp. BCC 1067 represents a family of fungal PKSs that can synthesize 2-pyrone-containing compounds.

A novel aspartic protease with HIV-1 reverse transcriptase inhibitory activity from fresh fruiting bodies of the wild mushroom Xylaria hypoxylon.

A novel aspartic protease with HIV-1 RT inhibitory activity was isolated and characterized from fruiting bodies of the wild mushroom Xylaria hypoxylon. The purification protocol comprised distilled water homogenization and extraction step, three ion exchange chromatographic steps (on DEAE-cellulose, Q-Sepharose, and CM-cellulose in succession), and final purification was by FPLC on Superdex 75. The protease was adsorbed on all the three ion exchangers. It was a monomeric protein with a molecular mass of 43 kDa as estimated by SDS-PAGE and FPLC. Its N-terminal amino acid sequence was HYTELLSQVV, which exhibited no sequence homology to other proteases reported. The activity of the protease was adversely affected by Pepstatin A, indicating that it is an aspartic protease. The protease activity was maximal or nearly so in the pH range 6-8 and in the temperature range 35-60°C. The purified enzyme exhibited HIV-1 RT inhibitory activity with an IC50 value of 8.3 µM, but was devoid of antifungal, ribonuclease, and hemagglutinating activities.

The wood rot ascomycete Xylaria polymorpha produces a novel GH78 glycoside hydrolase that exhibits α-L-rhamnosidase and feruloyl esterase activities and releases hydroxycinnamic acids from lignocelluloses.

Soft rot (type II) fungi belonging to the family Xylariaceae are known to substantially degrade hardwood by means of their poorly understood lignocellulolytic system, which comprises various hydrolases, including feruloyl esterases and laccase. In the present study, several members of the Xylariaceae were found to exhibit high feruloyl esterase activity during growth on lignocellulosic materials such as wheat straw (up to 1,675 mU g(-1)) or beech wood (up to 80 mU g(-1)). Following the ester-cleaving activity toward methyl ferulate, a hydrolase of Xylaria polymorpha was produced in solid-state culture on wheat straw and purified by different steps of anion-exchange and size-exclusion chromatography to apparent homogeneity (specific activity, 2.2 U mg(-1)). The peptide sequence of the purified protein deduced from the gene sequence and verified by de novo peptide sequencing shows high similarity to putative α-L-rhamnosidase sequences belonging to the glycoside hydrolase family 78 (GH78; classified under EC 3.2.1.40). The purified enzyme (98 kDa by SDS-PAGE, 103 kDa by size-exclusion chromatography; pI 3.7) converted diverse glycosides (e.g., α-L-rhamnopyranoside and α-L-arabinofuranoside) but also natural and synthetic esters (e.g., chlorogenic acid, hydroxycinnamic acid glycoside esters, veratric acid esters, or p-nitrophenyl acetate) and released free hydroxycinnamic acids (ferulic and coumaric acid) from arabinoxylan and milled wheat straw. These catalytic properties strongly suggest that X. polymorpha GH78 is a multifunctional enzyme. It is the first fungal enzyme that combines glycosyl hydrolase with esterase activities and may help this soft rot fungus to degrade lignocelluloses.

Investigation of the pH-dependent electron transfer mechanism of ascomycetous class II cellobiose dehydrogenases on electrodes.

Cellobiose dehydrogenase (CDH) is capable of direct electron transfer (DET) on various carbon and thiol-modified gold electrodes. As a result, these systems have been utilized as biocatalyst in biosensors and biofuel cell anodes. Class I CDHs, from basidiomycetous fungi, are highly specific to cellulose or lactose, and DET is only observed at pH values below 5.5. To extend the applicability of CDH-based electrodes, the catalytic properties and the behavior on electrode surfaces of ascomycetous class II CDHs from Chaetomium attrobrunneum, Corynascus thermophilus, Dichomera saubinetii, Hypoxylon haematostroma, Neurospora crassa, and Stachybotrys bisbyi were investigated. We found that class II CDHs have diverse properties but generally show a lower substrate specificity than class I CDHs by converting also glucose and maltose. Intramolecular electron transfer (IET) and DET at neutral and alkaline pH were observed and elucidated by steady-state kinetics, pre-steady-state kinetics, and electrochemical measurements. The CDHs ability to interact with the electron acceptor cytochrome c and to communicate with electrode surfaces through DET at various pH conditions was used to classify the investigated enzymes. In combination with stopped-flow measurements, a model for the kinetics of the pH-dependent IET is developed. The efficient glucose turnover at neutral/alkaline pH makes some of these new CDHs potential candidates for glucose biosensors and biofuel cell anodes.