Zumrut B. Ogel

Submerged cultivation of scytalidium thermophilum on complex lignocellulosic biomass for endoglucanase production.

Scytalidium thermophilum endoglucanase production was analyzed on lignocellulosic biomass in submerged cultures at 45 degrees C and 155 rpm for 8 days. Endoglucanase, adsorbability of endoglucanase onto avicel, as well as exoglucanase, and filter paper activities were determined and compared with those on microcrystalline cellulose (avicel) as the main source of carbon. Lentil bran and sunflower seed bagasse yielded c. 1.5 fold more endoglucanase and avicel-adsorbable endoglucanase activity than avicel, and activities on grass clippings were similar. Grass clippings yielded the highest percentage of avicel-adsorbable endoglucanase among all lignocellulosic substrates tested. By the time when endoglucanase activities reached maximal levels, exoglucanase activities on lentil bran, sunflower seed bagasse and grass clippings were c. 1.5-3 fold lower than those on avicel, although a significant difference in filter paper activities was not observed. On lignocellulosic biomass, maximum levels of endoglucanase activity were reached within 3-4 days, and within 6-7 days on avicel.

Cloning, Expression and Characterization of Endo-β-1,4-Mannanase from Aspergillus fumigatus in Aspergillus sojae and Pichia pastoris

To be utilized in biomass conversion, including ethanol production and galactosylated oligosaccharide synthesis, namely prebiotics, the gene of extracellular endo‐β‐1,4‐mannanase (EC 3.2.1.78) of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was expressed first in Aspergillus sojae and then in Pichia pastoris under the control of the glyceraldehyde triphosphate dehydrogenase (gpdA) and the alcohol oxidase (AOX1) promoters, respectively. The highest production of mannanase (352 U mL−1) in A. sojae was observed after 6 days of cultivation. In P. pastoris, the highest mannanase production was observed 10 h after induction with methanol (61 U mL−1). The fold increase in mannanase production was estimated as ∼12‐fold and ∼2‐fold in A. sojae and P. pastoris, respectively, when compared with A. fumigatus. Both recombinant enzymes showed molecular mass of about 60 kDa and similar specific activities (∼350 U mg−1 protein). Temperature optima were at 60°C and 45°C, and maximum activity was at pH 4.5 and 5.2 for A. sojae and P. pastoris, respectively. The enzyme from P. pastoris was more stable retaining most of the activity up to 50°C, whereas the enzyme from A. sojae rapidly lost activity above 40°C.

Cellulose-triggered sporulation in the galactose oxidase-producing fungus Cladobotryum (Dactylium) dendroides NRRL 2903 and its re-identification as a species of Fusarium

The production of extracellular galactose oxidase is limited to a few fungal species, including the important plant pathogens Fusarium graminearum and F. moniliforme . The best-studied enzyme is the one produced by the mycoparasitic fungus Cladobotryum (Dactylium) dendroides NRRL 2903. The NRRL 2903 strain was first mis-identified as Polyporus circinatus and later re-determined as Dactylium dendroides , although sporulation was never observed and the fungus was regarded as sterile. Upon growth at 25 °C, 50 rpm, in liquid medium containing 2% cellulose as the sole carbon source, and in the presence of 0·5–0·75% yeast extract, conidial production was induced in NRRL 2903, which was re-identified as Fusarium sp. The only other known commercial strain of Cladobotryum (Dactylium) dendroides able to produce galactose oxidase, ATCC 46032, also produced fusiform conidia upon growth in cellulose-containing medium, and was shown to be genetically identical to the NRRL 2903 strain. Genetic comparison with six different representative strains of Cladobotryum dendroides (teleomorph: Hypomyces rosellus ), and four strains of the closely related Hypomyces aurantius , based on the analysis of the presence or absence of a homologous galactose oxidase gene ( gaoA ), RAPD-PCR and RFLP analysis, confirm the distinct nature of the NRRL 2903 strain and Cladobotryum dendroides . Despite the resemblance of NRRL 2903 conidia and conidiophores to those of Fusarium chlamydosporum genetic comparison, with three different strains, suggests NRRL 2903 cannot be re-identified as F. chlamydosporum . Two of the strains, however, contain a region in their genome that is highly homologous to the galactose oxidase gene ( gaoA ), and one strain exhibits extracellular galactose oxidase activity but only partial homology to the gaoA gene of NRRL 2903.

Avicel-adsorbable endoglucanase production by the thermophilic fungus Scytalidium thermophilum type culture Torula thermophila.

Scytalidium thermophilum type culture Torula thermophila was isolated from mushroom compost and the total cellulase, endoglucanase, Avicel-adsorbable endoglucanase activities, as well as the fungal biomass generation and cellulose utilisation were analyzed in shake flask cultures with Avicel (microcrystalline cellulose) as the carbon source. Results were compared with an industrial strain of Scytalidium thermophilum type culture Humicola insolens. The pH and temperature optima for endoglucanase activities during enzyme assays were also analyzed for both organisms and determined to be pH 6.0 and 65 degrees C for type culture Torula thermophila, and pH 6.5 and 60 degrees C for type culture Humicola insolens. Analysis of the effect of growth temperature showed that type culture T. thermophila can grow and produce cellulases in the range of 35 to 55 degrees C although 40 to 50 degrees C seemed to favor growth and cellulase production. Although 45 degrees C was found optimal for fungal growth, both the specific endoglucanase and Avicel-adsorbable endoglucanase activities (U/mg protein) as well as the percentage of Avicel-adsorbable endoglucanase activity reached maxima at 50 degrees C and were higher as compared to type culture H. insolens. Results indicate that type culture T. thermophila, with further optimisations, is of potential use in the industrial production of cellulases.

Analysis of cellulase and polyphenol oxidase production by southern pine beetle associated fungi

In this study, the production of extracellular enzymes by fungi associated with southern pine beetle was investigated for the first time. Cellulase and polyphenol oxidase production were analyzed for three beetle associated fungi. Only the mutualistic symbiont Entomocorticium sp. A was found to produce cellulases and polyphenol oxidase. In time course analyses of cellulase production in batch cultures, Entomocorticium sp. A showed maximum activity of 0.109 U/ml and 0.141 U/ml for total cellulase and endoglucanase activity respectively. Polyphenol oxidase production was simultaneous with fungal growth. Characterization of polyphenol oxidase by activity staining suggests that the enzyme is a tyrosinase/catechol oxidase. Enzyme assays in the presence of polyphenol oxidase inhibitors support the results of the activity staining.

Oxidation of phenolic compounds by the bifunctional catalase–phenol oxidase (CATPO) from Scytalidium thermophilum

The thermophilic fungus Scytalidium thermophilum produces a novel bifunctional catalase with an additional phenol oxidase activity (CATPO); however, its phenol oxidation spectrum is not known. Here, 14 phenolic compounds were selected as substrates, among which (+)-catechin, catechol, caffeic acid, and chlorogenic acid yielded distinct oxidation products examined by reversed-phase HPLC chromatography method. Characterization of the products by LC-ESI/MS and UV–vis spectroscopy suggests the formation of dimers of dehydrocatechin type B (hydrophilic) and type A (hydrophobic), as well as oligomers, namely, a trimer and tetramer from (+)-catechin, the formation of a dimer and oligomer of catechol, a dimer from caffeic acid with a caffeicin-like structure, as well as trimeric and tetrameric derivatives, and a single major product from chlorogenic acid suggested to be a dimer. Based on the results, CATPO oxidizes phenolic compounds ranging from simple phenols to polyphenols but all having an ortho-diphenolic structure in common. The enzyme also appears to have stereoselectivity due to the oxidation of (+)-catechin, but not that of epicatechin. It is suggested that CATPO may contribute to the antioxidant mechanism of the fungus and may be of value for future food and biotechnology applications where such a bifunctional activity would be desirable.

Optimization of culture conditions for Aspergillus sojae expressing an Aspergillus fumigatus α-galactosidase

Using Response Surface Methodology, carbon and nitrogen sources and agitation speed for cultivation of Aspergillus sojae expressing the α-galactosidase gene, aglB of Aspergillus fumigatus IMI 385708 were optimized. Compared to cultivation in modified YpSs medium, cultivation in 250-mL Erlenmeyer flasks agitated at 276 rpm and containing 100 mL of optimized medium consisting of 10.5% molasses (w/v) and 1.3% NH(4)NO(3) (w/v), 0.1% K(2)HPO(4), and 0.005% MgSO(4)·7H(2)O achieved a 4-fold increase in α-galactosidase production (10.4 U/mL). These results suggest the feasibility of industrial large scale production of an α-galactosidase known to be valuable in galactomannan modification.

Pattern analysis for the prediction of fungal pro-peptide cleavage sites

Support vector machines (SVMs) have many applications in investigating biological data from gene expression arrays to understanding EEG signals of sleep stages. In this paper, we have developed an application that will support the prediction of the pro-peptide cleavage site of fungal extracellular proteins which display mostly a monobasic or dibasic processing site. Many of the secretory proteins and peptides are synthesized as inactive precursors and they become active after post-translational processing. A collection of fungal pro-protein sequences are used as a training data set. A specifically designed kernel is expressed as an application of the well-known Gaussian kernel via feature spaces defined for our problem. Rather than fixing the kernel parameters with cross validation or other methods, we introduce a novel approach that simultaneously performs model selection together with the test of accuracy and testing confidence levels. This leads us to higher accuracy at significantly reduced training times. The results of the server ProP1.0 which predicts pro-peptide cleavage sites are compared with the results of this study. A similar mathematical approach may be adapted to pro-peptide cleavage prediction in other eukaryotes.

Crystallization and preliminary X-ray analysis of a bifunctional catalase-phenol oxidase from Scytalidium thermophilum.

Catalase-phenol oxidase from Scytalidium thermophilum is a bifunctional enzyme: its major activity is the catalase-mediated decomposition of hydrogen peroxide, but it also catalyzes phenol oxidation. To understand the structural basis of this dual functionality, the enzyme, which has been shown to be a tetramer in solution, has been purified by anion-exchange and gel-filtration chromatography and has been crystallized using the hanging-drop vapour-diffusion technique. Streak-seeding was used to obtain larger crystals suitable for X-ray analysis. Diffraction data were collected to 2.8 A resolution at the Daresbury Synchrotron Radiation Source. The crystals belonged to space group P2(1) and contained one tetramer per asymmetric unit.

Structure, Recombinant Expression and Mutagenesis Studies of the Catalase with Oxidase Activity from Scytalidium Thermophilum

Scytalidium thermophilum produces a catalase with phenol oxidase activity (CATPO) that catalyses the decomposition of hydrogen peroxide into oxygen and water and also oxidizes various phenolic compounds. A codon-optimized catpo gene was cloned and expressed in Escherichia coli. The crystal structures of native and recombinant S. thermophilum CATPO and two variants, H82N and V123F, were determined at resolutions of 2.7, 1.4, 1.5 and 1.9 Å, respectively. The structure of CATPO reveals a homotetramer with 698 residues per subunit and with strong structural similarity to Penicillium vitale catalase. The haem component is cis-hydroxychlorin γ-spirolactone, which is rotated 180° with respect to small-subunit catalases. The haem-binding pocket contains two highly conserved water molecules on the distal side. The H82N mutation resulted in conversion of the native d-type haem to a b-type haem. Kinetic studies of the H82N and V123F mutants indicate that both activities are likely to be associated with the haem centre and suggest that the secondary oxidase activity may be a general feature of catalases in the absence of hydrogen peroxide.