Purification and characterization of a thermostable α-galactosidase from Thielavia terrestris NRRL 8126 in solid state fermentation.

PubMed

Saad, Rawia R; Fawzi, Eman M

2012-03-01

Several seeds and husks of some plants belonging to leguminosae, Graminae, Compositae and Palmae were evaluated as carbon substrates to produce α-galactosidase (α-Gal) by the thermophilic fungus, Thielavia terrestris NRRL 8126 in solid substrate fermentation. The results showed that Cicer arietinum (chick pea seed) was the best substrate for α-Gal production. The crude enzyme was precipitated by ammonium sulphate (60%) and purified by gel filtration on sephadex G-100 followed by ion exchange chromatography on DEAE-Cellulose. The final purification fold of the enzyme was 30.42. The temperature and pH optima of purified α-Gal from Thielavia terrestris were 70 °C and 6.5, respectively. The enzyme showed high thermal stability at 70 °C and 75 °C and the half-life of the α-Gal at 90 °C was 45 min. Km of the purified enzyme was 1.31 mM. The purified enzyme was inhibited by Ag2+, Hg2+, Zn2+, Ba2+, Mg2+, Mn2+ and Fe2+ at 5 mM and 10 mM. Also, EDTA, sodium arsenate, L-cysteine and iodoacetate inhibited the enzyme activity. On the other hand, Ca2+, Cu2+, K+ and Na+ slightly enhanced the enzyme activity at 5 mM while at 10 mM they caused inhibition. The molecular weight of the α-Gal was estimated to be 82 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for biotechnological and medicinal applications.

Degradation of bisphenol A and acute toxicity reduction by different thermo-tolerant ascomycete strains isolated from arid soils.

PubMed

Mtibaà, Rim; Olicón-Hernández, Dario Rafael; Pozo, Clementina; Nasri, Moncef; Mechichi, Tahar; González, Jesus; Aranda, Elisabet

2018-07-30

Four different laccase-producing strains were isolated from arid soils and used for bisphenol A (BPA) degradation. These strains were identified as Chaetomium strumarium G5I, Thielavia arenaria CH9, Thielavia arenaria HJ22 and Thielavia arenaria SM1(III) by internal transcribed spacer 5.8 S rDNA analysis. Residual BPA was evaluated by HPLC analysis during 48 h of incubation. A complete removal of BPA was observed by the whole cell fungal cultures within different times, depending on each strain. C. strumarium G5I was the most efficient degrader, showing 100% of removal within 8 h of incubation. The degradation of BPA was accompanied by the production of laccase and dye decolorizing peroxidase (DyP) under degradation conditions. The presence of aminobenzotriazole (ABT) as an inhibitor of cytochrome P450s monooxygenases (CYP) demonstrated a slight decrease in BPA removal rate, suggesting the effective contribution of CYP in the conversion. The great involvement of laccase in BPA transformation together with cell-associated enzymes, such as CYP, was supported by the identification of hydroxylated metabolites by ultra-high performance liquid chromatography-mass spectroscopy (UHPLC-MS). The metabolic pathway of BPA transformation was proposed based on the detected metabolites. The acute toxicity of BPA and its products was investigated and showed a significant reduction, except for T. arenaria SM1(III) that did not caused reduction of toxicity (IC 50 < 8%), possibly due to the presence of toxic metabolites. The results of the present study point out the potential application of the isolated ascomycetes in pollutant removal processes, especially C. strumarium G5I as an efficient degrader of BPA. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berka, Randy M.; Grigoriev, Igor V.; Otillar, Robert

2011-10-02

Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanismsmore » in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.« less

Molecular characterization of a thermophilic endo-polygalacturonase from Thielavia arenaria XZ7 with high catalytic efficiency and application potential in the food and feed industries.

PubMed

Tu, Tao; Meng, Kun; Huang, Huoqing; Luo, Huiying; Bai, Yingguo; Ma, Rui; Su, Xiaoyun; Shi, Pengjun; Yang, Peilong; Wang, Yaru; Yao, Bin

2014-12-31

Thermophilic endo-polygalacturonases with high catalytic efficiency are of great interest in the food and feed industries. This study identified an endo-polygalacturonase gene (pg7fn) of glycoside hydrolase family 28 in the thermophilic fungus Thielavia arenaria XZ7. Recombinant PG7fn produced in Pichia pastoris is distinguished from other enzyme counterparts by its high functional temperature (60 °C) and specific activity (34382 ± 351 U/mg toward polygalacturonic acid). The enzyme exhibited good pH stability (pH 3.0-8.0) and resistance to pepsin and trypsin digestion and had a significant effect on disaggregation of soybean meal. Addition of 1 U/g PG7fn increased the pectin bioavailability by 19.33%. The excellent properties described above make PG7fn valuable for applications in the food and feed industries. Furthermore, a comparative study showed that N-glycosylation improved the thermostability and catalytic efficiency of PG7fn.

Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berka, Randy M.; Grigoriev, Igor V.; Otillar, Robert

2011-05-16

Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanismsmore » in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.« less

Comparative metagenomics approaches to characterize the soil fungal communities of western coastal region, Saudi Arabia

PubMed Central

Fuller, Michael P.

2017-01-01

A total of 145007 reads were obtained from pyrosequencing for all the 4 samples. The total count ranged from 11,301,014 (Mecca old road) to 23,503,512 bp (Thuwal). A total of 460 fungal species belonging to 133 genera, 58 families, 33 orders, 13 classes and 4 phyla was identified across the four sites. The most abundant phylum at all four sites was Ascomycota followed by Basidiomycota. Four phyla (Ascomycota—99.31%, Basidiomycota—0.59%, Chytridiomycota—0.04%, Glomeromycota—0.03%) were detected in Khulais. Except for Glomeromycota, all phyla were detected at Mecca old road (Ascomycota—74.26%, Basidiomycota—25.71%, Chytridiomycota—0.01%) and Thuwal (Ascomycota—99.59%, Basidiomycota—0.40%, Chytridiomycota—0.002%); while only Ascomycota—90.98% and Basidiomycota—9.01% were detected in Asfan road. At the class level, Sordariomycetes was predominantly observed at Asfan road—59.88%, Khulais—68.26% and Thuwal—94.84%; while Pezizomycetes was dominant at Mecca old road—56.01%, was absent at Asfan road. Agaricomycetes was present only at Mecca old road—25.73%; while Tremellomycetes—5.77%, Malasseizomycetes—2.13% and Microbotryomycetes—1.10% were found only at Asfan road. The phylogenetic trees revealed that clear genus level differences are visible across all the four sites, with an overall predominance of Thielavia followed by Madurella, Aspergillus, and Gelasinospora. Chaetomium sp., Aspergillus caespitosus and Aspergillus sp. were found in moderate (Mecca old road and Thuwal) to abundant (Asfan road and Khulais) quantities. Thielavia sp., Thielavia hyalocarpa and Madurella sp. are found in moderate quantities at Khulais and Mecca old road, while in abundant levels at Asfan road and Thuwal. Fusarium equisati and F. oxysporum were detected at Thuwal and Khulais. Sordaria araneosa was present at Khulais, while Malasseiza globosa species was detected in moderate quantities across all sites except Khulais. PMID:28934322

Comparative metagenomics approaches to characterize the soil fungal communities of western coastal region, Saudi Arabia.

PubMed

Moussa, Tarek A A; Al-Zahrani, Hassan S; Almaghrabi, Omar A; Abdelmoneim, Tamer S; Fuller, Michael P

2017-01-01

A total of 145007 reads were obtained from pyrosequencing for all the 4 samples. The total count ranged from 11,301,014 (Mecca old road) to 23,503,512 bp (Thuwal). A total of 460 fungal species belonging to 133 genera, 58 families, 33 orders, 13 classes and 4 phyla was identified across the four sites. The most abundant phylum at all four sites was Ascomycota followed by Basidiomycota. Four phyla (Ascomycota-99.31%, Basidiomycota-0.59%, Chytridiomycota-0.04%, Glomeromycota-0.03%) were detected in Khulais. Except for Glomeromycota, all phyla were detected at Mecca old road (Ascomycota-74.26%, Basidiomycota-25.71%, Chytridiomycota-0.01%) and Thuwal (Ascomycota-99.59%, Basidiomycota-0.40%, Chytridiomycota-0.002%); while only Ascomycota-90.98% and Basidiomycota-9.01% were detected in Asfan road. At the class level, Sordariomycetes was predominantly observed at Asfan road-59.88%, Khulais-68.26% and Thuwal-94.84%; while Pezizomycetes was dominant at Mecca old road-56.01%, was absent at Asfan road. Agaricomycetes was present only at Mecca old road-25.73%; while Tremellomycetes-5.77%, Malasseizomycetes-2.13% and Microbotryomycetes-1.10% were found only at Asfan road. The phylogenetic trees revealed that clear genus level differences are visible across all the four sites, with an overall predominance of Thielavia followed by Madurella, Aspergillus, and Gelasinospora. Chaetomium sp., Aspergillus caespitosus and Aspergillus sp. were found in moderate (Mecca old road and Thuwal) to abundant (Asfan road and Khulais) quantities. Thielavia sp., Thielavia hyalocarpa and Madurella sp. are found in moderate quantities at Khulais and Mecca old road, while in abundant levels at Asfan road and Thuwal. Fusarium equisati and F. oxysporum were detected at Thuwal and Khulais. Sordaria araneosa was present at Khulais, while Malasseiza globosa species was detected in moderate quantities across all sites except Khulais.

Influence of surface charge, binding site residues and glycosylation on Thielavia terrestris cutinase biochemical characteristics

PubMed Central

Shirke, Abhijit N.; Basore, Danielle; Holton, Samantha; Su, An; Baugh, Evan; Butterfoss, Glenn L.; Makhatadze, George

2016-01-01

Cutinases are esterases of industrial importance for applications in recycling and surface modification of polyesters. The cutinase from Thielavia terrestris (TtC) is distinct in terms of its ability to retain its stability and activity in acidic pH. Stability and activity in acidic pHs are desirable for esterases as the pH of the reaction tends to go down with the generation of acid. The pH stability and activity are governed by the charged state of the residues involved in catalysis or in substrate binding. In this study, we performed the detailed structural and biochemical characterization of TtC coupled with surface charge analysis to understand its acidic tolerance. The stability of TtC in acidic pH was rationalized by evaluating the contribution of charge interactions to the Gibbs free energy of unfolding at varying pHs. The activity of TtC was found to be limited by substrate binding affinity, which is a function of the surface charge. Additionally, the presence of glycosylation affects the biochemical characteristics of TtC owing to steric interactions with residues involved in substrate binding. PMID:26758295

A family 5 β-mannanase from the thermophilic fungus Thielavia arenaria XZ7 with typical thermophilic enzyme features.

PubMed

Lu, Haiqiang; Zhang, Huitu; Shi, Pengjun; Luo, Huiying; Wang, Yaru; Yang, Peilong; Yao, Bin

2013-09-01

A novel β-mannanase gene, man5XZ7, was cloned from thermophilic fungus Thielavia arenaria XZ7, and successfully expressed in Pichia pastoris. The gene (1,110 bp) encodes a 369-amino acid polypeptide with a molecular mass of approximately 40.8 kDa. The deduced sequence of Man5XZ7 consists of a putative 17-residue signal peptide and a catalytic module belonging to glycoside hydrolase (GH) family 5, and displays 76 % identity with the experimentally verified GH 5 endo-β-1,4-mannanase from Podospora anserina. Recombinant Man5XZ7 was optimally active at 75 °C and pH 5.0 and exhibited high activity at a wide temperature range (>50.0 % activity at 50-85 °C). Moreover, it had good adaptability to acidic to basic pH (>74.1 % activity at pH 4.0-7.0 and 25.6 % even at pH 9.0) and good stability from pH 3.0 to 10.0. These enzymatic properties showed that Man5XZ7 was a new thermophilic and alkali-tolerant β-mannanase. Further amino acid composition analysis indicated that Man5XZ7 has several characteristic features of thermophilic enzymes.

Inhibitory effect of OPC-15161, a component of fungus Thielavia minor, on proliferation and extracellular matrix production of rat cultured hepatic stellate cells.

PubMed

Sugawara, H; Ueno, T; Torimura, T; Inuzuka, S; Tanikawa, K

1998-03-01

A component of fungus Thielavia minor, OPC-15161, has been shown to inhibit the proliferation and extracellular matrix production of extracellular matrix-producing mesangial cells in the kidney in vivo. In this study, we examined the effects of OPC-15161 on the proliferation and extracellular matrix production of rat cultured hepatic stellate cells (HSCs). To determine the effect of OPC-15161 on proliferation of HSCs, the cell number and the uptake of [3H]thymidine were investigated in the presence and absence of interleukin-1beta (IL-1beta). IL-1beta significantly increased the uptake of [3H]thymidine in the HSCs, and the addition of OPC-15161 inhibited the uptake in a dose-dependent manner. The cell number of HSCs was also increased by IL-1beta, which was inhibited by OPC-15161. Production of extracellular matrix by OPC-15161 was studied by the production of [3H]-hydroxyproline in the presence and absence of transforming growth factor-beta1 (TGF-beta1). TGF-beta1 significantly increased the production of [3H]-hydroxyproline in the cells, whereas the addition of OPC-15161 inhibited this effect dose dependently. We also investigated the effects of OPC-15161 on Ca2+ mobilization and measured D-myo-inositol 1,4,5-triphosphate (IP3) in the HSCs. IL-1beta induced the increase of intracellular Ca2+ and IP3 concentrations in the HSCs, which were decreased by OPC-15161. Based on these results, we conclude that OPC-1 5161 inhibited the proliferation and production of hydroxyproline in cultured rat HSCs, and thus, it may have a role in prevention of liver fibrosis in vivo.

Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions.

PubMed

McClendon, Shara D; Batth, Tanveer; Petzold, Christopher J; Adams, Paul D; Simmons, Blake A; Singer, Steven W

2012-07-28

Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents. Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of ionic liquid pretreated switchgrass (Panicum virgatum) revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails. T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for biomass deconstruction, without strain development or genetic modifications. Therefore, T. aurantiacus provides an excellent platform to develop a thermophilic fungal system for enzyme production for the conversion of biomass to biofuels.

Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions

PubMed Central

2012-01-01

Background Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents. Results Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of ionic liquid pretreated switchgrass (Panicum virgatum) revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails. Conclusions T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for biomass deconstruction, without strain development or genetic modifications. Therefore, T. aurantiacus provides an excellent platform to develop a thermophilic fungal system for enzyme production for the conversion of biomass to biofuels. PMID:22839529

A promising strain of Streptomyces sp. with agricultural traits for growth promotion and disease management.

PubMed

Alam, Mansoor; Dharni, Seema; Abdul-Khaliq; Srivastava, Santosh Kumar; Samad, Abdul; Gupta, Mahesh Kumar

2012-08-01

A bacterial strain, Streptomyces sp. CIMAP- A1 was isolated from Geranium rhizosphere and identified by morphological, physiological, biochemical and molecular characters (16S rDNA gene sequence). Phylogenetically, it was found most closely related to S. vinacendrappus, strain NRRL-2363 with 99% sequence similarity. The strain had potential antagonistic activity (in vitro) against wide range of phytopathogenic fungi like Stemphylium sp., Botrytis cinerea, Sclerotinia sclerotiorum, Colletotrichum spp., Curvularia spp., Corynespora cassicola and Thielavia basicola. The extracellular secondary metabolites produced by the strain in the culture filtrates significantly inhibited the spore germination, growth of germ tube of the germinated spores and radial growth of Alternaria alternata, Colletotrichum acutatum, Curvularia andropogonis and Fusarium moniliforme. The extraction of culture filtrate with solvents and purification by following VLC and PTLC methods always yielded a 10th fraction antifungal compound showing activity against wide range of phytopathogenic fungi. The strain was able to produce siderophores and indole-3-acetic acid. The strain was found to enhance the growth and biomass production of Geranium. It increased 11.3% fresh shoot biomass of Geranium and 21.7% essential oil yield.

Cellobiose dehydrogenase of Chaetomium sp. INBI 2-26(-): structural basis of enhanced activity toward glucose at neutral pH.

PubMed

Vasilchenko, Liliya G; Karapetyan, Karen N; Yershevich, Olga P; Ludwig, Roland; Zamocky, Marcel; Peterbauer, Clemens K; Haltrich, Dietmar; Rabinovich, Mikhail L

2011-05-01

Cellobiose dehydrogenase (CDH) is an extracellular fungal flavocytochrome specifically oxidizing cellooligosaccharides and lactose to corresponding (-lactones by a variety of electron acceptors. In contrast to basidiomycetous CDHs, CDHs of ascomycetes also display certain activity toward glucose. The objective of this study was to establish the structural reasons of such an activity of CDH from mesophilic ascomycete Chaetomium sp. INBI 2-26 (ChCDH). The complete amino acid sequence of ChCDH displayed high levels of similarity with the amino acid sequences of CDHs from the thermophilic fungi Thielavia heterotallica and Myriococcum thermophilum. Peptide mass fingerprinting of purified ChCDH provided evidence for the oxidation of methionine residues in the FAD-domain. Comparative homology modeling of the structure of the ChCDH FAD-domain in complex with the transition state analog based on the structure of the same complex of basidiomycetous CDH (1NAA) as template indicated possible structural reasons for the enhanced activity of ascomycetous CDHs toward glucose at neutral pH, which is a prerequisite for application of CDH in a variety of biocompatible biosensors and biofuel cells. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thermophilic growth and enzymatic thermostability are polyphyletic traits within Chaetomiaceae.

PubMed

van den Brink, Joost; Facun, Kryss; de Vries, Michel; Stielow, J Benjamin

2015-12-01

Thermophilic fungi have the potential to produce industrial-relevant thermostable enzymes, in particular for the degradation of plant biomass. Sordariales is one of the few fungal orders containing several thermophilic taxa, of which many have been associated with the production of thermostable enzymes. The evolutionary affiliation of Sordariales fungi, especially between thermophiles and non-thermophilic relatives, is however poorly understood. Phylogenetic analysis within the current study was based on sequence data, derived from a traditional Sanger and highly multiplexed targeted next generation sequencing approach of 45 isolates. The inferred phylogeny and detailed growth analysis rendered the trait 'thermophily' as polyphyletic within Chaetomiaceae (Sordariales, Sordariomycetes), and characteristic to: Myceliophthora spp., Thielavia terrestris, Chaetomium thermophilum, and Mycothermus thermophilus. Compared to mesophiles, the isolates within thermophilic taxa produced enzyme mixtures with the highest thermostability of known cellulase activities. Temperature profiles of the enzyme activities correlated strongly with the optimal growth temperatures of the isolates but not with their phylogenetic relationships. This strong correlation between growth and enzyme characteristics indicated that detailed analysis of growth does give predictive information on enzyme physiology. The variation in growth and enzyme characteristics reveals these fungi as an excellent platform to better understand fungal thermophily and enzyme thermostability. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Oxidoreductive Cellulose Depolymerization by the Enzymes Cellobiose Dehydrogenase and Glycoside Hydrolase 61▿†

PubMed Central

Langston, James A.; Shaghasi, Tarana; Abbate, Eric; Xu, Feng; Vlasenko, Elena; Sweeney, Matt D.

2011-01-01

Several members of the glycoside hydrolase 61 (GH61) family of proteins have recently been shown to dramatically increase the breakdown of lignocellulosic biomass by microbial hydrolytic cellulases. However, purified GH61 proteins have neither demonstrable direct hydrolase activity on various polysaccharide or lignacious components of biomass nor an apparent hydrolase active site. Cellobiose dehydrogenase (CDH) is a secreted flavocytochrome produced by many cellulose-degrading fungi with no well-understood biological function. Here we demonstrate that the binary combination of Thermoascus aurantiacus GH61A (TaGH61A) and Humicola insolens CDH (HiCDH) cleaves cellulose into soluble, oxidized oligosaccharides. TaGH61A-HiCDH activity on cellulose is shown to be nonredundant with the activities of canonical endocellulase and exocellulase enzymes in microcrystalline cellulose cleavage, and while the combination of TaGH61A and HiCDH cleaves highly crystalline bacterial cellulose, it does not cleave soluble cellodextrins. GH61 and CDH proteins are coexpressed and secreted by the thermophilic ascomycete Thielavia terrestris in response to environmental cellulose, and the combined activities of T. terrestris GH61 and T. terrestris CDH are shown to synergize with T. terrestris cellulose hydrolases in the breakdown of cellulose. The action of GH61 and CDH on cellulose may constitute an important, but overlooked, biological oxidoreductive system that functions in microbial lignocellulose degradation and has applications in industrial biomass utilization. PMID:21821740

The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable β-1,4-xylanase with exo- and endo-activity.

PubMed

García-Huante, Yolanda; Cayetano-Cruz, Maribel; Santiago-Hernández, Alejandro; Cano-Ramírez, Claudia; Marsch-Moreno, Rodolfo; Campos, Jorge E; Aguilar-Osorio, Guillermo; Benitez-Cardoza, Claudia G; Trejo-Estrada, Sergio; Hidalgo-Lara, María Eugenia

2017-01-01

A hyperthermophilic and thermostable xylanase of 82 kDa (TtXynA) was purified from the culture supernatant of T. terrestris Co3Bag1, grown on carboxymethyl cellulose (CMC), and characterized biochemically. TtXynA showed optimal xylanolytic activity at pH 5.5 and at 85 °C, and retained more than 90% of its activity at a broad pH range (4.5-10). The enzyme is highly thermostable with a half-life of 23.1 days at 65 °C, and active in the presence of several metal ions. Circular dichroism spectra strongly suggest the enzyme gains secondary structures when temperature increases. TtXynA displayed higher substrate affinity and higher catalytic efficiency towards beechwood xylan than towards birchwood xylan, oat-spelt xylan, and CMC. According to its final hydrolysis products, TtXynA displays endo-/exo-activity, yielded xylobiose, an unknown oligosaccharide containing about five residues of xylose and a small amount of xylose on beechwood xylan. Finally, this report represents the description of the first fungal hyperthermophilic xylanase which is produced by T. terrestris Co3Bag1. Since TtXynA displays relevant biochemical properties, it may be a suitable candidate for biotechnological applications carried out at high temperatures, like the enzymatic pretreatment of plant biomass for the production of bioethanol.

Functional Evolution in Orthologous Cell-encoded RNA-dependent RNA Polymerases.

PubMed

Qian, Xinlei; Hamid, Fursham M; El Sahili, Abbas; Darwis, Dina Amallia; Wong, Yee Hwa; Bhushan, Shashi; Makeyev, Eugene V; Lescar, Julien

2016-04-22

Many eukaryotic organisms encode more than one RNA-dependent RNA polymerase (RdRP) that probably emerged as a result of gene duplication. Such RdRP paralogs often participate in distinct RNA silencing pathways and show characteristic repertoires of enzymatic activities in vitro However, to what extent members of individual paralogous groups can undergo functional changes during speciation remains an open question. We show that orthologs of QDE-1, an RdRP component of the quelling pathway in Neurospora crassa, have rapidly diverged in evolution at the amino acid sequence level. Analyses of purified QDE-1 polymerases from N. crassa (QDE-1(Ncr)) and related fungi, Thielavia terrestris (QDE-1(Tte)) and Myceliophthora thermophila (QDE-1(Mth)), show that all three enzymes can synthesize RNA, but the precise modes of their action differ considerably. Unlike their QDE-1(Ncr) counterpart favoring processive RNA synthesis, QDE-1(Tte) and QDE-1(Mth) produce predominantly short RNA copies via primer-independent initiation. Surprisingly, a 3.19 Å resolution crystal structure of QDE-1(Tte) reveals a quasisymmetric dimer similar to QDE-1(Ncr) Further electron microscopy analyses confirm that QDE-1(Tte) occurs as a dimer in solution and retains this status upon interaction with a template. We conclude that divergence of orthologous RdRPs can result in functional innovation while retaining overall protein fold and quaternary structure. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional Evolution in Orthologous Cell-encoded RNA-dependent RNA Polymerases*

PubMed Central

Qian, Xinlei; Hamid, Fursham M.; El Sahili, Abbas; Darwis, Dina Amallia; Wong, Yee Hwa; Bhushan, Shashi; Makeyev, Eugene V.; Lescar, Julien

2016-01-01

Many eukaryotic organisms encode more than one RNA-dependent RNA polymerase (RdRP) that probably emerged as a result of gene duplication. Such RdRP paralogs often participate in distinct RNA silencing pathways and show characteristic repertoires of enzymatic activities in vitro. However, to what extent members of individual paralogous groups can undergo functional changes during speciation remains an open question. We show that orthologs of QDE-1, an RdRP component of the quelling pathway in Neurospora crassa, have rapidly diverged in evolution at the amino acid sequence level. Analyses of purified QDE-1 polymerases from N. crassa (QDE-1Ncr) and related fungi, Thielavia terrestris (QDE-1Tte) and Myceliophthora thermophila (QDE-1Mth), show that all three enzymes can synthesize RNA, but the precise modes of their action differ considerably. Unlike their QDE-1Ncr counterpart favoring processive RNA synthesis, QDE-1Tte and QDE-1Mth produce predominantly short RNA copies via primer-independent initiation. Surprisingly, a 3.19 Å resolution crystal structure of QDE-1Tte reveals a quasisymmetric dimer similar to QDE-1Ncr. Further electron microscopy analyses confirm that QDE-1Tte occurs as a dimer in solution and retains this status upon interaction with a template. We conclude that divergence of orthologous RdRPs can result in functional innovation while retaining overall protein fold and quaternary structure. PMID:26907693

Phylogenetic findings suggest possible new habitat and routes of infection of human eumyctoma.

PubMed

de Hoog, G Sybren; Ahmed, Sarah A; Najafzadeh, Mohammad J; Sutton, Deanna A; Keisari, Maryam Saradeghi; Fahal, Ahmed H; Eberhardt, Ursala; Verkleij, Gerard J; Xin, Lian; Stielow, Benjamin; van de Sande, Wendy W J

2013-01-01

Eumycetoma is a traumatic fungal infection in tropical and subtropical areas that may lead to severe disability. Madurella mycetomatis is one of the prevalent etiologic agents in arid Northeastern Africa. The source of infection has not been clarified. Subcutaneous inoculation from plant thorns has been hypothesized, but attempts to detect the fungus in relevant material have remained unsuccessful. The present study aims to find clues to reveal the natural habitat of Madurella species using a phylogenetic approach, i.e. by comparison of neighboring taxa with known ecology. Four species of Madurella were included in a large data set of species of Chaetomium, Chaetomidium, Thielavia, and Papulaspora (n = 128) using sequences of the universal fungal barcode gene rDNA ITS and the partial LSU gene sequence. Our study demonstrates that Madurella species are nested within the Chaetomiaceae, a family of fungi that mainly inhabit animal dung, enriched soil, and indoor environments. We hypothesize that cattle dung, ubiquitously present in rural East Africa, plays a significant role in the ecology of Madurella. If cow dung is an essential factor in inoculation by Madurella, preventative measures may involve the use of appropriate footwear in addition to restructuring of villages to reduce the frequency of contact with etiologic agents of mycetoma. On the other hand, the Chaetomiaceae possess a hidden clinical potential which needs to be explored.