Chaetomium Thermophilum Research Articles

Nuclear Pore Structure: Warming up the Core

Structural determination of the nuclear pore complex has been limited by the complexity and size of this cellular megalith. By taking advantage of exceptionally stable nucleoporins from the thermophilic fungus Chaetomium thermophilum, Amlacher et al. (2011) provide new insight into a core element of the nuclear pore scaffold.

Insight into Structure and Assembly of the Nuclear Pore Complex by Utilizing the Genome of a Eukaryotic Thermophile

Despite decades of research, the structure and assembly of the nuclear pore complex (NPC), which is composed of ∼30 nucleoporins (Nups), remain elusive. Here, we report the genome of the thermophilic fungus Chaetomium thermophilum (ct) and identify the complete repertoire of Nups therein. The thermophilic proteins show improved properties for structural and biochemical studies compared to their mesophilic counterparts, and purified ctNups enabled the reconstitution of the inner pore ring module that spans the width of the NPC from the anchoring membrane to the central transport channel. This module is composed of two large Nups, Nup192 and Nup170, which are flexibly bridged by short linear motifs made up of linker Nups, Nic96 and Nup53. This assembly illustrates how Nup interactions can generate structural plasticity within the NPC scaffold. Our findings therefore demonstrate the utility of the genome of a thermophilic eukaryote for studying complex molecular machines.

Expression of a novel thermostable Cu, Zn-superoxide dismutase from Chaetomium thermophilum in Pichia pastoris and its antioxidant properties

A new superoxide dismutase (SOD) gene from the thermophilic fungus Chaetomium thermophilum (Ctsod) was cloned and expressed in Pichia pastoris and its gene product was characterized. The specific activity of the purified CtSOD was 2,170U/mg protein. The enzyme was inactivated by KCN and H(2)O(2) but not by NaN(3), confirming that it belonged to the type of Cu, ZnSOD. The amino acid residues involved in coordinating copper and zinc were conserved. The recombinant CtSOD exhibited optimum activity at pH 6.5 and 60°C. The enzyme retained 65% of the maximum activity at 70°C for 60min and the half-life was 22 and 7min at 80 and 90°C, respectively. The recombinant yeast exhibited higher stress resistance than the control yeast cells to salt and superoxide-generating agents, such as paraquat and menadione.

Expression, purification and crystallization of Chaetomium thermophilum Cu,Zn superoxide dismutase.

Cu,Zn superoxide dismutase (Cu,ZnSOD) from the thermophilic fungus Chaetomium thermophilum was expressed in Pichia pastoris and purified. Crystals were grown in over 120 conditions but only those produced with 1.4 M sodium potassium phosphate pH 8.2 as precipitant were suitable for structural studies. Data were collected to 1.9 A resolution at 100 K from a single crystal using a synchrotron-radiation source. The crystals belonged to space group P6(1)/P6(5), with unit-cell parameters a=90.2, c=314.5 A and eight molecules in the asymmetric unit. Elucidation of the crystal structure will provide insights into the active site of the enzyme and a better understanding of the structure-activity relationship, assembly and thermal stability of Cu,ZnSODs.

Two novel thermostable chitinase genes from thermophilic fungi: Cloning, expression and characterization

Two chitinase genes, Tachit1 from Thermoascus aurantiacus var. levisporus and Ctchit1 from Chaetomium thermophilum were isolated. Tachit1 and Ctchit1 encode putative single-domain proteins (TaCHIT1 and CtCHIT1) of 399 and 402 amino acid residues, respectively. The catalytic domains of TaCHIT1 and CtCHIT1 are similar to those of other fungal chitinases in family 18 of glycosyl hydrolases. TaCHIT1 and CtCHIT1 have a molecular weight of about 48.4 and 47.3 kDa, respectively when produced in recombinant Pichia pastoris. The enzymes exhibited optimum catalytic activity at pH 8.0 and 50 °C for TaCHIT1 and at pH 5.5 and 60 °C for CtCHIT1. TaCHIT1 retained 95.3% of its activity after 60 min at 50 °C. CtCHIT1 was stable at 50 °C and retained 96.7% of its activity after 60 min incubation at 60 °C. The TaCHIT1 and CtCHIT1 produced Glc-NAc2 as the major product, when colloidal chitin was used as the substrate. The enzyme could not hydrolyze pNp-(GlcNAc), but hydrolyzed colloidal chitin, powdery chitin and chitosan. These features make these proteins potentially useful for applications requiring chitin hydrolysis at elevated temperatures.

Heterologous expression of a gene for thermostable xylanase from Chaetomium thermophilum in Pichia pastoris GS115

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml(-1) while that of recombinant without intron (xyn669) was 1.26 U ml(-1) after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5-8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.

Production and characterization of endo-β-1,4- glucanase from thermophilic fungus

Chaetomium thermophile produces substantial extra-cellular cellulase on several cellulosic substrates (wheat straw, wheat bran and corn straw etc.) including simple sugars. 2% (w/v) wheat straw, wheat bran and corn straw were used as carbon source and urea was used as a nitrogen source. Experiment was conducted in a 20-l stirred tank reactor under the following conditions: pH 5.0, stirring speed 300 rpm, temperature 45 o C and aeration 50% (DO). The samples were withdrawn after every 12 h regularly and enzyme activity was assayed. Endo-β-1,4-glucanase activities on wheat straw, wheat bran and corn straw were 2.51, 1.62 and 5.10 U/ml, respectively and extra-cellular protein contents were 1.48, 1.17 and 2.88 mg/ml, respectively. Specific activity of enzyme after ammonium sulphate precipitation and Fast Protein Liquid Chromatography (FPLC) was 2.1 and 3.3 U/mg of protein, respectively. SDS-PAGE showed only a single band at 53 kDa. The optimum temperature and pH of the CMCase were 60 o C and from 6.0 - 7.5, respectively.

Cloning, expression and characterization of the serine protease gene fromChaetomium thermophilum

Microbial proteases play an essential role in scientific research and commercial applications. This study is to clone, sequence, and express a thermostable protease gene from the thermophilic fungi Chaetomium thermophilum and to generate yeast strains expressing C. thermophilum protease suitable for industrial applications. Degenerate primers were designed based on the conserved domain of other identified serine proteases and cDNA fragment of C. thermophilum gene pro was obtained through reverse transcriptase-polymerase chain reaction (RT-PCR). The full-length cDNA of 2007 bp was generated using RACE amplification. The cDNA contains an open reading frame of 1596 bp encoding 532 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with the catalytic domains of the subtilisin serine proteases. The C. thermophilum gene pro was expressed in Escherichia coli BL21 (DE3) and Pichia pastoris, respectively and soluble protein was obtained in P. pastoris. The expressed protease was secreted into the culture medium by the yeast in a functional active form and the purified recombinant protease exhibits optimum catalytic activity at pH 8.0 and 60 degrees C. The enzyme is stable at 60 degrees C. The integration of gene pro into P. pastoris genome is stable after 10 generations and the yeast transformants showed a consistent protease expression. Gene pro encoding a serine protease from C. thermophilum was cloned, sequenced, and overexpressed successfully in P. pastoris. The expressed protease was purified and the properties of the recombinant protease are characterized. Chaetomium thermophilum is a soil-borne thermophilic fungus and the protease cloned from it is stable in a high temperature and a wide rage of pH. The overexpression of the enzyme in a mesophilic micro-organism offers a potential value for scientific research and commercial applications.

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases

As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH-7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate-limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate-binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4-10 degrees C) and more active (two- to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45 degrees C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70 degrees C, however, was the 2-module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three-dimensional homology models of these enzymes.

Purification and characterization of a thermostable MnSOD from the thermophilic fungus Chaetomium thermophilum

A thermostable superoxide dismutase (SOD) from the culture supernatant of a thermophilic fungus Chaetomium thermophilum strain CT2 was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on deae-sepharose, phenyl-sepharose hydrophobic interaction chromatography. The pure SOD had a specific activity of 115.77 U/mg of protein and was purified 7.49-fold, with a yield of 14.4%. The molecular mass of a single band of the enzyme was estimated to be 23.5 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 94.4 kDa, indicating that this enzyme was composed of four identical subunits of 23.5 kDa each. The SOD was found to be inhibited by NaN3, but not by KCN and H2O2. Atomic absorption spectrophotometric analysis showed that the content of Mn was 2.05 μg/mg of protein and Fe was not detected in the purified enzyme. These results suggested that the SOD in C. thermophilum was the manganese superoxide dismutase type. n-terminal amino acid sequencing (10 residues) was kx (x is uncertain) tlpdlkyd. The n-terminal amino acid sequencing homologies to other MnSod also indicated that it was a manganese-containing superoxide dismutase. The SOD exhibited maximal activity at pH 7.5 and optimum temperature at 60 C. It was thermostable at 50 and 60 C and retained 60% activity after 60 min at 70 C. The half-life of the SOD at 80 C was approximately 25 min and even retained 20% activity after 30 min at 90 C.

Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris

Chaetomium thermophilum is a soil-borne thermophilic fungus whose molecular biology is poorly understood. Only a few genes have been cloned from the Chaetomium genus. This study attempted to clone, to sequence and to express a thermostable glucoamylase gene of C. thermophilum. First strand cDNA was prepared from total RNA isolated from C. thermophilum and the glucoamylase gene amplified by using PCR. Degenerate primers based on the N-terminal sequences of the purified glucoamylase according to our previous works and a cDNA fragment encoding the glucoamylase gene was obtained through RT-PCR. Using RACE-PCR, full-length cDNA of glucoamylase gene was cloned from C. thermophilum. The full-length cDNA of the glucoamylase was 2016 bp and contained a 1797-bp open reading frame encoding a protein glucoamylase precursor of 599 amino acid residues. The amino-acid sequence from 31 to 45 corresponded to the N-terminal sequence of the purified protein. The first 30 amino acids were presumed to be a signal peptide. The alignment results of the putative amino acid sequence showed the catalytic domain of the glucoamylase was high homology with the catalytic domains of the other glucoamylases. The C. thermophilum glucoamylase gene was expressed in Pichia pastoris, and the glucoamylase was secreted into the culture medium by the yeast in a functionally active form. The recombinant glucoamylase purified was a glycoprotein with a size of about 66 kDa, and exhibited optimum catalytic activity at pH 4.5-5.0 and 65 degrees C. The enzyme was stable at 60 degrees C, the enzyme activity kept 80% after 60 min incubation at 70 degrees C. The half-life was 40 and 10 min under incubation at 80 and 90 degrees C respectively. A new thermostable glucoamylase gene of C. thermophilum was cloned, sequenced, overexpressed successfully in P. pastoris. Because of its thermostability and overexpression, this glucoamylase enzyme offers an interesting potential in saccharification steps in both starch enzymatic conversion and in alcohol production.

Production in Trichoderma reesei of three xylanases from Chaetomium thermophilum: a recombinant thermoxylanase for biobleaching of kraft pulp

Three endoxylanase genes were cloned from the thermophilic fungus Chaetomium thermophilum CBS 730.95. All genes contained the typical consensus sequence of family 11 glycoside hydrolases. Genomic copies of Ct xyn11A, Ct xyn11B, and Ct xyn11C were expressed in the filamentous fungus T. reesei under the control of the strong T. reesei cel7A (cellobiohydrolase 1, cbh1) promoter. The molecular masses of the Ct Xyn11A, Ct Xyn11B, and Ct Xyn11C proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were 27, 23, and 22 kDa, respectively. Ct Xyn11A was produced almost as efficiently as the homologous xylanase II from a corresponding single-copy transformant strain. Ct Xyn11B production level was approximately half of that of Ct Xyn11A. The amount of Ct Xyn11C was remarkably lower. Ct Xyn11A had the highest temperature optimum and stability of the recombinant xylanases and the highest activity at acid-neutral pH (pH 5-7). It was the most suitable for industrial bleaching of kraft pulp at high temperature.

Purification and partial characterization of β-1,3-glucanase from Chaetomium thermophilum

A thermostable extracellular β-1,3-glucanase from Chaetomium thermophilum was purified to homogeneity by fractional ammonium sulfate precipitation, Pheny1-Sepharose hydrophobic interaction chromatography, ion exchange chromatography on DEAE-Sepharose and gel filtration on Sephacryl S-100. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 76.3 kDa. The enzyme exhibited optimum catalytic activity at pH 6.0 and 60 °C. It was thermostable at 50 °C, and retained 90% activity after 60 min at 60 °C. The half-life at 65 °C, 70 °C and 80 °C was 55 min, 21.5 min, and 5 min, respectively. The N-terminal amino acid sequence (8 residues) of the enzyme was HWLGDIPH. The HPLC analysis showed that the only enzymatic product formed from laminarin by the purified β-1,3-glucanase was glucose, indicating that the enzyme is an exo-β-1,3-glucanase (EC 3.2.1.58).

Optimization of xylanase production by Chaetomium thermophilum in wheat straw using response surface methodology

A 3 2 central composite experimental design was performed with the aim of optimizing xylanase production by Chaetomium thermophilum IMI 291753 grown on wheat straw in submerged cultures. Various carbon and nitrogen sources were consecutively optimized, and wheat straw and sodium nitrate were the selected substrates to test the effect of two variables on xylanase production. A second-order quadratic model and a response surface method showed that the optimum conditions for xylanase production were 3.9% (w/v) wheat straw and 0.7% (w/v) sodium nitrate.

Identification and characterization of diverse xylanases from thermophilic and thermotolerant fungi

Thirteen fungal isolates included in this study expressed multiple xylanase isoforms as observed by xylan zymograms of polyacrylamide gel electrophoresis (PAGE) and isoelectrofocussing (IEF) fractionated proteins. Eighty-three xylanases produced by these thermophilic and thermotolerant strains were detected using the IEF profiling technique. Xylanases identified on the basis of their isoelectric points (pI) were functionally diverse and exhibited differential catalytic activities against various xylan types (birch wood xylan, larch wood xylan, oat spelt xylan, rye arabino xylan and wheat arabino xylan) as well as debranched arabinan. Thermophilic isolates, Chaetomium thermophilum , Humicola insolens , Melanocarpus sp., Malbranchea sp. and Thermoascus aurantiacus , were found to produce alkaline active xylanases that showed a bleach boosting effect on Decker pulp resulting in increased brightness (1.60-2.04 ISO units).

Efficiency of New Fungal Cellulase Systems in Boosting Enzymatic Degradation of Barley Straw Lignocellulose

This study examined the cellulytic effects on steam-pretreated barley straw of cellulose-degrading enzyme systems from the five thermophilic fungi Chaetomium thermophilum, Thielavia terrestris, Thermoascus aurantiacus, Corynascus thermophilus, and Myceliophthora thermophila and from the mesophile Penicillum funiculosum. The catalytic glucose release was compared after treatments with each of the crude enzyme systems when added to a benchmark blend of a commercial cellulase product, Celluclast, derived from Trichoderma reesei and a beta-glucosidase, Novozym 188, from Aspergillus niger. The enzymatic treatments were evaluated in an experimental design template comprising a span of pH (3.5-6.5) and temperature (35-65 degrees C) reaction combinations. The addition to Celluclast + Novozym 188 of low dosages of the crude enzyme systems, corresponding to 10 wt % of the total enzyme protein load, increased the catalytic glucose yields significantly as compared to those obtained with the benchmark Celluclast + Novozyme 188 blend. A comparison of glucose yields obtained on steam-pretreated barley straw and microcrystalline cellulose, Avicel, indicated that the yield improvements were mainly due to the presence of highly active endoglucanase activity/activities in the experimental enzyme preparations. The data demonstrated the feasibility of boosting the widely studied T. reeseicellulase enzyme system with additional enzymatic activity to achieve faster lignocellulose degradation. We conclude that this supplementation strategy appears feasible as a first step in identifying truly promising fungal enzyme sources for fast development of improved, commercially viable, enzyme preparations for lignocellulose degradation.

A novel α-glucosidase from Chaetomium thermophilum var. coprophilum that converts maltose into trehalose: Purification and partial characterisation of the enzyme

A constitutive extracellular α-glucosidase from Chaetomium thermophilum var. coprophilum was purified and characterised. The enzyme exhibited a carbohydrate content of 14% and an apparent molecular mass of 107 kDa, estimated by gel filtration. Analysis by SDS-PAGE revealed two polypeptide bands of 22 and 26 kDa, suggesting that the enzyme was constituted of two subunits of 22 kDa and two subunits of 26 kDa. Optima of pH and temperature were 7.0 and 60 °C, respectively. The enzyme hydrolysed maltose, amylose, starch, maltooligosaccharides and isomaltose, in order of preference. The enzyme exhibited transglycosylation activity producing trehalose and maltooligosaccharides from 5% maltose. At early stages of the transglycosylation reaction trehalose was the predominant product; at later stages maltooligosaccharides were also accumulated. Trehalose inhibited both glycosyl hydrolase and transglycosylase activities. To our knowledge this is the first report of a α-glucosidase from a filamentous fungus that in addition to glycosyl hydrolase activity produces trehalose by transglycosylation.

Purification and Characterization of a Xylanase Produced by Chaetomium thermophile NIBGE

Xylanase was produced by growing Chaetomium thermophile NIBGE in a submerged liquid culture using wheat straw and urea as carbon and nitrogen sources respectively. The xylanase was purified to electrophoretic homogeneity after ammonium sulphate precipitation, anion exchange chromatography by FPLC and gel filtration. The molecular mass of this xylanase BII was 50 kDa. The pH and temperature optima were 6.5 and 70 °C respectively. The xylanase BII showed reasonable stability at high pH and 65 °C temperature. Some metal ions and EDTA caused little inhibition at low concentrations but complete inhibition was observed at concentrations higher than 2 mM. The Km and Vmax values with oat spelt xylan as the substrate were found to be 12.5 mg/ml and 83.3 IU/mg protein, respectively. Liberation of reducing sugars from commercial paper pulp samples suggest the feasibility of a biopulping process using this xylanase.

Determination of thioxylo‐oligosaccharide binding to family 11 xylanases using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and X‐ray crystallography

Noncovalent binding of thioxylo-oligosaccharide inhibitors, methyl 4-thio-alpha-xylobioside (S-Xyl2-Me), methyl 4,4II-dithio-alpha-xylotrioside (S-Xyl3-Me), methyl 4,4II,4III-trithio-alpha-xylotetroside (S-Xyl4-Me), and methyl 4,4II,4III,4IV-tetrathio-alpha-xylopentoside (S-Xyl5-Me), to three family 11 endo-1,4-beta-xylanases from Trichoderma reesei (TRX I and TRX II) and Chaetomium thermophilum (CTX) was characterized using electrospray ionization Fourier transform ion cyclotron resonance (FT-ICR) MS and X-ray crystallography. Ultra-high mass-resolving power and mass accuracy inherent to FT-ICR allowed mass measurements for noncovalent complexes to within |DeltaM|average of 2 p.p.m. The binding constants determined by MS titration experiments were in the range 10(4)-10(3) M-1, decreasing in the series of S-Xyl5-Me>or=S-Xyl4-Me>S-Xyl3-Me. In contrast, S-Xyl2-Me did not bind to any xylanase at the initial concentration of 5-200 microM, indicating increasing affinity with increasing number of xylopyranosyl units, with a minimum requirement of three. The crystal structures of CTX-inhibitor complexes gave interesting insights into the binding. Surprisingly, none of the inhibitors occupied any of the aglycone subsites of the active site. The binding to only the glycone subsites is nonproductive for catalysis, and yet this has also been observed for other family 11 xylanases in complex with beta-d-xylotetraose [Wakarchuk WW, Campbell RL, Sung WL, Davoodi J & Makoto Y (1994) Protein Sci3, 465-475, and Sabini E, Wilson KS, Danielsen S, Schulein M & Davies GJ (2001) Acta CrystallogrD57, 1344-1347]. Therefore, the role of the aglycone subsites remains controversial despite their obvious contribution to catalysis.

Biochemical characterisation of the trehalase of thermophilic fungi: An enzyme with mixed properties of neutral and acid trehalase

The trehalases from some thermophilic fungi, such as Humicola grisea, Scytalidium thermophilum, or Chaetomium thermophilum, possess mixed properties in comparison with those of the two main groups of trehalases: acid and neutral trehalases. Such as acid trehalases these enzymes are highly thermostable extracellular glycoproteins, which act at acidic pH. However, these enzymes are activated by calcium or manganese, and as a result inhibited by chelators and by ATP, properties typical of neutral trehalases. Here we extended the biochemical characterisation of these enzymes, by assaying their activity at acid and neutral pH. The acid activity (25-30% of total) was assayed in McIlvaine buffer at pH 4.5. Under these conditions the enzyme was neither activated by calcium nor inhibited by EDTA or ATP. The neutral activity was estimated in MES buffer at pH 6.5, after subtracting the activity resistant to EDTA inhibition. The neutral activity was activated by calcium and inhibited by ATP. On the other hand, the acid activity was more thermostable than the neutral activity, had a higher temperature optimum, exhibited a lower K(m), and different sensitivity to several ions and other substances. Apparently, these trehalases represent a new class of trehalases. More knowledge is needed about the molecular structure of this protein and its corresponding gene, to clarify the structural and evolutionary relationship of this trehalase to the conventional trehalases.