Oxidative cleavage of cellulose in the horse gut.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) belonging to the auxiliary activity 9 family (AA9) are widely found in aerobic fungi. These enzymes are O2-dependent copper oxidoreductases that catalyze the oxidative cleavage of cellulose. However, studies that have investigated AA9 LPMOs of aerobic fungi in the herbivore gut are scare. To date, whether oxidative cleavage of cellulose occurs in the herbivore gut is unknown. RESULTS: We report for the first time experimental evidence that AA9 LPMOs from aerobic thermophilic fungi catalyze the oxidative cleavage of cellulose present in the horse gut to C1-oxidized cellulose and C1- and C4-oxidized cello-oligosaccharides. We isolated and identified three thermophilic fungi and measured their growth and AA9 LPMO expression at 37 °C in vitro. We also assessed the expression and the presence of AA9 LPMOs from thermophilic fungi in situ. Finally, we used two recombinant AA9 LPMOs and a native AA9 LPMO from thermophilic fungi to cleave cellulose to yield C1-oxidized products at 37 °C in vitro. CONCLUSIONS: The oxidative cleavage of cellulose occurs in the horse gut. This finding will broaden the known the biological functions of the ubiquitous LPMOs and aid in determining biological significance of aerobic thermophilic fungi.

Crystal Structure of a GH3 ß-Glucosidase from the Thermophilic Fungus Chaetomium thermophilum.

Beta-glucosidases (ß-glucosidases) have attracted considerable attention in recent years for use in various biotechnological applications. They are also essential enzymes for lignocellulose degradation in biofuel production. However, cost-effective biomass conversion requires the use of highly efficient enzymes. Thus, the search for new enzymes as better alternatives of the currently available enzyme preparations is highly important. Thermophilic fungi are nowadays considered as a promising source of enzymes with improved stability. Here, the crystal structure of a family GH3 ß-glucosidase from the thermophilic fungus Chaetomium thermophilum (CtBGL) was determined at a resolution of 2.99 Å. The structure showed the three-domain architecture found in other ß-glucosidases with variations in loops and linker regions. The active site catalytic residues in CtBGL were identified as Asp287 (nucleophile) and Glu517 (acid/base). Structural comparison of CtBGL with Protein Data Bank (PDB)-deposited structures revealed variations among glycosylated Asn residues. The enzyme displayed moderate glycosylation compared to other GH3 family ß-glucosidases with similar structure. A new glycosylation site at position Asn504 was identified in CtBGL. Moreover, comparison with respect to several thermostability parameters suggested that glycosylation and charged residues involved in electrostatic interactions may contribute to the stability of the enzyme at elevated temperatures. The reported CtBGL structure provides additional insights into the family GH3 enzymes and could offer new ideas for further improvements in ß-glucosidases for more efficient use in biotechnological applications regarding cellulose degradation.

Crystal structure and biological implications of a glycoside hydrolase family 55 ß-1,3-glucanase from Chaetomium thermophilum.

Crystal structures of a ß-1,3-glucanase from the thermophilic fungus Chaetomium thermophilum were determined at 1.20 and 1.42Å resolution in the free and glucose-bound form, respectively. This is the third structure of a family 55 glycoside hydrolase (GH55) member and the second from a fungus. Based on comparative structural studies and site-directed mutagenesis, Glu654 is proposed as the catalytic acid residue. The substrate binding cleft exhibits restricted access on one side, rendering the enzyme as an exo-ß-1,3-glucanase as confirmed also by thin layer chromatography experiments. A lack of stacking interactions was found at the substrate binding cleft, suggesting that interactions at positions -1, +1 and +2 are sufficient to orientate the substrate. A binding pocket was identified that could explain binding of branched laminarin and accumulation of laminaritriose.

Crystal structure and biochemical characterization of a manganese superoxide dismutase from Chaetomium thermophilum.

A manganese superoxide dismutase from the thermophilic fungus Chaetomium thermophilum (CtMnSOD) was expressed in Pichia pastoris and purified to homogeneity. Its optimal temperature was 60°C with approximately 75% of its activity retained after incubation at 70°C for 60min. Recombinant yeast cells carrying C. thermophilum mnsod gene exhibited higher stress resistance to salt and oxidative stress-inducing agents than control yeast cells. In an effort to provide structural insights, CtMnSOD was crystallized and its structure was determined at 2.0Å resolution. The overall architecture of CtMnSOD was found similar to other MnSODs with highest structural similarities obtained against a MnSOD from the thermotolerant fungus Aspergillus fumigatus. In order to explain its thermostability, structural and sequence analysis of CtMnSOD with other MnSODs was carried out. An increased number of charged residues and an increase in the number of intersubunit salt bridges and the Thr:Ser ratio were identified as potential reasons for the thermostability of CtMnSOD.

Role of a major facilitator superfamily transporter in adaptation capacity of Penicillium funiculosum under extreme acidic stress.

Fungal species present in extreme low pH environments are expected to have adapted for tolerance to high H(+) concentrations. However, their adaptability mechanism is unclear. In this study, we isolated an acid-tolerant strain of Penicillium funiculosum, which can grow actively at pH 1.0 and thrived in pH 0.6. A major facilitator superfamily transporter (PfMFS) was isolated from an acid-sensitive random insertional mutant (M4) of the fungus. It encodes a putative protein of 551 residues and contains 14 transmembrane-spanning segments. A targeted mutant (M7) carrying an inactivated copy of PfMFS showed an obvious reduction of growth compared with the wild type (WT) and complementation of M7 with PfMFS restored the wild-type level of growth at pH 1.0. Further data showed that the wild-type showed higher intracellular pH than M7 in response to pH 1. Subcellular localization showed that PfMFS was a cell membrane protein. Homology modeling showed structural similarity with an MFS transporter EmrD from Escherichiacoli. These results demonstrate that the PfMFS transporter is involved in the acid resistance and intracellular pH homeostasis of P. funiculosum.

Transcription Factor VM1G_06867: A Requirement for Growth, Pathogenicity, Development, and Maintenance of Cell Wall Integrity in Valsa mali.

Apple canker disease, caused by Valsa mali, is one of the most serious apple tree diseases in China. VmSom1 is an important transcription factor that acts on the cyclic adenosine signaling pathway (cAMP/PKA), regulating the growth, development, morphological differentiation, and pathogenic forces of the pathogen. We perform transcriptome analysis of the VmSom1 deletion mutant and the wild-type strain 11-175 and identify a significantly differentially expressed gene, VM1G_06867, a zinc finger motif transcription factor in V. mali. In this study, we obtain the VM1G_06867 gene using the single deletion mutant via homologous recombination. To determine the relationship between VmSom1 and VM1G_06867, we also obtain a double deletion mutant ΔVmSom1/06867. Compared to the wild-type strain 11-175, the single deletion mutant VM1G_06867 shows a drastic reduction in growth rate and forms more pycnidia on the PDA medium. Additionally, the growth of the mutant is inhibited by SDS, Congo red, and fluorescent brighteners. In comparison to the single deletion mutant VmSom1, the double deletion mutant ΔVmSom1/06867 shows no significant change in growth or conidiation and is unable to produce conidia. The growth rate is significantly increased in Congo red, NaCl, and Sorbitol mediums. These results demonstrate that VM1G_06867 plays important roles in growth, pathogenicity, asexual development, and maintenance of cell wall integrity. VM1G_06867 can recover osmotic stress and cell wall integrity defects caused by the deletion of VmSom1, as well as restore the loss of pathogenicity caused by the deletion of the VmSom1 gene, but not completely.

Directed evolution and structural prediction of cellobiohydrolase II from the thermophilic fungus Chaetomium thermophilum.

Cellulases can be engineered with enhanced properties for broad use in scientific and industrial applications. In this study, the wild-type cbh2 gene of the thermophilic fungus Chaetomium thermophilum encoding cellobiohydrolase II (CBHII) was mutagenized through in vitro directed evolution. The resulting Pichia pastoris yeast library was screened, and two transformants were selected for enhanced CBHII activities that were not attributed to increased gene copy numbers. The optimum fermentation times of the two mutant transformants were shortened to 4-5 days after methanol induction compared to 6 days for the wild-type. The optimum reaction temperature (60 °C) and pH level (5 or 6) of the mutant CBHII proteins, designated CBHIIX16 and CBHIIX305, were higher than those of wild-type CBHII (50 °C and pH 4). Kept at 80 °C for 1 h, CBHIIX16 and CBHIIX305 retained >50% of their activities, while the wild-type CBHII lost all activity. Sequence analysis of CBHIIX16 and CBHIIX305 revealed that they contained five and six mutated amino acids, respectively. Structural modeling confirmed the presence of carbohydrate binding type-1 and catalytic domains, where the hydrogen bond numbers between the 227th and 203rd amino acids were increased, which perhaps contributed to the elevated enzyme stability. Therefore, the two CBHII mutants selected for increased enzymatic activities also demonstrated elevated optimum reaction temperature and pH levels and enhanced thermal stability. These properties may be beneficial in practical applications for CBHII.

Cloning and functional analysis of a novel chitinase gene Trchi1 from Trichothecium roseum.

Chitinases produced by mycoparasites play an important role in disease control in plants. To explore the functions of chitinases in Trichothecium roseum, we cloned a new chitinase gene named Trchi1 from T. roseum by RT (reverse transcription)-PCR techniques. The T. roseum gene, Trchi1, contains an 1278-bp ORF that shares 76 % similarity with chitinase from Bionectria ochroleuca (ABV57861 3G6L_A). A plant expression vector, containing the Trchi1 gene driven by the CaMV35S promoter, was constructed and transformed into tobacco via Agrobacterium tumefaciens. Southern blot analysis showed that Trchi1 was integrated into the tobacco genome. Total chitinase activity in Trchi1-transgenic tobacco leaves was enhanced 2.2- to 5.8- times with respect to non-transgenic leaves. Transgenic tobacco plants transformed with the Trchi1 gene had increased resistance to Alternaria alternata and Colletotrichum nicotianae.

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in thermophilic fungus Thermomyces lanuginosus.

OBJECTIVE: To establish a stable transformation system of the thermophilic fungus Thermomyces lanuginosus for its insertional mutagenesis. METHODS: Agrobacterium tumefaciens-mediated transformation (ATMT) was applied to establish transformation system of T. lanuginosus. Southern blotting of hph gene and cloning of transforming DNA (T-DNA) flanking sequences were used to determine insert number and site of T-DNA in the fungal genome, respectively. RESULTS: A reliable transformation method is established for T. lanuginosus. Specifically, pre-germinating spores of T. lanuginosus used at co-cultivated period was a prerequisite. T. lanuginosus germinating spores co-cultivated with Agrobacterium tumefaciens at 28 degrees C for 48 h achieved the highest transformation efficiency. Addition of Acetosyringone (AS) during pre-culture of A. tumefaciens and co-cultivation of T. lanuginosus germinating spores with A. tumefaciens was essentially required, and the best results were obtained with AS at the concentration of 500 microM. Southern blotting analysis showed that majority of transformants (79.2%) contained a single insertion of T-DNA. Thermal asymmetric interlaced PCR (TAIL-PCR) analysis showed random insertion of T-DNA in the fungal genome. Using the transformation system, some stable phenotypic mutants of T. lanuginosus were obtained. CONCLUSION: We report, for the first time, a simple and efficient method for transforming T. lanuginosus by using ATMT. This approach provides a tool for insertional mutagenesis gene tagging in this thermophilic fungus.

An Aminobutyric Acid Transaminase in Zea mays Interacts With Rhizoctonia solani Cellulase to Participate in Disease Resistance.

Corn sheath blight, caused by AG1-IA, a fusion group of Rhizoctonia solani, which acts as a kind of necrotrophic fungal pathogen, poses a global threat to the production of Zea mays. Although cellulase plays a crucial role in R. solani infections, how plants respond to it is still poorly understood. In this study, we identified a gamma-aminobutyric acid transaminase (GABA-T), ZmGABA-T, in Z. mays that interacts with a cell wall-degrading enzyme (CWDE), EG1, in the cell membrane, using yeast two-hybrid assay, co-immunoprecipitation (Co-IP), and bimolecular fluorescence complementation assays. We found that the combination of EG1 and ZmGABA-T suppressed the allergic necrosis induced by EG1. We also found that the substrate of GABA-T-GABA, can inhibit the transcription of EG1. Transient expression of ZmGABA-T inhibited R. solani infection in Nicotiana benthamiana. The homolog in Oryza sativa, OsGABA-T, could also interact with EG1 to suppress the allergic necrosis induced by EG1. The OsGABA-T knocked out plants displayed enhanced susceptibility to R. solani and showed larger lesions. In conclusion, our results suggest that ZmGABA-T inhibits allergic necrosis induced by EG1 based on the combination with EG1, producing resistance to R. solani infection.

Analysis of lytic polysaccharide monooxygenase activity in thermophilic fungi by high-performance liquid chromatography-refractive index detector.

Introduction: Most current methods for analysing the activity of LPMO are based on the quantification of H2O2, a side product of LPMO; however, these methods cannot assay the LPMO activity of thermophilic fungi because of the low thermostability of H2O2. Therefore, we present a high-performance liquid chromatography-refractive index detector (HPLC-RID) method to assay the LPMO activity of the thermophilic fungus Thermoascus aurantiacus. Results: According to the established method, the specific activities of nTaAA9A C1 and C4 oxidation were successfully analysed and were 0.646 and 0.574 U/mg, respectively. By using these methods, we analyzed the C1 and C4 oxidation activities of the recombinant TaAA9A (rTaAA9A) and mutated rTaAA9A (Y24A, F43A, and Y212A) expressed in Pichia pastoris. The specific activities of rTaAA9A C1 and C4 oxidation were 0.155 and 0.153 U/mg, respectively. The specific activities of Y24A, F43A, and Y212A C1 and C4 oxidation were 0.128 and 0.125 U/mg, 0.194 and 0.192 U/mg, and 0.097 and 0.146 U/mg, respectively. Discussion: In conclusion, the method can assay the LPMO activity of thermophilic fungi and directly target C1 and C4 oxidation, which provides an effective activity assay method for LPMOs of thermophilic fungi.

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 2,170 U/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 60 min and the half-life was 22 and 7 min 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.

Novel Proteome and N-Glycoproteome of the Thermophilic Fungus Chaetomium thermophilum in Response to High Temperature.

Thermophilic fungi are eukaryotic species that grow at high temperatures, but little is known about the underlying basis of thermophily at cell and molecular levels. Here the proteome and N-glycoproteome of Chaetomium thermophilum at varying culture temperatures (30, 50, and 55°C) were studied using hydrophilic interaction liquid chromatography enrichment and high-resolution liquid chromatography-tandem mass spectroscopy analysis. With respect to the proteome, the numbers of differentially expressed proteins were 1,274, 1,374, and 1,063 in T50/T30, T55/T30, and T55/T50, respectively. The upregulated proteins were involved in biological processes, such as protein folding and carbohydrate metabolism. Most downregulated proteins were involved in molecular functions, including structural constituents of the ribosome and other protein complexes. For the N-glycoproteome, the numbers of differentially expressed N-glycoproteins were 160, 176, and 128 in T50/T30, T55/T30, and T55/T50, respectively. The differential glycoproteins were mainly involved in various types of N-glycan biosynthesis, mRNA surveillance pathway, and protein processing in the endoplasmic reticulum. These results indicated that an efficient protein homeostasis pathway plays an essential role in the thermophily of C. thermophilum, and N-glycosylation is involved by affecting related proteins. This is the novel study to reveal thermophilic fungi's physiological response to high-temperature adaptation using omics analysis, facilitating the exploration of the thermophily mechanism of thermophilic fungi.

Crystal Structure of a Cu,Zn Superoxide Dismutase From the Thermophilic Fungus Chaetomium thermophilum.

BACKGROUND: Thermophilic fungi have recently emerged as a promising source of thermostable enzymes. Superoxide dismutases are key antioxidant metalloenzymes with promising therapeutic effects in various diseases, both acute and chronic. However, structural heterogeneity and low thermostability limit their therapeutic efficacy. OBJECTIVE: Although several studies from hypethermophilic superoxide dismutases (SODs) have been reported, information about Cu,Zn-SODs from thermophilic fungi is scarce. Chaetomium thermophilum is a thermophilic fungus that could provide proteins with thermophilic properties. METHODS: The enzyme was expressed in Pichia pastoris cells and crystallized using the vapor-diffusion method. X-ray data were collected, and the structure was determined and refined to 1.56 Å resolution. Structural analysis and comparisons were carried out. RESULTS: The presence of 8 molecules (A through H) in the asymmetric unit resulted in four different interfaces. Molecules A and F form the typical homodimer which is also found in other Cu,Zn- SODs. Zinc was present in all subunits of the structure while copper was found in only four subunits with reduced occupancy (C, D, E and F). CONCLUSION: The ability of the enzyme to form oligomers and the elevated Thr:Ser ratio may be contributing factors to its thermal stability. Two hydrophobic residues that participate in interface formation and are not present in other CuZn-SODs may play a role in the formation of new interfaces and the oligomerization process. The CtSOD crystal structure reported here is the first Cu,Zn-SOD structure from a thermophilic fungus.

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.

The gene fpk1, encoding a cAMP-dependent protein kinase catalytic subunit homolog, is required for hyphal growth, spore germination, and plant infection in Fusarium verticillioides.

Fusarium verticillioides is an important pathogen of maize responsible for ear rots, stalk rots and seeding blight worldwide. During the past decade F. verticillioides has caused several severe epidemics of maize seeding blight in many areas of china, which lead to significant losses. In order to understand molecular mechanisms regulating fungal development and pathogenicity in the pathogen, we isolated and characterized the gene fpk1 (GenBank accession NO. EF405959) encoding catalytic subunit of cAMP-dependent protein kinase, which include 1854 bp DNA sequence from ATG to TAA, with 1680 bp coding region, three intron (their length: 66bp, 54bp and 54bp), and the predicated protein had 559 aa. The mutantdeltafpk1, which was disrupted of fpk1 gene, showed reduced vegetative growth, fewer and shorter aerial mycelia, strongly impaired conidiation and reduced spore germination rate. After germinating, the fresh hypha was stubby and lack of branch. When inoculated in susceptible maize varieties, the infection of the mutantdeltafpk1 was delayed and the infection efficiency was reduced than that of the wild-type. All this indicated that the gene fpk1 participated in hyphal growth, conidiophore producing, spore germination and virulence in F. verticillioides.

Identification and Characterization of a Novel Hyperthermostable Bifunctional Cellobiohydrolase- Xylanase Enzyme for Synergistic Effect With Commercial Cellulase on Pretreated Wheat Straw Degradation.

The novel cellobiohydrolase gene ctcel7 was identified from Chaetomium thermophilum, and its recombinant protein CtCel7, a member of glycoside hydrolase family 7, was heterologously expressed in Pichia pastoris and biochemically characterized. Compared with commercial hydrolases, purified CtCel7 exhibited superior bifunctional cellobiohydrolase and xylanase activities against microcrystalline cellulose and xylan, respectively, under optimal conditions of 60°C and pH 4.0. Moreover, CtCel7 displayed remarkable thermostability with over 90% residual activity after heat (60°C) treatment for 180 min. CtCel7 was insensitive to most detected cations and reagents and preferentially cleaved the ß-1,4-glycosidic bond to generate oligosaccharides through the continuous saccharification of lignocellulosic substrates, which are crucial for various practical applications. Notably, the hydrolysis effect of a commercial cellulase cocktail on pretreated wheat straw was substantively improved by its combination with CtCel7. Taken together, these excellent properties distinguish CtCel7 as a robust candidate for the biotechnological production of biofuels and biobased chemicals.

Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing N-glycosylation sites.

BACKGROUND: Endoglucanase has been extensively employed in industrial processes as a key biocatalyst for lignocellulosic biomass degradation. Thermostable endoglucanases with high catalytic activity at elevated temperatures are preferred in industrial use. To improve the activity and thermostability, site-directed mutagenesis was conducted to modify the N-glycosylation sites of the thermostable ß-1,4-endoglucanase CTendo45 from Chaetomium thermophilum. RESULTS: In this study, structure-based rational design was performed based on the modification of N-glycosylation sites in CTendo45. Eight single mutants and one double mutant were constructed and successfully expressed in Pichia pastoris. When the unique N-glycosylation site of N88 was eliminated, a T90A variant was active, and its specific activity towards CMC-Na and ß-d-glucan was increased 1.85- and 1.64-fold, respectively. The mutant R67S with an additional N-glycosylation site of N65 showed a distinct enhancement in catalytic efficiency. Moreover, T90A and R67S were endowed with extraordinary heat endurance after 200 min of incubation at different temperatures ranging from 30 to 90 °C. Likewise, the half-lives (t 1/2) indicated that T90A and R67S exhibited improved enzyme thermostability at 80 °C and 90 °C. Notably, the double-mutant T90A/R67S possessed better hydrolysis activity and thermal stability than its single-mutant counterparts and the wild type. CONCLUSIONS: This study provides initial insight into the biochemical function of N-glycosylation in thermostable endoglucanases. Moreover, the design approach to the optimization of N-glycosylation sites presents an effective and feasible strategy to improve enzymatic activity and thermostability.

Polysaccharide monooxygenase-catalyzed oxidation of cellulose to glucuronic acid-containing cello-oligosaccharides.

BACKGROUND: Polysaccharide monooxygenases (PMOs) play an important role in the enzymatic degradation of cellulose. They have been demonstrated to able to C6-oxidize cellulose to produce C6-hexodialdoses. However, the biological function of C6 oxidation of PMOs remains unknown. In particular, it is unclear whether C6-hexodialdoses can be further oxidized to uronic acid (glucuronic acid-containing oligosaccharides). RESULTS: A PMO gene, Hipmo1, was isolated from Humicola insolens and expressed in Pichia pastoris. This PMO (HiPMO1), belonging to the auxiliary activity 9 (AA9) family, was shown to able to cleave cellulose to yield non-oxidized and oxidized cello-oligosaccharides. The enzyme oxidizes C6 positions in cellulose to form glucuronic acid-containing cello-oligosaccharides, followed by hydrolysis with beta-glucosidase and beta-glucuronidase to yield glucose, glucuronic acid, and saccharic acid. This indicates that HiPMO1 can catalyze C6 oxidation of hydroxyl groups of cellulose to carboxylic groups. CONCLUSIONS: HiPMO1 oxidizes C6 of cellulose to form glucuronic acid-containing cello-oligosaccharides followed by hydrolysis with beta-glucosidase and beta-glucuronidase to yield glucose, glucuronic acid, and saccharic acid, and even possibly by beta-eliminative cleavage to produce unsaturated cello-oligosaccharides. This study provides a new mechanism for cellulose cleavage by C6 oxidation of HiPMO1.

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 microg/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.