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.     

Purification and partial characterization of β-1,3-glucanase from <Emphasis Type="Italic">Chaetomium thermophilum</Emphasis>

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).     

A novel,thermostable manganese-containing superoxide dismutase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

A new, thermostable superoxide dismutase (SOD) from Bacillus licheniformis M20, isolated from Bulgarian mineral springs, was purified 11-fold with 11% recovery of activity. From native PAGE and SDS-PAGE, the enzyme was composed of two subunits of 21.5 kDa each. The SOD was inhibited only by NaN₃, which suggested that this SOD is of the manganese superoxide dismutase type. The purified enzyme had maximum activity at pH 8 and 55°C. The half-life of the SOD was 10 min at 95°C.     

Purification,Characterization, and Molecular Cloning of a Thermostable Superoxide Dismutase from Thermoascus aurantiacus

A thermostable superoxide dismutase [(SOD) EC 1.15.1.1] from a Thermoascus aurantiacus var. levisporus was purified to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) homogeneity by a series of column chromatographies. The molecular mass of a single band of the enzyme was estimated to be 16.8 kDa by SDS–PAGE. The molecular mass was estimated to be 33.2 kDa by gel filtration on Sephacryl S-100, indicating that the enzyme was composed of two identical subunits of 16.8 kDa each. N-terminal amino acid sequencing (seven residues) yielded VKAVAVL. Using RACE-PCR, a Cu, Zn-SOD gene was cloned from T. aurantiacus var. levisporus. The sequence was 705 bp and contained a 468 bp ORF encoding a Cu, Zn-SOD of 155 amino acid residues.     

Cloning,expression, and characterization of thermostable Manganese superoxide dismutase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 μg/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN₃, but not by KCN or H₂O₂. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55°C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60°C and the half-life at 80°C was approximately 40 min.     

Purification and some properties of β-mannanase from Bacillus sp.

-Mannanase produced by Bacillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NH₄)₂ SO₄ precipitation, adsorption to konjak gel, and column chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 70°C and was stable for at least 1 h between pH 5 and 10 and below 60°C. Its activity was completely inhibited by Hg²⁺. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose.The authors are with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University. Utsunomiya, Tochigi 321, Japan     

Biochemical characterization of a 27 kDa 1,3-β-d-glucanase from Trichoderma asperellum induced by cell wall of Rhizoctonia solani

Trichoderma asperellum produces two extracellular 1,3-β-d-glucanase upon induction with cell walls from Rhizoctonia solani. A minor 1,3-β-d-glucanase was purified to homogeneity by ion exchange chromatography on Q-Sepharose and gel filtration on Sephacryl S-100. A typical procedure provided 13.8-fold purification with 70% yield. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 27 kDa. The enzyme exhibited optimum catalytic activity at pH 3.6 and 45 °C. It was thermostable at 40 °C, and retained 75% activity after 60 min at 45 °C. The K_m and V_max values for 1,3-β-d-glucanase, using laminarin as substrate, were 0.323 mg ml⁻¹ and 0.315 U min⁻¹, respectively. The enzyme was strongly inhibited by Hg²⁺ and SDS. The enzyme was only active toward glucans containing β-1,3-linkages. Peptide sequences showed similarity with two endo-1,3(4)-β-d-glucanases from Aspergillus fumigatus Af293when compared against GenBank non-redundant database.     

Purification and some properties of superoxide dismutase from<Emphasis Type="Italic"> Deinococcus radiophilus</Emphasis>, the UV-resistant bacterium

The superoxide dismutase (SOD, EC 1.15.1.1) of Deinococcus radiophilus, a bacterium extraordinarily resistant to UV, ionizing radiations, and oxidative stress, was purified 1,920-fold with a 58% recovery yield from the cell-free extract of stationary cells by steps of ammonium sulfate fractionation and Superdex G-75 gel-filtration chromatography. A specific activity of the purified enzyme preparation was ca. 31,300 U mg^–1 protein. D. radiophilus SOD is Mn/FeSOD, judging by metal analysis and its insensitivity to cyanide and a partial sensitivity to H₂O₂. The molecular weights of the purified enzyme estimated by gel chromatography and polyacrylamide gel electrophoresis are 51.5±1 and 47.1±5 kDa, respectively. The SOD seems to be a homodimeric protein with a molecular mass of 26±0.5 kDa per monomer. The purified native SOD showed very acidic pI of ca. 3.8. The enzyme was stable at pH 5.0–11.0, but quite unstable below pH 5.0. SOD was thermostable up to 40°C, but a linear reduction in activity above 50°C. Inhibition of the purified SOD activity by -naphthoquinone-4-sulfonic acid, -diazobenzene sulfonic acid, and iodine suggests that lysine, histidine, and tyrosine residues are important for the enzyme activity. The N-terminal peptide sequence of D. radiophilus Mn/FeSOD (MAFELPQLPYAYDALEPHIDA(>D) is strikingly similar to those of D. radiodurans MnSOD and Aerobacter aerogenes FeSOD.Communicated by G. Antranikian     

Production and purification of a calcium-dependent protease from Bacillus cereus BG1

The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg²⁺ and Ba²⁺, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L^–1) starch 5, CaCl₂ 2, yeast extract 2, K₂HPO₄ 0.2 and KH₂PO₄ 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl₂.     

Lipid peroxidation and superoxide dismutase activity in relation to photoinhibition induced by chilling in moderate light

The effect of a chilling stress, at a moderate photon flux density for a few hours, on the peroxidation of membrane lipids and on superoxide dismutase (SOD) activity was compared in leaf slices of chilling-sensitive and chilling-insensitive plants. The aim was to determine if susceptibility to chill-temperature photoinhibition could be related to either damage to membrane lipids by superoxide and-or a decrease in activity of chloroplast SOD. Plants used were Nerium oleander L., grown at 45° C, and Cucumis sativus L., both susceptible to chill-temperature photoinhibition, and N. oleander, grown at 20° C and Spinacia oleracea L., both insensitive to chill-temperature photoinhibition. Lipid peroxidation was assessed by measuring the concentration of malondialdehyde (MDA). Leaf slices from all plants showed a basal level of MDA which decreased by about 15% when the leaf slices were chilled in the light. The level of MDA was not increased by the addition of either KHCO₃ or methyl viologen during chilling but it was increased, up to threefold, by the addition of Rose Bengal, which produces singlet oxygen. Chloroplast SOD activity was assessed in leaf extracts as the cyanide-sensitive production of H₂O₂ in a system which produced superoxide. Activity of SOD was similar in all the plants and was altered little by chilling. The results show that for the plants tested, chilling at a moderate photon flux density for 5 h does not increase the susceptibility of cell membranes to peroxidative damage nor does it decrease the activity of SOD. It was concluded that the susceptibility of chilling-sensitive plants to chill-temperature photoinhibition cannot be explained on the basis of differences in the vulnerability of membrane lipids to damage by superoxide or differences in SOD activity.Abbreviations Chl chlorophyll - MDA malondialdehyde - MV methyl viologen - O ₂ ^- superoxide - 20°-oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - PFD photon flux density - SOD superoxide dismutase Deceased     

Purification, characterization, and molecular cloning of a thermostable superoxide dismutase from Thermoascus aurantiacus

A thermostable superoxide dismutase [(SOD) EC 1.15.1.1] from a Thermoascus aurantiacus var. levisporus was purified to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) homogeneity by a series of column chromatographies. The molecular mass of a single band of the enzyme was estimated to be 16.8 kDa by SDS-PAGE. The molecular mass was estimated to be 33.2 kDa by gel filtration on Sephacryl S-100, indicating that the enzyme was composed of two identical subunits of 16.8 kDa each. N-terminal amino acid sequencing (seven residues) yielded VKAVAVL. Using RACE-PCR, a Cu, Zn-SOD gene was cloned from T. aurantiacus var. levisporus. The sequence was 705 bp and contained a 468 bp ORF encoding a Cu, Zn-SOD of 155 amino acid residues.     

Thermostable esterase from Thermoanaerobacter tengcongensis: high-level expression, purification and characterization

The lipA gene encoding a thermostable esterase was cloned from Thermoanaerobacter tengcongensis and overexpressed in Escherichia coli. The recombinant esterase, with a molecular mass of approx. 43 kDa determined by SDS-PAGE, was purified to homogeneity through Sephadex G-100 gel filtration. The purified enzyme actively hydrolyzed tributyrin but not olive oil. Maximum activity was observed on p-nitrophenyl (NP)-propionate (C3) and p-NP-butyrate (C4), with little activity towards p-NP-palmitate (C16). The esterase was optimally active at 70 °C (over 15 min) and at pH 9. It is highly thermostable, with a residual activity greater than 80% after incubation at 50 °C for more than 10 h. The activity was not inhibited by 5 mM EDTA and PMSF, indicating the esterase is not a metalloenzyme and may contain a specific structure around the catalytic serine residue. In addition, it was stable for 1 h at 37 °C in 1% CHAPS and Triton X-100 but not stable in 1% Tween 20 or SDS.     

L-asparaginase of Thermus thermophilus: Purification,properties and identificaation of essential amino acids for its catalytic activity

L-asparaginase EC 3.5.1.1 was purified to homogeneity from Thermus thermophilus. The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 33 kDa, whereas by its mobility on Sephacryl S-300 superfine column was around 200 kDa, indicating that the enzyme at the native stage acts as hexamer. The purified enzyme showed a single band on acrylamide gel electrophoresis with pI = 6.0. The optimum pH was 9.2 and the Km for L-asparagine was 2.8 mM. It is a thermostable enzyme and it follows linear kinetics even at 77°C. Chemical modification experiments implied the existence of histidyl, arginyl and a carboxylic residues located at or near active site while serine and mainly cysteine seems to be necessary for active form.     

Purification and properties of mannanase from Bacillus subtilis

Extracellular mannanase from Bacillus subtilis NM-39, an isolate from Philippine soil, was purified about 240-fold with a yield of 7.3% by ammonium sulphate fractionation, DEAE-Toyopearl chromatography and Sephacryl S-200 gel filtration. Its M _r was 38 kDa and it had a pI of 4.8 and optimum activity at pH 5.0 and 55°C. It was stable at pH 4 to 9 and below 55°C. The amino acid composition of the enzyme was in the order Gly>Glx>Ser and Asx>Ala.N.S. Mendoza and L.M. Joson are with Industrial Technology Development Institute, Department of Science and Technology, Manila, Philippines. M. Arai and T. Kawaguchi are with Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 593, Japan; T. Yoshida is with Faculty of Engineering, Osaka University, Suita, Osaka 565, Japan.     

Purification and properties of extracellular phytase from Bacillus sp. KHU-10

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40°C without 10 mM CaCl₂ and pH 6.0-9.5 and 60°C with 10 mM CaCl₂. About 50% of its original activity remained after incubation at 80°C or 10 min in the presence of 10 mM CaCl₂. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The K _m value for sodium phytate was 50 M. Its activity was inhibited by EDTA and metal ions such as Ba²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Hg²⁺, and Mn²⁺ ions.     

Purification and biochemical characterization of a superoxide dismutase from the soluble fraction of the cyanobacterium, <Emphasis Type="Italic">Spirulina platensis</Emphasis>

A superoxide dismutase (SOD) was purified from Spirulina platensis sonicate. The SOD was purified to homogeneity (48-fold and 0.24% yield) through ammonium sulphate precipitation and DEAE-52 anion exchange chromatography. The SOD from S. platensis appeared to be a homodimer with a molecular weight of 30 kDa and a subunit MW of 15 kDa as determined by both native polyacrylamide gel electrophoresis and mass spectrometry. The enzyme activity was stable at pH 6.5–10.0 and 50 °C. Using group-specific chemical modifying reagents, the amino acids arginine, histidine, tryptophan, tyrosine and aspartic acid were identified to be essential for S. platensis SOD activity. The amino acid composition was found to lack methionine and cysteine. The inhibition of activity by H₂O₂ suggests that the enzyme may be an iron containing SOD.     

Manganese superoxide dismutase from a higher plant

A manganese-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from a higher plant for the first time. The enzyme was isolated fromPisum sativum leaf extracts by thermal fractionation, ammonium sulfate salting out, ion-exchange and gel-filtration column chromatography, and preparative polyacrylamide gel electrophoresis. Pure manganese superoxide dismutase had a specific activity of about 3,000 U mg^-1 and was purified 215-fold, with a yield of 1.2 mg enzyme per kg whole leaf. The manganese superoxide dismutase had a molecular weight of 94,000 and contained one g-atom of Mn per mol of enzyme. No iron and copper were detected. Activity reconstitution experiments with the pure enzyme ruled out the possibility of a manganese loss during the purification procedure. The stability of manganese superoxide dismutase at-20°C, 4°C, 25°C, 50°C, and 60°C was studied, and the enzyme was found more labile at high temperatures than bacterial manganese superoxide dismutases and iron superoxide dismutases from an algal and bacterial origin.Abbreviations NBT nitro blue tetrazolium - SOD superoxide dismutase (EC 1.15.1.1)     

Cloning and characterization of a thermostable superoxide dismutase from the thermophilic bacterium Rhodothermus sp. XMH10

A superoxide dismutase (SOD) gene was cloned from the thermophilic bacterium Rhodothermus sp. XMH10 for the first time and highly expressed in Escherichia coli. The Rhodothermus sp. XMH10 SOD (RhSOD) gene encodes 209 amino acids with a putative molecular weight of 23.6 kDa and a pI value of 5.53. The recombinant RhSOD was detected to be an iron type SOD and existed as a dimer on its natural status. Experiments revealed that this RhSOD showed high activity at 50–70 °C and pH 5.0. Compared to SODs from other thermophiles, it was highly thermostable, maintaining more than 90% of its activity after incubation at 70 °C for 12 h, only totally inactivated after more than 4-h incubation at 80 °C. It also showed much higher resistance to KCN, NaN₃ and H₂O₂ as compared to other SODs. Xin Wang and Haijie Yang contribute to this work equally.     

Studies on the <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> CCT-4518 laccase purified by hydrophobic interaction chromatography

A laccase from Pycnoporus sanguineus was purified by two steps using phenyl-Sepharose columm. A typical procedure provided 54.1-fold purification, with a yield of 8.37%, using syringaldazine as substrate. The molecular weight of the purified laccase was 69 and 68 kDa as estimated by 12% (w/v) SDS-PAGE gel and by gel filtration, respectively. The K _m values for the substrates ABTS, syringaldazine, and guaiacol were 58, 8.3, and 370 μM, respectively. The enzyme’s pH optimum for syringaldazine was 4.2 and optimal activity was 50°C. The enzyme showed to be thermostable because when kept at 50°C for 24 and 48 h it retained 93 and 76% activity. This laccase was inhibited by l-cysteine, β-mercaptoethanol, NaN₃, NaF, and HgCl₂.     

Purification of iron superoxide dismutase from the cyanobacterium Anabaena cylindrica Lemm. and localization of the enzyme in heterocysts by immunogold labeling

Iron superoxide dismutase (Fe-SOD; EC 1.15.1.1) was isolated from the nitrogen-fixing cyanobacterium Anabaena cylindrica Lemm. Polyacrylamide gel electrophoresis separated the purified protein into three closely running, enzymatically active bands. The molecular weight of the enzyme was estimated by gel filtration to be about 40 kDa. Polyclonal antibodies were produced by immunization of rabbits with the isolated enzyme, and were purified on a column of protein A-Sepharose. The Fe-SOD antibody reacted with the purified Fe-SOD and also specifically recognized the protein in extracts of A. cylindrica. In the extracts, anti-Fe-SOD did not cross-react with Mn-SOD, an enzyme which belongs to an SOD class displaying high homology of primary and three-dimensional structure with respect to Fe-SOD. Iron superoxide dismutase was localized in heterocysts by immunogold labeling and transmission electron microscopy. These results are the first in-situ evidence for the presence of SOD in the cells specialized for nitrogenase activity.Abbreviations ELISA enzyme-linked immunosorbent assay - SDS sodium dodecyl sulfate - SOD superoxide dismutase - PAGE polyacrylamide gel electrophoresis - pI isoelectric point This work was supported by a C.N.R. grant. We are grateful to Dr. A. De Martino for technical assistance.