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1.
Ning-Ning Song Yan Zheng Shi-Jin E Duo-Chuan Li 《Journal of microbiology (Seoul, Korea)》2009,47(1):123-130
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. 相似文献
2.
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 相似文献
3.
Krutika Desai Subramanian Sivakami 《World journal of microbiology & biotechnology》2007,23(12):1661-1666
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. 相似文献
4.
Cloning and characterization of a new manganese superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3 总被引:1,自引:0,他引:1
Yanbing Zhu Guohong Wang Hui Ni Anfeng Xiao Huinong Cai 《World journal of microbiology & biotechnology》2014,30(4):1347-1357
A new gene encoding a superoxide dismutase (SOD) was identified from a thermophile Geobacillus sp. EPT3 isolated from a deep-sea hydrothermal field in east Pacific. The open reading frame of this gene encoded 437 amino acid residues. It was cloned, overexpressed in Escherichia coli (DE3), and the recombinant protein was purified to homogeneity. Geobacillus sp. EPT3 SOD was of the manganese-containing SOD type, as judged by the insensitivity of the recombinant enzyme to both KCN and H2O2, and the activity analysis of Fe or Mn reconstituted SODs by polyacrylamide gel electrophoresis. The recombinant SOD was determined to be a homodimer with monomeric molecular mass of 59.0 kDa. In comparison with other Mn–SODs, the manganese-binding sites are conserved in the sequence (His260, His308, Asp392, His396). The recombinant enzyme had high thermostability at 50 °C. It retained 57 % residual activity after incubation at 90 °C for 1 h, which indicated that this SOD was thermostable. The enzyme also showed striking stability over a wide range of pH 5.0–11.0. At tested conditions, the recombinant SOD from Geobacillus sp. EPT3 showed a relatively good tolerance to some inhibitors, detergents, and denaturants, such as β-mercaptoethanol, dithiothreitol, phenylmethylsulfonyl fluoride, Chaps, Triton X-100, urea, and guanidine hydrochloride. 相似文献
5.
Identification of Three <Emphasis Type="Italic">Superoxide dismutase</Emphasis> Genes from a <Emphasis Type="Italic">Geobacillus</Emphasis> sp. 总被引:1,自引:0,他引:1
We report the characterization of three Superoxide dismutase (sod) genes isolated from a bacterium in the Geobacillus genus. We isolated the bacterium from high-temperature pond mud and used 16S rRNA gene sequence to confirm its identity in
the Geobacillus genus. The three genes Mn-sod, Fe/Mn-sod, and Cu/Zn-sod were cloned and analyzed. Their open reading frames are Mn-sod: 615 bp encoding a 204 amino acid protein; Fe/Mn-sod: 1,236 bp encoding a 411 amino acid protein; Cu/Zn-sod: 522 bp encoding a 173 amino acid protein. When these sod genes were expressed in Escherichia coli, only Mn-SOD was able to be purified. The activity of the purified Mn-SOD we got was about 2,730 U/mg. Studies of this Mn-SOD
showed that it was thermostable at 60°, had 70% activity at 80° after 2.5 h, and still had 30% activity at 90° after 2.5 h.
Mn-SOD activity required the ion Mn2+. Based on gel electrophoresis, we deduced that this Mn-SOD was a homotetramer. No activity was detected after the other two
genes (Fe/Mn-sod, Cu/Zn-sod) were expressed in Escherichia coli, but activities were detected when expressed in Pichia pastoris. 相似文献
6.
Purification,characterization and gene cloning of isoeugenol-degrading enzyme from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IE27 总被引:1,自引:0,他引:1
An isoeugenol-degrading enzyme was purified to homogeneity from Pseudomonas putida IE27, an isoeugenol-assimilating bacterium. The purified enzyme was a 55 kDa monomer and catalyzed the initial step of isoeugenol
degradation, the oxidative cleavage of the side chain double-bond of isoeugenol, to form vanillin. Another reaction product
of isoeugenol degradation besides vanillin was identified to be acetaldehyde. The values of Km and k
cat for isoeugenol were 175 μM and 5.18 s–1, respectively. The purified enzyme catalyzed the incorporation of an oxygen atom from either molecular oxygen or water into
vanillin, suggesting that the isoeugenol-degrading enzyme is a kind of monooxygenase. The gene encoding the isoeugenol-degrading
enzyme and its flanking regions were isolated from P. putida IE27. The amino acid sequence of the enzyme was similar to those of lignostilbene-α,β-dioxygenases, carotenoid monooxygenases
and 9-cis-epoxycarotenoid dioxygenases. 相似文献
7.
Yamahara T Shiono T Suzuki T Tanaka K Takio S Sato K Yamazaki S Satoh T 《The Journal of biological chemistry》1999,274(47):33274-33278
A novel extracellular Mn-superoxide dismutase (SOD) was isolated from a moss, Barbula unguiculata. The SOD was a glycoprotein; the apparent molecular mass of its native form was 120 kDa, as estimated by gel filtration chromatography, and that of its monomer was 22,072 Da, as estimated by time of flight mass spectroscopy. The protein had manganese with a stoichiometry of 0.80 Mn/monomer. The cDNA clone for a gene encoding the extracellular Mn-SOD was isolated. Sequence analysis showed that it has a strong similarity to germin (oxalate oxidase) and germin-like proteins (GLPs) of several plant species and possesses all the characteristic features of members of the germin family. The clone encoding this extracellular Mn-SOD was therefore designated B. unguiculata GLP (BuGLP). BuGLP had no oxalate oxidase activity. In addition, the cDNA for a gene encoding the moss mitochondrial Mn-SOD was isolated. Its amino acid sequence had little similarity to that of BuGLP, even though a close similarity was observed among the mitochondrial Mn-SODs of various organisms. BuGLP was the first germin-like protein that was really demonstrated to be a metalloprotein with Mn-SOD activity but no oxalate oxidase activity. 相似文献
8.
《Bioscience, biotechnology, and biochemistry》2013,77(4):1090-1093
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. 相似文献
9.
Iwasaki A Matsumoto K Hasegawa J Yasohara Y 《Applied microbiology and biotechnology》2012,93(4):1563-1573
A novel (R)-amine transaminase, which catalyzed (R)-enantioselective transamination of chiral amine, was purified to homogeneity from Arthrobacter sp. KNK168 (FERM BP-5228). The molecular mass of the enzyme was estimated to be 148 kDa by gel filtration and 37 kDa by sodium
dodecyl sulfate polyacrylamide gel electrophoresis, suggesting a homotetrameric structure. The enzyme catalyzed transamination
between amines and pyruvate stereo-specifically. The reaction on 1-methylbenzylamine was (R)-enantioselective. Pyruvate was the best amino acceptor, but the enzyme showed broad amino acceptor specificity for various
ketone and aldehyde compounds. The apparent K
ms for (R)-1-methylbenzylamine and pyruvate were 2.62 and 2.29 mM, respectively. The cloned gene of the enzyme consists of an open
reading frame (ORF) of 993 bp encoding a protein of 330 amino acids, with a calculated molecular weight of 36,288. The deduced
amino acid sequence was found to be homologous to those of the aminotransferases belonging to fold class IV of pyridoxal-5′-phosphate-dependent
enzymes, such as branched-chain amino acid aminotransferases. 相似文献
10.
Ivanka Petrova Boyadzhieva Miroslava Atanasova Elka Emanuilova 《Biotechnology letters》2010,32(12):1893-1896
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. 相似文献
11.
Superoxide dismutase (SOD) from the hyperthermophilic archaeon Pyrobaculum
aerophilum (a facultative aerobe) has been cloned and expressed in a mesophilic host (Escherichia coli) as a soluble tetrameric apoprotein. The purified apoprotein can be reconstituted with either Mn or Fe by heating the protein
with the appropriate metal salt at an elevated temperature (95 °C). Both Mn- and Fe-reconstituted P.
aerophilum SOD exhibit superoxide dismutase activity, with the Mn-containing enzyme having the higher activity. P.
aerophilum SOD is extremely thermostable and the reconstitution with Mn(II) can be performed in an autoclave (122 °C, 18 psi). The
Mn(III) optical absorption spectrum of Mn-reconstituted P.
aerophilum SOD is distinct from that of most other MnSODs and is unchanged upon addition of NaN3. The optical absorption spectrum of Fe-reconstituted P.
aerophilum SOD is typical of Fe-substituted MnSODs and authentic FeSOD and exhibits a pH-dependent transition with an effective pK
a value higher than that found for Fe-substituted MnSOD from either E.
coli or Thermus spp. Amino acid sequence analysis shows that the P.
aerophilum SOD is closely related to SODs from other hyperthermophilic archaea (Aeropyrum
pernix and Sulfolobus spp.), forming a family of enzymes distinct from the hyperthermophilic bacterial SOD from Aquifex
pyrophilus and from mesophilic SODs.
Received: 2 February 2000 / Accepted: 29 March 2000 相似文献
12.
Kurata A Uchimura K Shimamura S Kobayashi T Horikoshi K 《Applied microbiology and biotechnology》2007,77(2):311-319
The acpI gene encoding an alkaline protease (AcpI) from a deep-sea bacterium, Alkalimonas collagenimarina AC40T, was shotgun-cloned and sequenced. It had a 1,617-bp open reading frame encoding a protein of 538 amino acids. Based on analysis
of the deduced amino acid sequence, AcpI is a subtilisin-like serine protease belonging to subtilase family A. It consists
of a prepropeptide, a catalytic domain, and a prepeptidase C-terminal domain like other serine proteases from the genera Pseudomonas, Shewanella, Alteromonas, and Xanthomonas. Heterologous expression of the acpI gene in Escherichia coli cells yielded a 28-kDa recombinant AcpI (rAcpI), suggesting that both the prepropeptide and prepeptidase C-terminal domains
were cleaved off to give the mature form. Analysis of N-terminal and C-terminal amino acid sequences of purified rAcpI showed
that the mature enzyme would be composed of 273 amino acids. The optimal pH and temperature for the caseinolytic activity
of the purified rAcpI were 9.0–9.5 and 45°C in 100 mM glycine–NaOH buffer. Calcium ions slightly enhanced the enzyme activity
and stability. The enzyme favorably hydrolyzed gelatin, collagen, and casein. AcpI from A. collagenimarina AC40T was also purified from culture broth, and its molecular mass was around 28 kDa, indicating that the cleavage manner of the
enzyme is similar to that in E. coli cells. 相似文献
13.
Kroniger Werner; Rennenberg Heinz; Tadros Monier H.; Polle Andrea 《Plant & cell physiology》1995,36(1):191-196
A manganese-containing superoxide dismutase (SOD; EC 1.15.1.1
[EC]
)was purified to electrophoretic homogeneity from seeds of Norwayspruce (Picea abies L.). The apparent molecular mass of thepurified enzyme was 86 kDa, as determined by gel filtration.The subunit molecular mass, estimated by SDS-polyacrylamidegel electrophoresis, was 22 kDa both in the presence and inthe absence of 2-mercaptoethanol. Thus, the native enzyme isa homotetramer with subunits that were not linked by disulfidebonds. The isoelectric point of this Mn-SOD was 5.5. The specificactivity of the Mn-SOD was strongly pH-dependent and was 400units per nmol SOD at pH 7.8 and 30 units per nmol SOD at pH10.4. The first 25 amino acid residues in the amino terminalregion of spruce Mn-SOD exhibited a high degree of sequencehomology to those of Mn-SODs from other organisms. In Mn-deficientneedles the activity of Mn-SOD was only half of that in non-deficientneedles, whereas the activity of CuZn-SOD was doubled. (Received May 20, 1994; Accepted October 31, 1994) 相似文献
14.
Purification and characterization of thylakoid-bound Mn-superoxide dismutase in spinach chloroplasts 总被引:5,自引:0,他引:5
Thylakoid-bound superoxide dismutase (SOD; EC 1.15.1.1) was solubilized by Triton X-100 from spinach and purified to a homogeneous state. The molecular weight of thylakoid-bound SOD was 52000; the enzyme was composed of two equal subunits. Its activity was not sensitive to cyanide and hydrogen peroxide, and the isolated SOD contained Mn, but neither Fe nor Cu. Thus, the thylakoid-bound SOD is a Mn-containing enzyme. The subunit molecular weight of thylakoid Mn-SOD is the highest among Mn-SODs isolated so far, a fact which might reflect its binding to the membranes. 相似文献
15.
Superoxide dismutases (SODs) promote a conversion of harmful reactive oxygen species (ROS) to relatively moderate forms, resulting in the extension of lifespan in the nematode Caenorhabditis elegans under caloric restriction. The lifespan of the rotifer Brachionus plicatilis is also markedly extended by caloric restriction. We, therefore, cloned cDNA encoding SOD activated with Mn (Mn SOD) from B. plicatilis and examined its expression pattern in rotifers raised with energy restricted diet. The full length deduced amino acid sequence of the rotifer Mn SOD showed 61% identity with the C. elegans ortholog. Four amino acid residues that are essential to the binding of this enzyme to Mn were conserved in the rotifer Mn SOD. Subsequently we examined the mRNA expression patterns of Mn SOD using highly sensitive quantitative real-time PCR for various rotifer populations that are likely to differ in their lifespans in experiments on calorie restricted diets. The accumulated mRNA levels of Mn SOD were found to increase in supposedly long-lived rotifers. These results suggest that Mn SOD is possibly related to the aging of B. plicatilis. 相似文献
16.
Wang Y Osatomi K Nagatomo Y Yoshida A Hara K 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2011,158(4):289-296
Manganese-superoxide dismutase (Mn-SOD) from Japanese flounder (Paralichthys olivaceus) hepatopancreas has been purified with high purification (781-fold) and recovery (10.8%). The molecular mass of the purified enzyme was estimated to be 26kDa by SDS-PAGE under reducing conditions. In activity staining by native-PAGE, the Japanese flounder Mn-SOD gave three active bands and exhibited KCN-insensitive activity. In addition, the electrophoretic mobility of this enzyme was observed to be faster than that of Japanese flounder Cu,Zn-SOD. On the other hand, the N-terminal amino acid sequence of this Mn-SOD was determined to be 16 amino acid residues, and the sequence showed high homology to other Mn-SODs but not Japanese flounder Cu,Zn-SOD. Analysis of nucleotide and deduced amino acid sequences revealed that the Mn-SOD cDNA consisted of a 64bp 5'-non-coding region, a 675bp open reading frame encoding 225 amino acids, and a 465bp 3'-non-coding region. The first 27 amino acids containing a mitochondria-targeting signal were highly conserved among other Mn-SODs. 相似文献
17.
Abdollah Ghasemian Mojtaba Tabatabaei Yazdi Zargham Sepehrizadeh Zohreh Tabatabaei Yazdi Gholamreza Zarrini 《World journal of microbiology & biotechnology》2009,25(5):773-779
A 1.6 kb gene encoding a cholesterol oxidase (choR) from a local isolate, Rhodococcus sp. PTCC 1633 was cloned into pET23a and the highly expressed recombinant enzyme was purified from the cell lysate of IPTG-induced
Escherichia coli BL21(DE3)pLysS with one-step absorption on cholesterol. The purified protein had a molecular mass of 55 kDa, isoelectric
point at about pH 9.0 and absorption peaks at 280, 380 and 460 nm, indicating that the enzyme is a flavoprotein. The optimum
pH and temperature for the recombinant enzyme were 7.0 and 50°C, respectively. Steady-state kinetic revealed that the cholesterol
oxidase had a K
m
of 32 μM. This study is the first report concerning expression and one-step purification of a gene encoding cholesterol oxidase
from Rhodococcus spp. This study revealed that this enzyme is a type II cholesterol oxidase. 相似文献
18.
《Free radical research》2013,47(6):519-531
A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC. The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu, Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae. The enzyme subunit has a pl of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm. The non reduced enzyme has a relative molecular mass of 95 kDa. The enzyme remained active after prolonged incubation at 70°C and was pH insensitive in the range 7-11. Potassium cyanide and diethyldithiocarbamate, known Cu, Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM). SOD activity was also detectable in culture filtrate of A. fumigatus. This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms. 相似文献
19.
We purified phospholipase D (PLD) enzyme from peanut seeds, and the PLD enzyme eluted as two distinct peak fractions on Mono-Q chromatography, the first of which was characterized. N-terminal sequencing indicated that the N-terminus was blocked. The molecular mass of the purified enzyme was estimated to be 92 kDa by SDS-PAGE. The pH optimum of the enzyme was 5.0, and the K
m value against its substrate phosphatidylcholine (PC), in the presence of 10 mM CaCl2 and 4 mM deoxycholate, was estimated to be 0.072 mM. The enzyme catalyzed two reactions, i.e., hydrolysis of PC generating phosphatidic acid (PA) and choline, and transphosphatidylation of the PA-moiety in the PC molecule to the acceptor glycerol, generating phosphatidylglycerol. Furthermore, we cloned two types of full-length cDNA, Ahpld1 and Ahpld2, each encoding distinct PLD molecules having 794 and 807 residues, respectively. The partial amino acid sequence of the purified PLD was consistent with the deduced sequence of AhPLD2. 相似文献
20.
Pseudonocardia sp. strain K1 is the only Gram-positive bacterium among the bacteria aerobically metabolizing polyethylene glycol (PEG). Generally, PEG is metabolized by an oxidative pathway in which a terminal alcohol group of PEG is oxidized to aldehyde and to carboxylic acid and then an ether bond is oxidatively cleaved. As the cell-free extract of Pseudonocardia sp. strain K1 has PEG dehydrogenase, PEG aldehyde dehydrogenase and diglycolic acid (DGA) dehydrogenase (DGADH) activities, all of which are constitutively formed, the strain has a metabolic pathway similar to that so far known. We purified an ether bond-splitting enzyme as DGADH. The molecular mass of the enzyme was estimated to be 55 kDa; and it consisted of two identical subunits. The enzyme oxidatively cleaved both an ether bond of PEG 3000 dicarboxylic acid and DGA. The N-terminal amino acid sequence of the purified enzyme had high homology with various superoxide dismutases and the enzyme had also superoxide dismutase activity. The atomic absorption spectrum showed that approximately one atom of Fe was included in each subunit of the enzyme. DGADH activity increased in the cells grown in a PEG medium supplemented with FeCl3. Thus, we concluded that the enzyme purified from Pseudonocardia sp. strain K1 is a new ether bond-splitting enzyme. 相似文献