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1.
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
2.
A Streptomyces sp. was isolated that produced novel thermoalkalotolerant cellulase activity after growth on crystalline cellulose at 50°C.
Three major components of the cellulases (CMCase, Avicelase and cellobiase) were produced with maximal activities (11.8, 7.8
and 3.9 IU/ml) and maximum specific activities 357, 276 and 118 IU/mg protein, respectively, after 120 h growth. Maximum CMCase
activity was between 50 and 60°C measured over 3 h. The enzyme also retained 88% of its maximum activity at 70°C and pH 5,
and 80% of the activity at pH 10 and 50°C when assayed after 1 h. After incubation at 40°C for 1 h with commercial detergent
(Tide) at pH 11, 95% activity was retained. The enzyme mixture produced glucose from crystalline cellulose. 相似文献
3.
Marinomonas sp. NJ522, isolated from Antarctic sea ice, produces a cold-active iron superoxide dismutase (SOD; EC 1.15.1.1). The purified
SOD was dimeric and had an approx. Mr of 48 kDa. Highest activity was detected from pH 8 to 10 and at 40 °C (assayed over
10 min). Activity at 0 °C was nearly 35% of the maximum activity.
Received 25 August 2005; Revisions requested 30 August 2005 and 26 September 2005; Revisions received 12 September 2005 and
25 October 2005; Accepted 1 November 2005 相似文献
4.
Purification and Partial Characterization of Alkaline Xylanase from Escherichia coli Carrying pCX311
Hiroshi Honda Toshiaki Kudo Koki Horikoshi 《Bioscience, biotechnology, and biochemistry》2013,77(11):3165-3169
Xylanase produced by E. coli HB 101 carrying plasmid pCX311, which contains the xylanase A gene of alkalophilic Bacillus sp. strain C-125, was purified by ammonium sulfate precipitation, DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The purified enzyme had a molecular weight of 43,000. The pH and temperature optima for its activity were 6~10 and 70°C, respectively. The enzyme retained full activity after incubation at 50°C for 10 min. These enzymatic properties of the xylanase were almost the same as those of xylanase A. But this enzyme was less stable than xylanase A at low pHs. Furthermore, we could purify a larger amount of alkaline xylanase from E. coli than from alkalophilic Bacillus sp. strain C-125. 相似文献
5.
Peña-Montes C González A Castro-Ochoa D Farrés A 《Applied microbiology and biotechnology》2008,78(4):603-612
Aspergillus nidulans PW1 produces an extracellular carboxylesterase activity that acts on several lipid esters when cultured in liquid media containing
olive oil as a carbon source. The enzyme was purified by gel filtration and ion exchange chromatography. It has an apparent
MW and pI of 37 kDa and 4.5, respectively. The enzyme efficiently hydrolyzed all assayed glycerides, but showed preference toward short-
and medium-length chain fatty acid esters. Maximum activity was obtained at pH 8.5 at 40°C. The enzyme retained activity after
incubation at pHs ranging from 8 to11 for 12 h at 37°C and 6 to 8 for 24 h at 37°C. It retained 80% of its activity after
incubation at 30 to 70°C for 30 min and lost 50% of its activity after incubation for 15 min at 80°C. Noticeable activation
of the enzyme is observed when Fe2+ ion is present at a concentration of 1 mM. Inhibition of the enzyme is observed in the presence of Cu2+, Fe3+, Hg2+, and Zn2+ ions. Even though the enzyme showed strong carboxylesterase activity, the deduced N-terminal amino acid sequence of the purified
protein corresponded to the protease encoded by prtA gene. 相似文献
6.
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
7.
Guimin Zhang Liangwei Mao Yueju Zhao Yanfen Xue Yanhe Ma 《Biotechnology letters》2010,32(12):1915-1920
A xylanase gene (xyn10) from alkaliphilic Bacillus sp. N16-5 was cloned and expressed in Pichia pastoris. The deduced amino acid sequence has 85% identity with xylanase xyn10A from B. halodurans and contains two potential N-glycosylation sites. The glycosylated Xyn10 with MW 48 kDa can hydrolyze birchwood and oatspelt xylan. The enzyme had optimum
activity at pH 7 and 70°C, with the specific activity of 92.5U/mg. The Xyn10 retained over 90% residual activity at 60°C for
30 min but lost all activity at 80°C over 15 min. Most tested ions showed no or slight inhibition effects on enzyme activity. 相似文献
8.
Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon
and energy sources to produce acetate, CO2, H2, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of
40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen
sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and
Fe2+. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the
rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively.
The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K
m values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the
physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols. 相似文献
9.
A new xylanase from thermoacidophilic Alicyclobacillus sp. A4 with broad-range pH activity and pH stability 总被引:1,自引:0,他引:1
Yingguo Bai Jianshe Wang Zhifang Zhang Peilong Yang Pengjun Shi Huiying Luo Kun Meng Huoqing Huang Bin Yao 《Journal of industrial microbiology & biotechnology》2010,37(2):187-194
We have identified a highly pH-adaptable and stable xylanase (XynA4) from the thermoacidophilic Alicyclobacillus sp. A4, a strain that was isolated from a hot spring in Yunnan Province, China. The gene (xynA4) that encodes this xylanase was cloned, sequenced, and expressed in Escherichia coli. It encodes a 338-residue polypeptide with a calculated molecular mass of 42.5 kDa. The deduced amino acid sequence is most
similar to (53% identity) an endo-1,4-β-xylanase from Geobacillus stearothermophilus that belongs to family 10 of the glycoside hydrolases. Purified recombinant XynA4 exhibited maximum activity at 55°C and
pH 7.0, had broad pH adaptability (>40% activity at pH 3.8–9.4) and stability (retaining >80% activity after incubation at
pH 2.6–12.0 for 1 h at 37°C), and was highly thermostable (retaining >90% activity after incubation at 60°C for 1 h at pH
7.0). These properties make XynA4 promising for application in the paper industry. This is the first report that describes
cloning and expression of a xylanase gene from the genus Alicyclobacillus. 相似文献
10.
Madanala R Gupta V Deeba F Upadhyay SK Pandey V Singh PK Tuli R 《Biotechnology letters》2011,33(10):2057-2063
A gene from Withania somnifera (winter cherry), encoding a highly stable chloroplastic Cu/Zn superoxide dismutase (SOD), was cloned and expressed in Escherichia coli. The recombinant enzyme (specific activity of ~4,200 U mg−1) was purified and characterized. It retained ~90 and ~70% residual activities after 1 h at 80 and 95°C, respectively. At
95°C, thermal inactivation rate constant (K
d) of the enzyme was 2.46 × 10−3 min−1 and half-life of heat inactivation was 4.68 h. The enzyme was stable against a broad pH range (2.5–11.0). It also showed
a high degree of resistance to detergent, ethanol and protease digestion. This recombinant Cu/Zn SOD could therefore have
useful applications. 相似文献
11.
Production,purification and characterisation of a novel halostable xylanase from Bacillus sp. NTU-06
Bacillus sp. NTU-06 was used to produce xylanase, which is an important industrial enzyme used in the pulp and paper industry. The enzyme was purified by fast protein liquid chromatography (FPLC) and had a molecular mass of 24 kDa. The enzyme was active over a concentration range of 0–20% sodium chloride in culture broth, although its activity was optimal in 5% sodium chloride. A salinity stability test showed that 43% of the enzyme activity was retained after 4 h in 20% sodium chloride. Xylanase activity was maximal at pH 8.0 and 40°C. The enzyme was somewhat thermostable, retaining 20% of the original activity after incubation at 70°C for 4 h. The xylanase had Km and Vmax values of 3.45 mg mL−1 and 387.3 µmol min−1mg−1, respectively. The deduced internal amino acid sequence of Bacillus sp. NTU-06 xylanase resembled the sequence of beta-1,4-endoxylanase, which is a member of glycoside hydrolase family 11. Some of the novel characteristics that make this enzyme potentially effective in xylan biodegradation are discussed. 相似文献
12.
Anaerobic fungi belonging to the family Neocallimastigaceae are native inhabitants in the rumen of the most herbivores, such
as cattle, sheep and goats. A member of this unique group, Neocallimastix sp. GMLF2 was isolated from cattle feces and screened for its xylanase encoding gene using polymerase chain reaction. The
gene coding for a xylanase (xyn2A) was cloned in Escherichia coli and expression was monitored. To determine the enzyme activity, assays were conducted for both fungal xylanase and cloned
xylanase (Xyl2A) for supernatant and cell-associated activities. Optimum pH and temperature of the enzyme were found to be
6.5 and 50°C, respectively. The enzyme was stable at 40°C and 50°C for 20 min but lost most of its activity when temperature
reached 60°C for 5-min incubation time. Rumen fungal xylanase was mainly released to the supernatant of culture, while cloned
xylanase activity was found as cell-associated. Multiple alignment of the amino acid sequences of Xyl2A with published xylanases
from various organisms suggested that Xyl2A belongs to glycoside hydrolase family 11. 相似文献
13.
Fusarium sp. BLB, which produces a strongly fibrinolytic enzyme, was isolated from plant leaf (Hibiscus). Fibrinolytic alkaline protease was purified from a culture filtrate of Fusarium sp. BLB by precipitation with (NH4)2SO4 and column chromatography with CM-Toyopearl 650M and Superdex 75. The purified enzyme was homogeneous on sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was 27,000 by SDS-PAGE. Maximum activity of protease was
observed at pH 9.5 and 50°C. Purified protease was active between pH 2.5 and 11.5 and was found to be stable up to 50°C. The
enzyme derived from Fusarium sp. BLB is useful for thrombolytic therapy because this enzyme showed pH resistance. The activity was inhibited by diisopropylfluorophosphate
and phenylmethylsulfonyl fluoride. The N-terminal amino acid sequence of the enzyme showed a similarity to those of proteases
from Fusarium sp., Streptomyces griseus, Bos taurus bovine, Katsuwo pelamis digestive tract, and Lumbricus rubellus. 相似文献
14.
Two degenerate primers established from the alignment of highly conserved amino acid sequences of bacterial dihydropyrimidinases
(DHPs) were used to amplify a 330-bp gene fragment from the genomic DNA of Bacillus sp. TS-23 and the amplified DNA was successfully used as a probe to clone a dhp gene from the strain. The open reading frame of the gene consisted of 1422 bp and was deduced to contain 472 amino acids
with a molecular mass of 52 kDa. The deduced amino acid sequence exhibited greater than 45% identity with that of prokaryotic
d-hydantoinases and eukaryotic DHPs. Phylogenetic analysis showed that Bacillus sp. TS-23 DHP is grouped together with Bacillus stearothermophilus d-hydantoinase and related to dihydroorotases and allantoinases from various organisms. His6-tagged DHP was over-expressed in Escherichia coli and purified by immobilized metal affinity chromatography to a specific activity of 3.46 U mg−1 protein. The optimal pH and temperature for the purified enzyme were 8.0 and 60 °C, respectively. The half-life of His6-tagged DHP was 25 days at 50 °C. The enzyme activity was stimulated by Co2+ and Mn2+ ions. His6-tagged DHP was most active toward dihydrouracil followed by hydantoin derivatives. The catalytic efficiencies (kcat/Km) of the enzyme for dihydrouracil and hydantoin were 2.58 and 0.61 s−1 mM−1, respectively. 相似文献
15.
A moderately halophilic alkalitolerant Bacillus sp. Strain TSCVKK, with an ability to produce extracellular halophilic, alkalitolerant, surfactant, and detergent-stable alpha-amylase was
isolated from soil samples obtained from a salt-manufacturing industry in Chennai. The culture conditions for higher amylase
production were optimized with respect to NaCl, substrate, pH, and temperature. Maximum amylase production of 592 mU/ml was
achieved in the medium at 48 h with 10% NaCl, 1% dextrin, 0.4% yeast extract, 0.2% tryptone, and 0.2% CaCl2 at pH 8.0 at 30 °C. The enzyme activity in the culture supernatant was highest with 10% NaCl at pH 7.5 and 55 °C. The amylase
that was partially purified by acetone precipitation was highly stable in various surfactants and detergents. Glucose, maltose,
and maltooligosaccharides were the main end products of starch hydrolysis indicating that it is an alpha-amylase. 相似文献
16.
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 H2O2 but not by NaN3, 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. 相似文献
17.
Lee MH Yang SJ Kim JW Lee HS Kim JW Park KH 《Extremophiles : life under extreme conditions》2007,11(3):537-541
A gene that encodes the enzyme Pyrococcus furiosus cyclodextrin glucanotransferase (PFCGT) was cloned in Escherichia
coli. PFCGT was highly expressed in recombinant E. coli after compensation for codon usage bias using the pRARE plasmid. Purified PFCGT was extremely thermostable with an optimal
temperature and pH of 95°C and 5.0, respectively, retaining 97% of its activity at 100°C. Incubation at 60°C for 20 min during
the purification process led to a 1.5-fold increase in enzymatic activity. A time course assay of the PFCGT reaction with
starch indicated that cyclic α-1,4-glucans with DPs greater than 20 were produced at the beginning of the incubation followed
by an increase in β-CD. The major final product of PFCGT cyclization was β-CD, and thus the enzyme is a β-CGTase. 相似文献
18.
The effect of inoculation withAzospirillum brasilense (strain 7001) on soil Eh under anaerobic conditions (N2 flux) was examined during 144 h at 26°C and 230 h at 18°C. The Eh values of the control (not inoculated) soil decreased to
approximately 80 or 140 mV in both cases, whereas after 24 h of anaerobic incubation, the Eh of theAzospirillum-inoculated soil remained at higher values. After 144 and 230 h of anaerobic incubation, the denitrifying activity (measured
in anaerobiosis with excess of e- acceptor and donor) in the inoculated soil was seven and three times lower respectively, than in the non-inoculated soil.
This indicates thatAzospirillum may affect the soil Eh and consequently any highly Eh-dependent microbial activity, such as denitrification. 相似文献
19.
Effects of short-term heat stress on oxidative damage and responses of antioxidant system in Lilium longiflorum 总被引:2,自引:0,他引:2
This paper aims to determine the changes in reactive oxygen species (ROS) and the responses of the lily (Lilium longiflorum L.) antioxidant system to short-term high temperatures. Plants were exposed to three levels of heat stress (37°C, 42°C, 47°C)
for 10 h when hydrogen peroxide (H2O2) and superoxide (O2−) production rate along with membrane injury indexes, and changes in antioxidants were measured. Compared with the control
(20°C), electrolyte leakage and MDA concentration varied slightly after 10 h at 37°C and 42°C, while increased significantly
at 47°C. During 10 h at 37°C and 42°C, antioxidant enzyme activities, such as SOD, POD, CAT, APX and GR, were stimulated and
antioxidants (AsA and GSH concentrations) maintained high levels, which resulted in low levels of O2− and H2O2 concentration. However, after 10 h at 47°C, SOD, APX, GR activities and GSH concentration were similar to the controls, while
POD, CAT activities and AsA concentration decreased significantly as compared with the control, concomitant with significant
increase in O2− and H2O2 concentrations. In addition, such heat-induced effects on antioxidant enzymes were also confirmed by SOD and POD isoform,
as Cu/ZnSOD maintained high stability under heat stress and the intensity of POD isoforms reduced with the duration of heat
stress, especially at 47°C. It is concluded that in lily plants, the oxidative damage induced by heat stress was related to
the changes in antioxidant enzyme activities and antioxidants. 相似文献
20.
Aim: To determine the minimal conditions (temperature–time), necessary to achieve set sanitation targets for selected microbial indicators during the continuous thermal treatment of pig slurry. Methods and Results: The effectiveness of thermal treatment between 55 and 96°C was studied using Escherichia coli, enterococci, sulfite‐reducing Clostridia (SRC), mesophilic culturable bacteria (MCB), F+‐specific and somatic phages. Identification of SRC and MCB was performed using 16S rRNA gene analysis. Ten minutes at 70°C or 1 h at 60°C was sufficient to reduce the vegetative bacteria by 4–5 log10, but it had little effect on somatic phages nor on spore formers, dominated by Clostridium sp. At 96°C, somatic phages were still detected, but there was a reduction of 3·1 log10 for SRC and of 1·4 log10 for MCB. At 96°C, Clostridium botulinum was identified among the thermotolerant MCB. Conclusion: Only those hygienic risks relating to mesophilic vegetative bacteria can be totally eliminated from pig slurry treated at 60°C (60 min) or 70°C (<10 min). Significance and Impact of the Study: Hygiene standards based on the removal of the indicators E. coli and enterococci can easily be met by treatment as low as 60°C (enabling, a low‐cost treatment using heat recovery). However, even at 96°C, certain pathogens may persist. 相似文献