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1.
Hydrogenase was solubilized from the cytoplasmic membrane fraction of betaine-grown Sporomusa sphaeroides, and the enzyme was purified under oxic conditions. The oxygen-sensitive enzyme was partially reactivated under reducing conditions,
resulting in a maximal activity of 19.8 μmol H2 oxidized min–1 (mg protein)–1 with benzyl viologen as electron acceptor and an apparent K
m
value for H2 of 341 μM. The molecular mass of the native protein estimated by native PAGE and gel filtration was 122 and 130 kDa, respectively.
SDS-PAGE revealed two polypeptides with molecular masses of 65 and 37 kDa, present in a 1:1 ratio. The native protein contained
15.6 ± 1.7 mol Fe, 11.4 ± 1.4 mol S2–, and 0.6 mol Ni per mol enzyme. The hydrogenase coupled with viologen dyes, but not with other various artificial electron
carriers, FAD, FMN, or NAD(P)+. The amino acid sequence of the N-termini of the subunits showed a high degree of similarity to eubacterial membrane-bound
uptake hydrogenases. Washed membranes catalyzed a H2-dependent cytochrome b reduction at a rate of 0.18 nmol min–1 (mg protein)–1.
Received: 7 September 1995 / Accepted: 4 December 1995 相似文献
2.
Emi Miyamoto Yuri Tanioka Ayako Nishizawa-Yokoi Yukinori Yabuta Kouhei Ohnishi Haruo Misono Shigeru Shigeoka Yoshihisa Nakano Fumio Watanabe 《Archives of microbiology》2010,192(6):437-446
Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4°C with
10 μM 5′-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular
mass of the enzyme was calculated to be 149.0 kDa ± 5.0 kDa using Superdex 200 gel filtration. SDS–polyacrylamide gel electrophoresis
of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa ± 3.0 kDa, indicating that
the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5′-deoxyadenosylcobalamin
per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular
mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore,
we studied some properties and physiological function of the Euglena enzyme. 相似文献
3.
Uricase (urate: oxygen oxidoreductase; EC 1.7.3.3) from the rust Puccinia recondita was purified to electrophoretic homogeneity. Preparations with a specific activity of 8.4 U/mg were used for characterization
of the enzyme, which showed a strong similarity to other plant and fungal urate oxidases. The enzyme had a pH optimum of 9.0,
a K
m
of 35 μM for urate, and it was inhibited only by oxonate and xanthine. A molecular mass of 152 kDa was estimated for the
native protein. SDS-PAGE analysis revealed a striking difference to most urate oxidases, since two different-sized subunits
were detected. These results suggest that P. recondita uricase is a tetramer with two types of subunits.
Received: 21 February 2001 / Accepted: 30 July 2001 相似文献
4.
A gene-encoding imidase was isolated from Pseudomonas putdia YZ-26 genomic DNA using a combination of polymerase chain reaction and activity screening the recombinant. Analysis of the
nucleotide sequence revealed that an open reading frame (ORF) of 879 bp encoded a protein of 293 amino acids with a calculated
molecular weight of 33712.6 kDa. The deduced amino-acid sequence showed 78% identity with the imidase from Alcaligenes eutrophus 112R4 and 80% identity with N-terminal 20 amino-acid imidase from Blastobacter sp. A17p-4. Next, the ORF was subcloned into vector pET32a to form recombinant plasmid pEI. The enzyme was overexpressed
in Escherichia coli and purified to homogeneity by Ni2+–NTA column, with 75% activity recovery. The subunit molecular mass of the recombinant imidase as estimated by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis was approximately 36 kDa, whereas its functional unit was approximately 141 kDa
with four identical subunits determined by size-exclusion chromatography. The purified enzyme showed the highest activity
and affinity toward succinimide, and some other substrates, such as dihydrouracil, hydantoin, succinimide, and maleimde, were
investigated. 相似文献
5.
Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme 总被引:9,自引:0,他引:9
C. G. van Ginkel G. B. Rikken A. G. M. Kroon S. W. M. Kengen 《Archives of microbiology》1996,166(5):321-326
A novel enzyme that catalyzes the disproportionation of chlorite into chloride and oxygen was purified from a gram-negative
bacterium, strain GR-1 to homogeneity. A four-step purification procedure comprising Q-Sepharose, hydroxyapatite, and phenyl-Superose
chromatography and ultrafiltration resulted in a 13.7-fold purified enzyme with a final specific activity of 2.0 mmol min–1 (mg protein)–1. The dismutase obeyed Michaelis-Menten kinetics. The V
max and K
m calculated for chlorite were 2,200 U (mg protein)–1 and 170 μM, respectively. Dismutase activity was inhibited by hydroxylamine, cyanide, and azide, but not by 3-amino-1,2,4-triazole.
Chlorite dismutase had a molecular mass of 140 kDa and consisted of four 32-kDa subunits. The enzyme was red-colored and had
a Soret peak at 392 nm. Per subunit, it contained 0.9 molecule of protoheme IX and 0.7 molecule of iron. Chlorite dismutase
displayed maxima for activity at pH 6.0 and 30° C.
Received: 9 April 1996 / Accepted: 12 August 1996 相似文献
6.
Chunli Zheng Yanfei Zhang Yuandong Liu Anna Wu Lexian Xia Jia Zeng Jianshe Liu Guanzhou Qiu 《Current microbiology》2009,58(6):586-592
Adenosine 5′-phosphosulfate (APS) reductase is a key enzyme involved in the pathways of sulfate reduction and sulfide oxidation
in the biological sulfur cycle. In this study, the gene of APS reductase from Acidithiobacillus ferrooxidans was cloned and expressed in Escherichia coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. The molecular mass of the
recombinant APS reductase was determined to be 28 kDa using SDS-PAGE. According to optical and EPR spectra results of the
recombinant protein confirmed that the iron–sulfur cluster inserted into the active site of the protein. Site-directed mutation
for the enzyme revealed that Cys110, Cys111, Cys193, and Cys196 were in ligation with the iron–sulfur cluster. The [Fe4S4] cluster could be assembled in vitro, and exhibited electron transport and redox catalysis properties. As we know so far,
this is the first report of expression in E. coli of APS reductase from A. ferrooxidans. 相似文献
7.
8.
For the first time, a β-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on SDS-PAGE. The recombinant enzyme had a molecular
mass of 380 kDa with subunits of 97 kDa. The maximum activity was at pH 6.4 and 50 °C over a 5 min assay. The purified enzyme
was stable from pH 5.6–8.0, had a half life of 1 h at 45 °C. The β-glucosidase had a Km of 0.2 mM for p-nitrophenyl-β-D-glucopyranoside. 相似文献
9.
Pereira MG Guimarães LH Furriel RP Polizeli Mde L Terenzi HF Jorge JA 《Journal of microbiology (Seoul, Korea)》2011,49(5):809-815
The thermophilic fungus Malbranchea pulchella var. sulfurea produced good amounts of extracellular trehalase activity when grown for long periods on starch, maltose or
glucose as the main carbon source. Studies with young cultures suggested that the main role of the extracellular acid trehalase
is utilizing trehalose as a carbon source. The specific activity of the purified enzyme in the presence of manganese (680
U/mg protein) was comparable to that of other thermophilic fungi enzymes, but many times higher than the values reported for
trehalases from other microbial sources. The apparent molecular mass of the native enzyme was estimated to be 104 kDa by gel
filtration and 52 kDa by SDS-PAGE, suggesting that the enzyme was composed by two subunits. The carbohydrate content of the
purified enzyme was estimated to be 19 % and the pi was 3.5. The optimum pH and temperature were 5.0–5.5 and 55° C, respectively.
The purified enzyme was stimulated by manganese and inhibited by calcium ions, and insensitive to ATP and ADP, and 1 mM silver
ions. The apparent KM values for trehalose hydrolysis by the purified enzyme in the absence and presence of manganese chloride were 2.70±0.29 and
2.58±0.13 mM, respectively. Manganese ions affected only the apparent Vmax, increasing the catalytic efficiency value by 9.2-fold. The results reported herein indicate that Malbranchea pulchella produces a trehalase with mixed biochemical properties, different from the conventional acid and neutral enzymes and also
from trehalases from other thermophilic fungi. 相似文献
10.
Mika Murata Masaru Nagai Makoto Takao Akira Suzuki Takuo Sakai Takao Terashita 《Mycoscience》2001,42(5):479-482
Trehalase from the culture filtrate ofLentinula edodes was purified and characterized. Molecular masses were estimated to be 158 kDa and 79–91 kDa by gel filtration and SDS-PAGE
under the reduced condition, respectively. The enzyme was composed of two identical subunits and contained carbohydrate molecules.
The optimum temperature and pH were obtained at around 40°C and pH 5.0, respectively. The enzyme was stable up to 40°C and
in a range pH of 4–10 at 30°C. It cleaved α-1,1 linkages of trehalose, but not α-1,4, α-1,6 or β-1,4 glycosyl linkages, and
was defined as an acid trehalase. 相似文献
11.
The chemolithoautotroph, Arthrobacter sp.15b oxidizes arsenite to arsenate using a membrane bound arsenite oxidase. The enzyme arsenite oxidase is purified to
its homogeneity and identified using MALDI-TOF MS analysis. Upon further characterization, it was observed that the enzyme
is a heterodimer showing native molecular mass as ~100 kDa and appeared as two subunits of ~85 kDa LSU and 14 kDa SSU on SDS–PAGE.
The V
max and K
m values of the enzyme was found to be 2.45 μM (AsIII)/min/mg) and 26 μM, respectively. The purified enzyme could withstand
wide range of pH and temperature changes. The enzyme, however, gets deactivated in the presence of 1 mM of DEPC suggesting
the involvement of histidine at the binding site of the enzyme. The peptide analysis of large sub unit of the enzyme showed
close match with the arsenite oxidases of Burkholderia sp. YI019A and arsenite oxidase, Mo-pterin containing subunit of Alcaligenes
faecalis. The small subunit, however, differed from other arsenite oxidases and matched only with 2Fe–2S binding protein of Anaplasma
phagocytophilum. This indicates that Rieske subunits containing the iron–sulfur clusters present in the large as well as small subunits of
the enzyme are integral part of the protein. 相似文献
12.
A C S Rizzatti J A Jorge H F Terenzi C G V Rechia M L T M Polizeli 《Journal of industrial microbiology & biotechnology》2001,26(3):156-160
A β-D-xylosidase was purified from cultures of a thermotolerant strain of Aspergillus phoenicis grown on xylan at 45°C. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-100. The
purified enzyme was a monomer of molecular mass 132 kDa by gel filtration and SDS-PAGE. Treatment with endoglycosidase H resulted
in a protein with a molecular mass of 104 kDa. The enzyme was a glycoprotein with 43.5% carbohydrate content and exhibited
a pI of 3.7. Optima of temperature and pH were 75°C and 4.0–4.5, respectively. The activity was stable at 60°C and had a K
m of 2.36 mM for p-nitrophenyl-β-D-xylopiranoside. The enzyme did not exhibit xylanase, cellulase, galactosidase or arabinosidase activities. The purified enzyme
was active against natural substrates, such as xylobiose and xylotriose. Journal of Industrial Microbiology & Biotechnology (2001) 26, 156–160.
Received 23 June 2000/ Accepted in revised form 29 September 2000 相似文献
13.
A fragment coding for a putative extracellular α-amylase, from the genomic library of the yeast Saccharomycopsis fibuligera KZ, has been subcloned into yeast expression vector pVT100L and sequenced. The nucleotide sequence revealed an ORF of 1,485 bp
coding for a 494 amino acid residues long protein with 99% identity to the α-amylase Sfamy from S. fibuligera HUT 7212. The S. fibuligera KZ α-amylase (Sfamy KZ) belongs to typical extracellular fungal α-amylases classified in the glycoside hydrolase family 13,
subfamily 1, as supported also by clustering observed in the evolutionary tree. Sfamy KZ, in addition to the essential GH13
α-amylase three-domain arrangement (catalytic TIM barrel plus domains B and C), does not contain any distinct starch-binding
domain. Sfamy KZ was expressed as a recombinant protein in Saccharomyces cerevisiae and purified to electrophoretic homogeneity. The enzyme had a molecular mass 53 kDa and contained about 2.5% of carbohydrate.
The enzyme exhibited pH and temperature optima in the range of 5–6 and 40–50 °C, respectively. Stable adsorption of the enzyme
to starch granules was not detected but a low degradation of raw starch in a concentration-dependent manner was observed. 相似文献
14.
Valeria Calandrelli Agata Gambacorta Ida Romano Vito Carratore Licia Lama 《World journal of microbiology & biotechnology》2008,24(10):2269-2275
A novel thermo-alkali-stable catalase–peroxidase from Oceanobacillus oncorhynchi subsp. incaldaniensis subsp. nov., strain 20AG, was purified and characterized. The protein purified from the cells resulted in 110-fold purification
with a specific activity of 35,000 U/mg. The enzyme consisted of four identical subunits of 72 kDa as determined by SDS-PAGE
and the total molecular mass measured by gel filtration was 280 kDa. The heme content was determined to be 1 heme per homodimer.
The enzyme showed a Soret peak at 406 nm in the oxidized form and was easily reduced by dithionite. The enzyme showed an appreciable
peroxidase activity in addition to high catalase activity. The behaviour of this heme-enzyme was typical of the class of prokaryotic
catalase–peroxidases, which are sensitive to cyanide and insensitive to the eukaryotic catalase inhibitor 3-amino-1,2,4-triazole.
The enzyme was active over a temperature range from 30 to 60°C and a pH range from 5 to 10, with an optimum pH about 9.0 and
an optimum temperature of 40°C. The enzyme was stable in the pH range of 5.0 to 10.0 after 1 h of treatment at 40°C. The enzyme
was stable for 24 h at 40°C with a half-life of 4 h 60°C. The enzyme had a K
m of 24 mM for hydrogen peroxide. The amino terminal amino acid sequence of the catalase–peroxidase from strain 20AG was SEKRKMTTAFGA
and it showed no homology with other catalases. 相似文献
15.
Bin Wu Licheng Wu Daijie Chen Zhijun Yang Minyu Luo 《Journal of industrial microbiology & biotechnology》2009,36(3):451-459
A novel fibrinolytic enzyme from Fusarium sp. CPCC 480097, named Fu-P, was purified to electrophoretic homogeneity using ammonium sulfate precipitation and ion exchange
and gel filtration chromatography. Fu-P, a single protein had a molecular weight of 28 kDa, which was determined by SDS-PAGE
and gel filtration chromatography. The isoelectric point of Fu-P determined by isoelectric focusing electrophoresis (IEF)
was 8.1, and the optimum temperature and pH value were 45°C and 8.5, respectively. Fu-P cleaved the α-chain of fibrin (ogen)
with high efficiency, and the β-chain and γ-γ (γ-)-chain with lower efficiency. Fu-P activity was inhibited by EDTA and PMSF,
and the enzyme exhibited a high specificity for the chymotrypsin substrate S-2586. Fu-P was therefore identified as a chymotrypsin-like
serine metalloprotease. The first 15 amino acids of the N-terminal sequence of Fu-P were Q-A-S–S-G-T-P-A-T-I-R-V-L-V–V and
showed no homology with that of other known fibrinolytic enzymes. This protease may have potential applications in thrombolytic
therapy and in thrombosis prevention. 相似文献
16.
Mabrouk SB Messaoud EB Ayadi D Jemli S Roy A Mezghani M Bejar S 《Molecular biotechnology》2008,38(3):211-219
A gene encoding maltogenic amylase from acidic Bacillus sp. US149 (maUS149) was cloned, sequenced and over-expressed in Escherichia coli. The nucleotide sequence analysis revealed an open reading frame (ORF) of 1749 bp encoding a protein of 582 residues. The
alignment of deduced amino acid sequence revealed a relatively low homology with the already reported maltogenic amylases.
In fact, its highest identity, of only 60%, was found with the maltogenic amylase of Thermus sp. IM6501. The recombinant enzyme (MAUS149) was found to be intracellular and was purified to homogeneity from the cell
crude extract with a yield of 23%. According to PAGE analysis, under reducing and non-reducing conditions, the recombinant
enzyme has an apparent molecular weight of 135 kDa and is composed of two identical subunits of 67.5 kDa each. The maximum
activity was obtained at 40°C and pH 6.5. MAUS149 could be classified as a maltogenic amylase since it produces mainly maltose
from starch, maltose and glucose from β-cyclodextrin, and panose from pullulan. 相似文献
17.
Hydrogenase enzyme from the unicellular marine green alga Tetraselmis kochinensis NCIM 1605 was purified 467 fold to homogeneity. The molecular weight was estimated to be approximately 89kDa by SDS-PAGE. This enzyme consists of two subunits with molecular masses of approximately 70 and approximately 19kDa. The hydrogenase was found to contain 10g atoms of Fe and 1g of atom of Ni per mole of protein. The specific activity of hydrogen evolution was 50micromol H(2)/mg/h of enzyme using reduced methyl viologen as an electron donor. This hydrogenase enzyme has pI value approximately 9.6 representing its alkaline nature. The absorption spectrum of the hydrogenase enzyme showed an absorption peak at 425nm indicating that the enzyme had iron-sulfur clusters. The total of 16 cysteine residues were found per mole of enzyme under the denaturing condition and 20 cysteine residues in reduced denatured enzyme indicating that it has two disulfide bridges. 相似文献
18.
A proline iminopeptidase (EC. 3.4.11.5) was isolated from shoots of 3 day old seedlings. The purification procedure consisted
of 5 steps: acid precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on Sepharose CL 6B, twice repeated
hydrophoic chromatography on Phenyl-Sepharose HP. The enzyme was purified 404.8-fold, with the specific activity of 8.5 units
mg−1 of protein with recovery yield of 3%. The purified enzyme had a molecular mass of 225 kDa estimated by gel filtration and
55.4 kDa by SDS PAGE. This indicates that native enzyme is composed of four subunits. The enzyme was specific for proline
β-naphtylamide among various amino acid β-naphtylamides.
An optimal activity was observed at 37 °C at pH 7.75. The enzyme was thermostable up to 37 °C for 30 min. The enzyme was strongly
inhibited by pHMB, E-64, heavy metal ions and partially by PMSF, DFP. The results suggest that cysteine and serine residues
may participate in the enzyme activity. 相似文献
19.
E S Guo F Liu S Chen J Wang Y Li D 《Bioscience, biotechnology, and biochemistry》2007,71(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
20.
Lectins are carbohydrate-binding proteins present in a wide variety of plants and animals, which serve various important physiological
functions. A soluble β-galactoside binding lectin has been isolated and purified to homogeneity from buffalo brain using ammonium
sulphate precipitation (40–70%) and gel permeation chromatography on Sephadex G50–80 column. The molecular weight of buffalo brain lectin (BBL) as determined by SDS-PAGE under reducing and non-reducing conditions
was 14.2 kDa, however, with gel filtration it was 28.5 kDa, revealing the dimeric form of protein. The neutral sugar content
of the soluble lectin was estimated to be 3.3%. The BBL showed highest affinity for lactose and other sugar moieties in glycosidic
form, suggesting it to be a β-galactoside binding lectin. The association constant for lactose binding as evidenced by Scatchard
analysis was 6.6 × 103 M−1 showing two carbohydrate binding sites per lectin molecule. A total inhibition of lectin activity was observed by denaturants
like guanidine HCl, thiourea and urea at 6 M concentration. The treatment of BBL with oxidizing agent destroyed its agglutination
activity, abolished its fluorescence, and shifted its UV absorption maxima from 282 to 250 nm. The effect of H2O2 was greatly prevented by lactose indicating that BBL is more stable in the presence of its specific ligand. The purified
lectin was investigated for its brain cell aggregation properties by testing its ability to agglutinate cells isolated from
buffalo and goat brains. Rate of aggregation of buffalo brain cells by purified protein was more than the goat brain cells.
The data from above study suggests that the isolated lectin may belong to the galectin-1 family but is glycosylated unlike
those purified till date. 相似文献