共查询到20条相似文献,搜索用时 15 毫秒
1.
A ferredoxin-dependent nitrite reductase from Spinacea oleracea was purified approximately 180-fold, with a specific activity of 285 units/mg protein. This purified enzyme also had methyl viologen-dependent nitrite reductase activity, with a specific activity of 164 units/mg protein. After disc electrophoresis with polyacrylamide gel, the purified enzyme showed one major and one minor protein band.The molecular weight of the enzyme was estimated to be 86,000 from Ultrogel filtration. This purified enzyme in oxidized form had absorption peaks at 278, 390, 573 and 690 nm. The absorbance ratios, A390: A278 and A673: A390 were 0.61 and 0.37, respectively.By applying the purified enzyme to DEAE-Sephadex A–50 column chromatography, the ferredoxin-dependent nitrite reductase activity was selectively decreased. However, the methyl viologen-dependent nitrite reductase activity was increased, with a specific activity of 391 units/mg protein. This modified enzyme was homogeneous by disc electrophoresis with polyacrylamide gel. 相似文献
2.
Isocitrate lyase has been purified to homogeneity, as determined by SDS-polyacrylamide gel electrophoresis and subsequent silver staining, fromEscherichia coli D5H3G7. The enzyme was found to have a subunit molecular weight of 48,000 and a native molecular weight of 188,000 as determined by gel filtration chromatography. Thus, the enzyme appears to have tetrameric structure. The isoelectric point was determined to be 4.6, and the enzyme displayed a pH optimum at 7.3. The Km of isocitrate lyase forthreo-Ds-isocitrate was determined to be 8 M. The purification procedure is highly reproducible and results in a 39% net yield of purified protein. 相似文献
3.
Marcelo Desimone Martina Krüger Tim Wessel Marco Wehofsky Ralf Hoffmann Edgar Wagner 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2000,737(1-2)
Aminopeptidases catalyze the cleavage of amino acids from the amino terminus of protein or peptide substrates. Although some aminopeptidase activities have been found in plant chloroplasts, the identity of these proteins remains unclear. In this work, we report the purification to apparent homogeneity of a soluble aminopeptidase from isolated barley chloroplasts which preferentially degraded alanyl-p-nitroanilide (Ala-pNA). After organelle isolation in a density gradient and precipitation of soluble proteins with ammonium sulfate, the proteins were purified in three consecutive steps including hydrophobic interaction, gel permeation and ion-exchange chromatographies. The purified enzyme appeared as a single band with a Mr of 84 000 in sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The Mr of the native enzyme was estimated to be 93 000 by gel permeation chromatography, suggesting that the protein is a monomer. Mass spectrometry analysis of tryptic digests indicates that the primary structure of the protein has not been reported previously. The enzyme was characterized as a metalloprotease as it could be totally inhibited by 1,10-phenanthroline. Strong inhibition could also be observed using the specific aminopeptidase inhibitors amastatin and bestatin. Besides Ala-pNA, the purified protein could also cleave with decreasing activity glycyl-pNA, leucyl-pNA, lysyl-pNA, methionyl-pNA and arginyl-pNA. The possible physiological role of this enzyme in the chloroplast stroma is discussed. 相似文献
4.
Jinender Jain Anil Kumar Davender Singh 《Preparative biochemistry & biotechnology》2013,43(8):718-724
AbstractA novel protease-resistant and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 was purified 36-fold to homogeneity with a combination of ammonium sulfate precipitation followed by Q-Sepharose and Sephadex G-50 chromatographic techniques. The estimated molecular mass of the purified phytase was 46?kDa by electrophoresis with optimal activity at pH 7.0 and 70?°C. About 19% of original activity was maintained at 80?°C for 10?min. Phytase activity was stimulated in presence of surfactants like Tween-20, Tween-80, and Triton X-100 and metal ions like Ca+2, K+, and Co+2 and it was inhibited by SDS and Mg+2, Al+2, and Fe+2. Purified enzyme showed specificity to different salts of phytic acid and values of Km and Vmax were 0.293?mM and 11.49 nmoles s?1, respectively for sodium phytate. The purified enzyme was resistant to proteases (trypsin and pepsin) that resulted in amelioration of food nutrition with simultaneous release of inorganic phosphate, reducing sugars, and soluble protein. 相似文献
5.
Galactinol: raffinose-6-galactosyltransferase (EC 2.4.1.67), a stachyose synthase, was extracted from mature leaves of Cucumis melo cv. Ranjadew and was purified to homogeneity by (NH4)2SO4 precipitation, ion-exchange chromatography, gel-filtration and non-denaturing polyacrylamide gel electrophoresis. A specific activity of 516 kat · mg-1 and a 160-fold purification was achieved. The pH optimum of the enzyme reaction was found to be 6.8 in sodium-phosphate buffer, and the temperature optimum 32° C. The purified enzyme was very sensitive towards SH-poisons but its reaction was hardly affected by changes in the ion composition of the assay medium. The two-substrate enzyme was specific for galactinol and raffmose; uridine-diphosphate galactose and p-nitrophenyl--d-galactoside as well as melibiose were not accepted by the purified enzyme. Stachyose synthesis was competitively inhibited by concentrations >4 mM raffinose as well as 2.5 mM galactinol. The K
m values determined under non-saturating conditions were 3.3 mM for raffinose and 7.7 mM for galactinol. Myoinositol was a strong competitive inhibitor with a K
i of 1.8mM. Galactinol was hydrolyzed in the absence of raffinose with a K
m of 0.8 mM. The pure enzyme is a protein with a molecular weight of at least 95 kDa and an isoelectric point of 5.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two subunits of 45 and 50 kDa. Polyclonal antibodies from rabbit were obtained which were specific for the native enzyme but cross-reacted with other proteins separated under denaturing conditions.Abbreviations DEAE
diethylaminoethyl
- DTT
dithiothreitol
- FPLC
fast protein liquid chromatography
- HPLC
high-performance liquid chromatography
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecyl sulfate
This work was supported by Deutsche Forschungsgemeinschaft. The gift of galactinol by Dr. T. Schweizer (Nestlé, Switzerland) is gratefully acknowledged. 相似文献
6.
A multifunctional Ca2+/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca2+/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca2+/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca2+/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca2+/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca2+/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca2+/calmodulin-dependent protein kinase of Candida albicans. 相似文献
7.
Purification and Characterization of Carbonic Anhydrase from Ağrı Balık Lake Trout Gill (Salmo trutta labrax) and Effects of Sulfonamides on Enzyme Activity
下载免费PDF全文
![点击此处可从《Journal of biochemical and molecular toxicology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Ramazan Demirdag Veysal Comakli Muslum Kuzu Emrah Yerlikaya Murat Şentürk 《Journal of biochemical and molecular toxicology》2015,29(3):123-128
Carbonic anhydrase (CA) was purified from A?r? Bal?k Lake trout gill (fCA) by affinity chromatography on a sepharose 4B‐tyrosine‐sulfanilamide column. The fCA enzyme was purified with about a 303.9 purification factor, a specific activity 4130.4 EU (mg‐protein)–1, and a yield of 79.3 by using sepharose‐4B‐l tyrosine‐sulfanilamide affinity gel chromatography. The molecular weight determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was found to be about 29.9 kDa. The kinetic parameters, KM and Vmax were determined for the 4‐nitrophenyl acetate hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CA enzymes. The Ki constants for mafenide ( 1 ), p‐toluenesulfonamide ( 2 ), 2‐bromo‐benzene sulfonamide ( 3 ), 4‐chlorobenzene sulfonamide ( 4 ), 4‐amino‐6‐chloro‐1–3 benzenedisulfonamide ( 5 ), sulfamethazine ( 6 ), sulfaguanidine ( 7 ), sulfadiazine ( 8 ), and acetozazolamide ( 9 ) were in the range of 7.5–108.75 μM. 相似文献
8.
Koshi Arai Tetsu Ando Sadahiro Tatsuki Kenji Usui Yoshiko Ohguchi Matsuo Kurihara 《Bioscience, biotechnology, and biochemistry》2013,77(12):3165-3168
Lipoxygenase-3, the major component of the enzyme in rice grain, was purified 2980-fold with a yield of 7% from embryos. The purified enzyme had a specific activity of 280 μmol O2 formed/min per mg protein. This enzyme was inactivated by SH compounds, such as cysteine and glutathione. The inactivation was prevented by the addition of catalase or replacement of the air by N2 gas. These two treatments were also effective for the stable storage of the purified enzyme. The molecular weights measured by sodium dodecyl sulfate gel and gradient gel electrophoresis were 93,000 and 89,000, respectively, indicating that the enzyme is a single polypeptide chain. The purified enzyme contained 0.73 Fe atom per molecule. The absorption spectrum suggested that the enzyme is a non-heme iron protein. Some similarities in amino-acid composition were observed between rice, soybean, and pea lipoxygenases. The purified enzyme specifically produced 9-d-hydroperoxy-10,12(E,Z)-octadecadienoic acid when linoleic acid was used as a substrate. 相似文献
9.
Thermostable β‐galactosidase from Bacillus coagulans RCS3 was purified by successive column chromatography using DEAE‐cellulose and Sephadex G‐50. Immobilization of the purified enzyme was studied with DEAE‐cellulose and calcium alginate. The efficiency of β‐galactosidase retention was 87 % with DEAE‐cellulose (17 mg protein/mL of matrix) and 80 % with calcium alginate (2.2 mg protein/g bead). Comparative studies of immobilization displayed a shift in the optimum temperature from 65 °C to 70 °C provoked by DEAE‐cellulose, although no effect was observed with calcium alginate. The heat inactivation curve revealed an improvement in the stability (t1/2 of 14.5 h for the immobilized enzyme as compared to 2 h for the free enzyme at 65 °C) in a calcium alginate system. This immobilized enzyme has a wide pH stability range (6.5–11). β‐Galactosidase immobilized by DEAE‐cellulose and calcium alginate allowed a 57 and 70 % lactose hydrolysis, respectively, to be achieved within 48 h after repeated use for twenty times. 相似文献
10.
An intracellular glucoamylase (E.C. 3.2.1.3) was purified
to homogeneity from Lactobacillus amylovorus on a Fast Protein Liquid
Chromatography System (FPLC) with a Mono Q ion-exchanger and two Superose 12
gel filtration columns arranged in series. The enzyme activity was quantified
with a specific, chromogenic substrate, p-nitrophenyl-β-maltoside.
Preparative gel electrophoresis was then used to further purify active enzyme
fractions. Native polyacrylamide gel electrophoresis (Native-PAGE) and sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified
enzyme showed a single protein band of molecular weight 47 kDa. Glucoamylase
activity of the purified protein was confirmed by its ability to degrade
starch on a 0.025% starch-polyacrylamide gel stained with
I2/KI. Glucoamylase exhibited optimum catalytic activity at pH 6.0
and 45°C, and the enzyme had an isoelectric point near 4.39. The
glucoamylase contained high levels of hydrophilic amino acids, comparable to
fungal glucoamylases.
Received: 12 July 1996 / Accepted: 10 September 1996 相似文献
11.
The production of an extracellular chitin deacetylase (CDA) produced by Aspergillus flavus under solid-substrate fermentation (SSF) using wheat bran as substrate was optimized using statistical methods. The CDA production in SSF increased 1.79-fold in comparison to the unoptimized basal level medium. It was purified to a final purity of 3.94-fold by ammonium sulphate precipitation, ion-exchange chromatography, and gel-permeation chromatography (GPC) consecutively and further characterized. The molecular mass of the enzyme was estimated to be about 28?kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and GPC analysis. The optimum pH and temperature of the purified enzyme were pH 8.0 and 50?°C, respectively. Additionally, the effect of some cations and other chemical compounds on the CDA activity was studied. A marginal increase in enzyme activity was observed with metal ions mainly Mn2+ and Zn2+. No inhibition of the enzyme was observed by the end product, that is, acetate up to 70?mM concentration. The Km and kcat values of the enzyme were determined to be 9.45?mg mL?1 and 26.72?s?1 respectively, using colloidal chitin as substrate. Among various substrates tested, glycol chitin and colloidal chitin were deacetylated. 相似文献
12.
Brush border membrane trehalase was purified from monkey small intestine by a procedure which includes solubilisation by Triton
X-100, ammonium sulphate fractionation, and chromatography on DE-52 and hydroxyapatite. The purified enzyme had a specific
activity of 11 units/mg protein and was purified 140-fold. The enzyme showed a single protein band on Polyacrylamide gel electrophoresis.
It had aK
m value of 17.4 mM for trehalose and a Vmax of 1.33 units. Sucrose and Tris acted as competitive inhibitors of the enzyme. 相似文献
13.
X-Prolyl dipeptidyl peptidase, which hydrolysed X-Pro-Y almost specifically, has been purified to homogeneity from crude cell-free extracts ofLactobacillus casei subsp.casei LLG using fast protein liquid chromatography equipped with preparative and analytical anion exchange columns. The enzyme was purified to 274-fold by ammonium sulphate fractionation, and by two successive ion-exchange chromatographies with a recovery of 34%. The purified enzyme appeared as a single band on both native-polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulphate (SDS)-PAGE and had a molecular mass of 79 kDa. The pH and the temperature optima by the purified enzyme were 7.0 and 50°C, respectively. X-PDP was a serine-dependent enzyme, as both diisopropylfluorophosphate and phenylmethylsulphonylfluoride caused complete inhibition of the enzyme activity. The Michaelis constant (K
m
) and maximum reaction velocity (V
max
) values were 0.2 mm and 43 mm per milligram, respectively. 相似文献
14.
Extracellular pullulanase was purified and crystallized from the culture fluid of Aerobacter aerogenes. Pullulanase was purified by means of ammonium sulfate fraction, DEAE-cellulose column chromatography and Sephadex column chromatography. Crystalline pullulanase was formed when saturated ammonium sulfate solution was added to the purified enzyme solution. The crystalline enzyme appeared as colorless fine rods. On ultracentrifugation analysis, the enzyme showed a single sharp and symmetrical Schlieren peak. The sedimentation coefficient, s20,w was 4.39S. Polyacrylamide gel electrophoresis at pH 8.4 gave a main band with two sub-bands and the molecular weight of the main enzyme was estimated to be 66,000 from Polyacrylamide gel electrophoresis and to be 58,000 from sedimentation equilibrium. The optimum pH and temperature for the enzyme action were pH 6.5 and 50°C, respectively. 相似文献
15.
Takayuki Uwajima Naohiro Yoshikawa Osamu Terada 《Bioscience, biotechnology, and biochemistry》2013,77(12):2727-2733
A crystalline aminopeptidase obtained from the culture filtrate of Streptomyces peptidofaciens KY 2389 appeared to be homogeneous on ultracentrifugation and acrylamide gel electrophoresis. The sedimentation coefficient, s20, w., was determined to be 2.6 S. The molecular weight was estimated to be approximately 19,000 by sedimentation equilibrium studies. The amino acid analyses indicated that the enzyme was composed of 147 amino acid residues and contained no sulfhydryl group. The isoelectric point was found to be around pH 7.4 by isoelectric focusing on ampholites.The enzyme required Ca2+ for its maximal activity and was strongly inhibited by some metal-chelating agents such as ethylenediaminetetraacetic acid (EDTA) and o-phenanthroline. The EDTA-inactivated enzyme restored its activity almost completely by the addition of Ca2+ The crystalline preparation of aminopeptidase contained 1 g-atom of calcium and about 2 g-atoms of magnesium per mole of enzyme protein, and the calcium was essential for the activity of the enzyme. 相似文献
16.
Diisopropyl phosphorofluoridate (DFP) produces type I organophosphorus ester-induced delayed neurotoxicity in humans and sensitive animal species. This is accompanied by enhanced Ca2+/CaM-dependent protein kinase II (CaM-kinase II) activity, and [125I]calmodulin binding to CaM-kinase II in DFP-treated hen brain supernatant without increase in the enzyme quantity. We have purified CaM-kinase II from control and DFP-treated hen whole brains and compared various physical and biochemical properties. The two enzymes exhibited similar properties in many respects. However, there was a decrease in calcium-independent protein kinase II activity after autophosphorylation, and an increase in K0.5 for free calcium and calmodulin of enzyme purified from DFP-treated hen brains. This change in kinetic parameters may result in greater percentage of total CaM-kinase II present in unphosphorylated form, which is consistent with the increased autophosphorylation of CaM-kinase II and [125I]calmodulin binding in the brain supernatant of DFP-treated hens.Abbreviations used CaM
calmodulin
- CaM-kinase II
Ca2+/calmodulin-dependent protein kinase II
- MAP-2
microtubule associated protein-2
- DFP
diisopropylphosphorofluoridate
- DTT
dithiothreitol
- EDTA
ethylenediaminetetraacetic acid
- EGTA
ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid
- NEPHGE
nonequilibrium pH gradient electrophoresis
- OPIDN
organophosphorus ester-induced delayed neurotoxicity
- PIPES
1,4-piperazinediethanesulfonic acid
- PMSF
phenylmethylsulfonyl fluoride
- SDS-PAGE
sodium dedecyl sulfate-polyacrylamide gel electrophoresis
-
St. aureus V8 protease
Staphylococus aureus V8 protease
- TOCP
tri-O-cresyl phosphate
- TPCK
N-tosyl-I-phenylalanine chloromethyl ketone 相似文献
17.
Aims: The purification and biochemical properties of the 1,4‐β‐xylosidase of an oenological yeast were investigated. Methods and Results: An ethanol‐tolerant 1,4‐β‐xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G‐100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS‐PAGE. The activity of 1,4‐β‐xylosidase was optimum at pH 6·0 and at 35°C. The activity had a Km of 0·48 ± 0·06 mmol l?1 and a Vmax of 7·4 ± 0·1 μmol min?1 mg?1 protein for p‐nitrophenyl‐β‐d ‐xylopyranoside. Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4‐β‐xylosidase from P. membranifaciens to be used in the bioethanol production process. 相似文献
18.
Yasuyuki Takeda Hirokazu Matsui Masatoshi Tanida Shoichi Takao Seiya Chiba 《Bioscience, biotechnology, and biochemistry》2013,77(6):1633-1641
A glucoamylase was purified from the culture broth of Paecilomyces varioti AHU 9417 by precipitation with ethanol, chromatography on DEAE-Sepharose CL-6B, gel filtration on Bio-Gel P-150, and preparative disc electrophoresis. The enzyme was homogeneous by disc electrophoretic analysis. The molecular weight was estimated to be 6.9×104 by SDS-disc electrophoresis, and the optimum pH was 4.5. The enzyme preferentially hydrolyzes α-1,4-glucosidic linkages in a series of maltooligosaccharides, and is capable to slowly hydrolyzing nigerose, isomaltose, and panose, but is not active on kojibriose. Soluble starch, amylopectin, glycogen, and β-limit dextrin are rapidly degraded. Activity toward raw starches is very low, but rice and waxy corn raw starches are relatively subject to attack. The subsite affinities (Ai) of each subsite (i) in the active site of the enzyme were evaluated from the Michaelis constants (Km) and the molecular activities (k0) for a series of maltooligosaccharides according to the subsite theory. The active site was considered to be made up of about four subsites: A1?0.46 kcal/mol, A2?4.78 kcal/mol, A3?1.76 kcal/mol and A4?0.67 kcal/mol. 相似文献
19.
Etsuo Yamamoto 《Phytochemistry》1980,19(5):779-781
Dehydroquinate synthase of Phaseolus mungo seedlings was purified 4400-fold from the (NH4)2SO4 fraction of a crude extract, the specific activity being 810 nkat per mg protein. When the purified enzyme was subjected to electrophoresis with or without sodium dodecyl sulfate, a single band was observed. The MW of the enzyme was estimated to be 67 000 by Sephadex G-100 gel chromatography and the minimum MW of the enzyme 43 000 by gel electrophoresis with sodium dodecyl sulfate. Atomic absorption analysis revealed that the purified enzyme contained small amounts of copper. Cobalt was not detected, although it has been implicated as a cofactor requirement. 相似文献
20.
Purification and Characterization of a Dipeptidase from Streptococcus cremoris Wg2 总被引:13,自引:9,他引:4
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A dipeptidase was purified to homogeneity from a crude cell extract of Streptococcus cremoris Wg2 by DEAE-Sephacel column chromatography followed by preparative disc gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 49,000. The dipeptidase is capable of hydrolyzing a range of dipeptides, but not peptides with longer chains. The enzyme was shown to be a metallo-Mn2+ enzyme with a pH optimum of 8 and a temperature optimum of 50°C. The enzyme is strongly inhibited by thiol-reducing reagents but not by sulfhydryl reagents. Kinetic studies indicated that the enzyme has a relatively low affinity for leucyl-leucine and alanyl-alanine (Km, 1.6 and 7.9 mM, respectively) but can hydrolyze these substrates at very high rates (Vmax, 3,700 and 13,000 μmol/min per mg of protein, respectively). 相似文献