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1.
D. Mamma D. Hatzinikolaou D. Kekos H. Stamatis E. Kalogeris 《World journal of microbiology & biotechnology》2009,25(5):781-788
A thermostable endoglucanase (EndoI) was produced by the thermophilic fungus Thermoascus aurantiacus when grown on cellulosic materials under submerged culture (SC) and solid-state fermentation (SSF). In both cultivation techniques
a considerable amount of enzyme activity remained adsorbed onto solid particles, and this was taken into consideration when
modeling enzyme production. The results were compatible with the assumption that, following its synthesis, an amount of EndoI
was bound on substrate and gradually released into the liquid medium. Adsorption of the enzyme on crystalline cellulose was
confirmed in vitro by experiments with purified endoglucanase, which was isolated by anion exchange chromatography. The Langmuir
isotherm could efficiently describe the adsorption kinetics, and the estimated A
max and K
ad values compared with those obtained for cellulases bearing a binding domain. EndoI displayed high affinity for crystalline
cellulose and low binding capacity, which could be beneficial in textile processing. 相似文献
2.
NAD+-linked primary and secondary alcohol dehydrogenase activity was detected in cell-free extracts of propane-grown Rhodococcus rhodochrous PNKb1. One enzyme was purified to homogeneity using a two-step procedure involving DEAE-cellulose and NAD-agarose chromatography and this exhibited both primary and secondary NAD+-linked alcohol dehydrogenase activity. The Mr of the enzyme was approximately 86,000 with subunits of Mr 42,000. The enzyme exhibited broad substrate specificity, oxidizing a range of short-chain primary and secondary alcohols (C2–C8) and representative cyclic and aromatic alcohols. The pH optimum was 10. At pH 6.5, in the presence of NADH, the enzyme catalysed the reduction of ketones to alcohols. The K
m values for propan-1-ol, propan-2-ol and NAD were 12 mM, 18 mM and 0.057 mM respectively. The enzyme was inhibited by metal-complexing agents and iodoacetate. The properties of this enzyme were compared with similar enzymes in the current literature, and were found to be significantly different from those thus far described. It is likely that this enzyme plays a major role in the assimilation of propane by R. rhodochrous PNKb1.Abbreviations HPLC
high performance liquid chromatography
- DEAE
diethyl amino ethyl
- IEF
isoelectrofocusing
- NTG
nitrosoguanidine
- SDS-PAGE
sodium dodecylsulphate polyacrylamide gel electrophoresis
- pI
isoelectric point 相似文献
3.
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
4.
Takehiro Kawano Yasunori Kozutsumi Hiromu Takematsu Toshisuke Kawasaki Akemi Suzuki 《Glycoconjugate journal》1993,10(1):109-115
The hydroxylation of CMP-NeuAc has been demonstrated to be carried out by several factors including the soluble form of cytochromeb
5. In the present study, mouse liver cytosol was subjected to ammonium sulfate fractionation and cellulose phosphate column chromatography for the separation of two other essential fractions participating in the hydroxylation. One of the fractions, which bound to a cellulose phosphate column, was able to reduce the soluble cytochromeb
5, using NADH as an electron donor. The other fraction, which flowed through the column, was assumed to contain the terminal enzyme which accepts electrons from cytochromeb
5, activates oxygen, and catalyses the hydroxylation of CMP-NeuAc. Assay conditions for the quantitative determination of the terminal enzyme were established, and the activity of the enzyme in several tissues of mouse and rat was measured. The level of the terminal enzyme activity is associated with the expression ofN-glycolylneuraminic acid in these tissues, indicating that the expression of the terminal enzyme possibly regulates the overall velocity of CMP-NeuAc hydroxylation.Abbreviations CMP
cytidine 5-monophosphate
- NeuAc
N-acetylneuraminic acid
- NeuGc
N-glycolylneuraminic acid
- NADH
reduced nicotinamide adenine dinucleotide
- NADPH
reduced nicotinamide adenine dinucleotide phosphate
- DTT
dithiothreitol 相似文献
5.
Elaine O’Connell Patrick Murray Charles Piggott Franck Hennequart Maria Tuohy 《Journal of applied phycology》2008,20(5):557-565
A β-N-acetylglucosaminidase produced by a novel fungal source, the moderately thermophilic aerobic ascomycete Talaromyces emersonii, was purified to apparent homogeneity. Submerged fermentation of T. emersonii, in liquid medium containing algal fucoidan as the main carbon source, yielded significant amounts of extracellular N-acetylglucosaminidase activity. The N-acetylglucosaminidase present in the culture-supernatant was purified by hydrophobic interaction chromatography and preparative
electrophoresis. The enzyme is a dimer with molecular weight and pI values of 140 and 3.85, respectively. Substrate specificity
studies confirmed the glycan specificity of the enzyme for N-acetylglucosamine. Michaelis-Menten kinetics were observed during enzyme-catalyzed hydrolysis of the fluorescent substrate
methylumbelliferyl-β-D-N-acetylglucosaminide at 50°C, pH 5.0 (Km value of 0.5 mM). The purified N-acetylglucosaminidase displayed activity over broad ranges of pH and temperature, yielding respective optimum values of pH
5.0 and 75°C. The T. emersonii enzyme was less susceptible to inhibition by N-acetylglucosamine and other related sugars than orthologs from other sources. The enzyme was sensitive to Hg2+, Co2+ and Fe3+. 相似文献
6.
S. K. Roy S. K. Raha R. K. Sadhukhan S. L. Chakrabarty 《World journal of microbiology & biotechnology》1991,7(6):613-618
The extracellular -glucosidase has been purified from culture broth of Myceliophthora thermophila ATCC 48104 grown on crystalline cellulose. The enzyme was purified approximately 30-fold by (NH4)2SO4 precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G-200 and DEAE-Sephadex A-50. The molecular mass of the enzyme was estimated to be about 120 kD by both sodium dodecyl sulphate gel electrophoresis and gel filtration chromatography. It displayed optimal activity at pH 4.8 and 60°C. The purified enzyme in the absence of substrate was stable up to 60°C and pH between 4.5 and 5.5. The enzyme hydrolysed p-nitrophenyl--d-glucoside, cellobiose and salicin but not carboxymethyl cellulose or crystalline cellulose. The K
m of the enzyme was 1.6mm for p-nitrophenyl--d-glucoside and 8.0mm for cellobiose. d-Glucose was a competitive inhibitor of the enzyme with a K of 22.5mm. Enzyme K activity was inhibited by HgCl2, FeSO4, CuSO4, EDTA, sodium dodecyl sulphate, p-chloromercurobenzoate and iodoacetamide and was stimulated by 2-mercaptoethanol, dithiothreitol and glutathione. Ethanol up to 1.7 m had no effect on the enzyme activity.The authors are with the Department of Microbiology, Bose Institute, 93/1, A.P.C. Road, Calcutta 700 009, India. S.K. Raha is presently with the Department of Medicine, University of Saskatchewan, Saskatoon, Canada S7N OXO. 相似文献
7.
Vishal V. Dawkar Umesh U. Jadhav Amar A. Telke Sanjay P. Govindwar 《Biotechnology and Bioprocess Engineering》2009,14(3):361-368
Peroxidase was purified by an ion exchange chromatography followed by gel filtration chromatography from dye degrading Bacillus sp. strain VUS. The optimum pH and temperature of the enzyme activity was 3.0 and 65°C, respectively. This enzyme showed
more activity with n-propanol than other substrates tested viz. xylidine, 3-(3,4-dihydroxy phenyl) Lalanine (L-DOPA), hydroxyquinone, ethanol, indole, and veratrole. Km value of the enzyme
was 0.076 mM towards n-propanol under standard assay conditions. Peroxidase was more active in presence of the metal ions like Li2+, Co2+, K2+, Zn2+, and Cu2+ where as it showed less activity in the presence of Ca2+ and Mn2+. Inhibitors like ethylenediamine tetraacetic acid (EDTA), glutamine, and phenylalanine inhibited the enzyme partially, while
sodium azide (NaN3) completely. The crude as well as the purified peroxidase was able to decolourize different industrial dyes. This enzyme
decolourized various textile dyes and enhanced percent decolourization in the presence of redox mediators. Aniline was the
most effective redox mediator than other mediators tested. Gas chromatography-Mass spectrometry (GC-MS) confirmed the formation
of 7-Acetylamino-4-hydroxy-naphthalene-2-sulphonic acid as the final product of Reactive Orange 16 indicating asymmetric cleavage
of the dye. 相似文献
8.
Lei Kai Xiao-Hang Ma Xue-Lai Zhou Xiao-Ming Jia Xia Li Kang-Ping Guo 《World journal of microbiology & biotechnology》2008,24(3):401-406
In order to study the properties of a thermostable uricase produced by Microbacterium sp. strain ZZJ4-1, the enzyme was purified by ammonium sulfate precipitation and DEAE-cellulose ion exchange, hydrophobic
and molecular sieve chromatography. The molecular mass of the purified enzyme was estimated to be 34 kDa by SDS-PAGE. The
enzyme was stable between pH 7.0 and 10.00. The optimal reaction temperature of the enzyme was 30 °C at pH 8.5. The K
m
and K
cat
of the enzyme were 0.31 mM and 3.01 s−1, respectively. Fe3+ could enhance the enzyme activity, whereas Ag+, Hg2+, o-phenanthroline and SDS inhibited the activity of the enzyme considerably. After purification, the enzyme was purified 19.7-fold
with 31% yield. As compared with uricases from other microbial sources, the purified enzyme showed excellent thermostability
and other unique characteristics. The results of this work showed that strains of Microbacterium could be candidates for the production of a thermostable uricase, which has the potential clinical application in measurement
of uric acid. 相似文献
9.
Liu B Hong Y Wu L Li Z Ni J Sheng D Shen Y 《Extremophiles : life under extreme conditions》2007,11(5):733-739
A glycerate kinase (GK) gene (PH0495) from the hyperthermophilic archaeon Pyrococcus horikoshii, was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity by affinity chromatography and ion exchange chromatography. The enzyme
was likely a homodimer based on SDS-PAGE (47 kDa) and gel filtration chromatography (100 kDa) analysis. A radioisotope-labeling
examination method was initially used for the enzymatic activity detection, and the enzyme (GKph) was found to catalyze the formation of 2-phosphoglycerate using d-glycerate as the substrate. The enzyme exhibited unique phosphoryl donor specificity with maximal activity towards pyrophosphate.
The temperature and pH optima of the enzyme were 45°C and 7.0, respectively, and about half of the maximal activity remained
at 100°C. The enzyme was highly thermostable with almost no loss of activity at 90°C for 12 h. Based on sequence alignment
and structural comparison it was assigned to group I of the trichotomy of GKs.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
10.
A NAD-dependent (R)-2,3-butanediol dehydrogenase (EC 1.1.1.4), selectively catalyzing the oxidation at the (R)-center of 2,3-butanediol irrespective of the absolute configuration of the other carbinol center, was isolated from cell extracts of the yeast Saccharomyces cerevisiae. Purification was achieved by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, affinity chromatography on Matrex Gel Blue A and Superose 6 prep grade chromatography leading to a 70-fold enrichment of the specific activity with 44% yield. Analysis of chiral products was carried out by gas chromatographic methods via pre-chromatographic derivatization and resolution of corresponding diasteromeric derivatives. The enzyme was capable to reduce irreversibly diacetyl (2,3-butanediol) to (R)-acetoin (3-hydroxy-2-butanone) and in a subsequent reaction reversibly to (R,R)-2,3-butanediol using NADH as coenzyme. 1-Hydroxy-2-ketones and C5-acyloins were also accepted as substrates, whereas the enzyme was inactive towards the reduction of acetone and dihydroxyacetone. The relative molecular mass (M
r) of the enzyme was estimated as 140 000 by means of gel filtration. On SDS-polyacrylamide gel the protein decomposed into 4 (identical) subunits of M
r 35 000. Optimum pH was 6.7 for the reduction of acetoin to 2,3-butanediol and 7.2 for the reverse reaction.Abbreviations GC-MS
gas chromatography-mass spectrometry
- i.d.
internal diameter
-
M
r
relative molecular mass
- MTPA-Cl
-methoxy--trifluoromethylphenyl acetic acid chloride
- PEIC
1-phenylethylisocyanate 相似文献
11.
The dehydration of (R)-2-hydroxyglutarate to glutaconate is catalysed by a soluble enzyme system found in extracts of Acidaminococcus fermentans. The enzyme has to be activated by ATP, NADH and MgCl2 prior to the reaction which requires dithioerythritol, acetylphosphate and CoASH. Activity and stability of the enzyme depend on anaerobic conditions. Experiments with ATP labelled at different atoms indicated an adenylation or adenylphosphorylation during the activation. However, only the activity but not the label was removed by inactivators such as 0.1 mM 2,4-dinitrophenol or 1 mM hydroxylamine. In the presence of ATP, MgCl2 and dithioerythritol the dehydratase was purified 2-fold by chromatography on Sephacryl S-300, but on DEAE-Sephacel all activity was lost. ESR-spectra and chemical considerations led to the conclusion that a hydroxyradical plays a central role in the mechanism of the dehydration.Abbreviations Pipes
1,4-piperazineethanesulfonic acid
- DTE
dithiothritol
- ESR
electron spin resonance 相似文献
12.
Purification and properties of putrescine N-methyltransferase from transformed roots of Datura stramonium L. 总被引:1,自引:0,他引:1
Nicholas J. Walton Abigael C. J. Peerless Richard J. Robins Michael J. C. Rhodes Henry D. Boswell David J. Robins 《Planta》1994,193(1):9-15
Putrescine-N-methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrrolidine alkaloids, has been purified about 700-fold from root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes. The native enzyme had a molecular weight estimated by gel-permeation chromatography on Superose-6 of 40 kDa; sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fractions from Superose-6 chromatography revealed a band of 36 kDa molecular weight. Kinetic studies of the purified enzyme gave K
m values for putrescine and S-adenosyl-l-methionine of 0.31 mM and 0.10 mM, respectively, and K
i values for S-adenosyl-l-homocysteine and N-methylputrescine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2-hydroxybutane and 1,4-diamino-trans-but-2-ene. Little activity was observed with 1,4-diamino-cis-but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for substrate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhibitor of the enzyme, with a K
i of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discussed.Abbreviations PEG
polyethylene glycol
- PMT
putrescine-N-methyltransferase
- SAH
S-adenosyl-l-homocysteine
- SAM
S-adenosyl-l-methionine
- SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
We are grateful to C.R. Waspe, M.G. Hilton and P.D.G. Wilson for assistance with the provision of roots from fermenters. We thank W. Martin and S.D. Barr, Chemistry Department, University of Glasgow, and T.A. Smith, Long Ashton Research Station, Bristol, for the supply of compounds not commercially available, as indicated in the text. For helpful discussion and comment, we are grateful to A.J. Parr, W.R. McLauchlan and P. Bachmann. H.D.B, thanks the Science and Engineering Research Council for a research studentship and the Agricultural and Food Research Council Institute of Food Research for additional support. 相似文献
13.
Separation and purification of the tonoplast ATPase and pyrophosphatase from plants with constitutive and inducible Crassulacean acid metabolism 总被引:7,自引:0,他引:7
Tonoplast vesicles were isolated from Kalanchoe daigremontiana Hamet et Pierrer de la Bâthie and Mesembryanthemum crystallinum L., exhibiting constitutive and inducible crassulacean acid metabolism (CAM), respectively. Membrane-bound proteins were detergent-solubilized with 2% of Triton X-100. During CAM induction in M. crystallinum, ATPase activity increases four-fold, whereas pyrophosphatase activity decreases somewhat. With all plants, ATPase and pyrophosphatase could be separated by size-exclusion chromatography (SEC, Sephacryl S 400), and the ATPase was further purified by diethylaminoethyl-ion-exchange chromatography. Sodium-dodecyl-sulfate electrophoresis of the SEC fractions from K. daigremontiana containing maximum ATPase activity separates several protein bands, indicating subunits of 72, 56, 48, 42, 28, and 16 kDa. Purified ATPase from M. crystallinum in the C3 and CAM states shows a somewhat different protein pattern. With M. crystallinum, an increase in ATP-hydrolysis and changes in the subunit composition of the native enzyme indicate that the change from the C3 to the CAM state is accompanied by de-novo synthesis and by structural changes of the tonoplast ATPase.Abbreviations CAM
Crassulacean acid metabolism
- DTT
dithiothreitol
- kDa
kilodalton
- PAGE
polyacrylamide gel electrophoresis
- PPiase
pyrophosphatase
- SEC
size exclusion chromatography
- SDS
sodium dodecyl sulfate
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
14.
Yun Hee Choi Ki-Nam Uhm Hyung-Kwoun Kim 《Journal of Molecular Catalysis .B, Enzymatic》2008,55(3-4):157-163
The Rhodococcus erythropolis strain (N′4) possesses the ability to convert 4-chloro-3-hydroxybutyronitrile into the corresponding acid. This conversion was determined to be performed by its nitrile hydratase and amidase. Ammonium sulfate fractionation, DEAE ion exchange chromatography, and phenyl chromatography were used to partially purify nitrile hydratase from cell-free extract. A SDS-PAGE showed that the partially purified enzyme had two subunits and gel filtration chromatography showed that it consisted of four subunits of α2β2. The purified enzyme had a high specific activity of 860 U mg−1 toward methacrylonitrile. The enzyme was found to have high activity at low temperature range, with a maximum activity occurring at 25 °C and be stable in the presence of organic acids at higher temperatures. The enzyme exhibited a preference for aliphatic saturated nitrile substrates over aliphatic unsaturated or aromatic ones. It was inhibited by sulfhydryl, oxidizing, and serine protease inhibitors, thus indicating that essential cysteine and serine residues can be found in the active site.The purified nitrile hydratase was able to convert 4-chloro-3-hydroxybutyronitrile into the corresponding amide at 15 °C. GC analysis showed that the initial conversion rate of the reaction was 215 mg substrate consumed min−1 mg−1. This demonstrated that this enzyme could be used in conjunction with a stereoselective amidase to synthesize ethyl (S)-4-chloro-3-hydroxybutyrate, an intermediate for a hypercholesterolemia drug, Atorvastatin. 相似文献
15.
Malic enzyme (ME=L-malate: NADP oxidoreductase; E.C. 1.1.1.40) was extracted by Triton X-100-induced resolubilization of enzyme proteins which denaturize spontaneously upon homogenization of grape berry material. The purification procedure included fractionating with (NH4)2SO4, preparative IEF, and Sephadex G-100 chromatography. ME was identified by TLC of the radioactive product after supplementing the assay mixture with [14C]malate. Cofactor dependence, pH-optimum and affinities for substrates and cosubstrates were determined. Enzymic pI was found to be 5.8, the Hill coefficients range from 1 to 3. In malate decarboxylating direction at pH 7.4, grape ME displayed positive cooperativity toward the substrate, the curve approaching normal Michaelis-Menten-kinetics at pH 7.0. Substituting Mn2+ for Mg2+ not only increased maximal turnover rates, but also enzymic affinity for malate. These features were considered indicative of the regulatory properties of the enzyme. Their relevance for grape malate metabolism and fruit ripening is discussed.Abbreviations EDTA
ethylenediaminetetraacetic acid
- IFF
isoelectric focusing
- MDH
malate dehydrogenase
- ME
malic enzyme
- OAA
oxaloacetic acid
- PAG
polyacrylamide gel
- TCA
trichloroacetic acid
- TLC
thin layer chromatography 相似文献
16.
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
17.
The cytosolic pyruvate kinase (PKC, EC 2.7.1.40) and phosphoenolpyruvate carboxylase (PEP-Case, EC 4.1.1.31) from cotyledons of 6-d-old castor seedlings (Ricinus communis L.) have been partially purified and characterized. PKC was purified 370-fold to a specific activity of 20 mol · min 1·(mg protein)–1, and was shown to exist as a 237-kDa homotetramer. In addition, PKC displayed hyperbolic substrate saturation kinetics and demonstrated pH-dependent modulation by several metabolite effectors including glutamine, glutamate, arginine, malate and 2-oxoglutarate. Most were inhibitors at pH 6.9, while activation by glutamine, asparagine and arginine and only weak inhibition for the rest were observed at pH 7.5. PEPCase was purified 33-fold to a final specific activity of 1 mol · min–1 · (mg protein)–1. The subunit and native Mr for the enzyme were shown to be 100 and 367 kDa, respectively, suggesting a homotetrameric native structure. PEPCase displayed a typical pH activity profile with an alkaline optimum and activity decreasing rapidly below pH 7.0. The enzyme was potently inhibited by malate, isocitrate, aspartate and glutamate at pH 7.0, whereas inhibition by these compounds was considerably diminished at pH 7.5. A model depicting the regulation of glycolytic carbon flow during amino-acid and sucrose import by castor cotyledons is proposed.Abbreviations IgG
immunoglobulin G
- I50a
inhibitor concentration producing 50 inhibition of enzyme activity
- PKC and PKpa
cytosolic and plastidic isoenzymes of pyruvate kinase, respectively
- PEP
phosphoenolpyruvate
- PEPCase
phosphoenolpyruvate carboxylase
- 3-PGA
3-phosphoglycerate
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). 相似文献
18.
Thomas Jahns 《Antonie van Leeuwenhoek》1995,68(3):209-214
Urease (EC 3.5.1.5) catalyses the hydrolysis of urea to ammonia and carbon dioxide. The enzyme fromSporobolomyces roseus was enriched 780-fold and purified to apparent homogeneity using heat treatment, ion exchange chromatography on Q-Sepharose fast flow, hydrophobic interaction chromatography on Phenyl-Sepharose, size exclusion chromatography on Sephacryl S 300 HR, and ion exchange chromatography on MonoQ. Analysis of the purified enzyme by SDS-PAGE demonstrated the presence of subunits with a molecular weight of 90 (± 4) kDa. The M
r
of the native enzyme was estimated by size exclusion chromatography to be 340 (± 30) kDa, suggesting a tetrameric structure different from other ureases isolated so far from both prokaryotes and eukaryotes. The enzyme was heat-stable, showing no loss of activity after incubation at 70 °C for 15 min. The highest urease activities were observed after growth on media containing urea as the sole source of nitrogen. 相似文献
19.
Coenzyme A-linked acetaldehyde dehydrogenase (ACDH) of ethanol-grown cells of Acetobacterium woodii was purified to apparent homogeneity; a 28-fold purification was achieved with 13% yield. The enzyme proved to be oxygen-sensitive and was inactive in the absence of dithioerythritol. During the purification procedure addition of 1 mM MgCl2 was necessary to maintain enzyme activity. Alcohol dehydrogenase (ADH) activity was separated from ACDH during anion exchange chromatography using DEAE Sephacel. A part of the ACDH activity coeluted with ADH, but both could be separately eluted from a Cibacron Blue 3GA-Agarose column, revealing the same subunit structure and activity band for ACDH as found before and, thus, indicating an aggregation of the enzyme. The remaining ADH activity could be separated by gel filtration. For the native ACDH a molecular mass of 255 kDa was determined by polyacrylamide gel electrophoresis and of 272 kDa by gel filtration using Superose 12. The enzyme subunit sizes were 28 kDa and 40 kDa, respectively, indicating a 44 structure for the active form. The enzyme catalyzed the oxidation of several straight chain aldehydes although it was most active with acetaldehyde. NADH strongly inhibited oxidation of acetaldehyde whereas NADPH had no effect. The inhibition was noncompetitive.Non-standard abbrevations ACDH
acetaldehyde dehydrogenase
- ADH
alcohol dehydrogenase
- CHES
2-(N-cyclohexylamino)-ethanesulfonate
- DTE
dithioerythritol
- KP-buffer
25 mM K-PO4, pH 7.5, containing, 4 mM DTE
- MES
2-(N-morpholino)-ethanesulfonate
- TAPS
N-Tris-(hydroxymethyl)-methyl-3-aminopropa-nesulfonate 相似文献
20.
Jin Zhou Ju Chu Yong-Hong Wang Si-Liang Zhang Ying-Ping Zhuang Zhong-Yi Yuan 《World journal of microbiology & biotechnology》2008,24(6):789-796
An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)2SO4 fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed
the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular
weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric
point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5
and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature
stability was up to 45 °C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K
m of 120 and 330 μM and V
max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively. 相似文献