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1.
Heat-stable protease from Pseudomonas fluorescens T16: purification by affinity column chromatography and characterization 总被引:1,自引:0,他引:1
A heat-stable extracellular protease from Pseudomonas fluorescens T16, a psychrotroph, was purified by affinity column chromatography on a carbobenzoxy-D-phenylalanine-triethylene tetramine-Sepharose-4B column. The purified enzyme is a monomer with a molecular weight of 38,905 +/- 2,000. In an analytical ultracentrifuge, the Schlieren profile revealed a single symmetrical peak. The sedimentation coefficient was estimated to be 3.93S. Alpha-casein was the preferred substrate, with a Km of 0.05 mM. Heating crude enzyme and purified enzyme in buffer at 50, 90, and 120 degrees C resulted in a rapid initial loss of more than 50% of the initial activity followed by a gradual inactivation which exhibited first-order kinetics. The activation energy for the hydrolysis of casein was calculated to be 3.2 kcal/mol (13.4 kJ/mol). 相似文献
2.
trans-N-Deoxyribosylase (EC 2.4.2.6) is usually considered as a single protein catalyzing indifferently the transfer of the deoxyribosyl moiety to and from a purine or a pyrimidine base. Affinity chromatography of an extract from Lactobacillus helveticus with two types of ligands allowed the separation and purification of two distinct trans-N-deoxyribosylases. One catalyzes specifically the deoxyribosyl transfer to and from purine bases exclusively: trans-N-deoxyribosylase-I, the other catalyzes the transfer to and from pyrimidine and purine bases: trans-N-deoxyribosylase-II. A Tris inhibition study showed a markedly different susceptibility of the two enzymes. Preliminary results indicate that the purine-specific enzyme is a polymeric enzyme of molecular weight 86 000 (+/- 4000). 相似文献
3.
4.
The -glucosidase (-d-glucoside glucohydrolase, EC 3.2.1.20) of Pseudomonas fluorescens W was partially purified by (NH4)2SO4 fractionation, Sephadex G-200 and DEAE-cellulose column chromatography. The enzyme showed great specificity for maltose hydrolysis, with very little action against polymeric forms. Sucrose, isomaltose, -methylglucoside, and maltobionic acid were not hydrolyzed. Turanose was a strong competitive inhibitor, and glucose a weaker one. Tris (2-amino-2-hydroxymethylpropan-1:3-diol) inhibited enzyme activity significantly only at alkaline pH. Mercuric, cupric, and silver cations strongly inhibited, and EDTA (ethylenediaminetetraacetate) weakly inhibited the enzyme. The isolated enzyme was rather unstable even at 4° C, and was destroyed by freezing and lyophilization. Inositol and albumin had a slightly protective effect. Sulfhydryl-binding reagents strongly inhibited the enzyme.Abbreviations PNPG
paranitrophenyl--d-glucoside
- PCMB
parachloromercuribenzoate
- DEAE
diethylaminoethyl cellulose
- NEM
N-ethylmaleimide
- EDTA
ethylenediaminetetraacetate 相似文献
5.
Pseudomonas fluorescens RO98, a raw milk isolate, was inoculated into McKellar's minimal salts medium and incubated at 25 degrees C for 48 h to allow production of protease. A zinc-metalloacid protease was purified from the cell-free concentrate by anion exchange and gel filtration chromatography. The purified protease was active between 15 and 55 degrees C, and pH 4.5 and 9.0, and was stable to pasteurization. The enzyme had pH and temperature optima for activity of 5.0 and 35 degrees C, respectively. It was heat stable with a D55 of 41 min and a D62.5 of 18 h. Molecular weight of the enzyme was estimated to be 52 kDa by SDS PAGE and size exclusion chromatography. Values for kM of 144.28, 18.73, 110.20 and 35.23 micromol were obtained for whole, alpha-, beta- and kappa-casein, with a Vmax of 8.26, 0.09, 0.42 and 0.70 micromol mg-1 min-1, respectively. The enzyme hydrolysed kappa-casein preferentially when incubated with artificial casein micelles. 相似文献
6.
(NADPH)-cytochrome P-450 reductase was purified to apparent homogeneity by a procedure utilizing nicotinamide adenine dinucleotide phosphate (NADP)-Sepharose affinity column chromatography. The purified flavoprotein has a molecular weight of 79 700 and catalyzes cytochrome P-450 dependent drug metabolism, as well as reduction of exogenous electron acceptors. Aerobic titration of cytochrome P-450 reductase with NADPH indicates that an air-stable reduced form of the enzyme is generated by the addition of 0.5 mol of NADPH per mole of flavin, as judged by spectral characteristics. Further addition of NADPH causes no other changes in the absorbance spectrum. A Km value for NADPH of 5 micron was observed when either cytochrome P-450 or cytochrome c was employed as electron acceptor. A Km value of 8 +/- 2 micron was determined for cytochrome c and a Km of 0.09 +/- 0.01 micron was estimated for cytochrome P-450. 相似文献
7.
A ribonuclease from cobra snake venom was isolated and purified to homogeneity using antibody-affinity chromatography, increasing the yield fourfold. The purified enzyme showed cytidylic acid specificity, as reported earlier. Further, the effects of temperature, pH, metal ions, inhibitors, and urea on the enzyme activity were studied. Snake venom RNase exhibited salt-dependent reversible association-dissociation behaviour. Immunological studies indicate that this enzyme shares one of the antigenic sites of RNase A. The partial N-terminal sequence of the enzyme showed considerable homology with phospholipases from snake venom; however, the enzyme itself did not show any phospholipase activity. 相似文献
8.
Tsutom Takegami Kazuo Yoshida 《Biochemical and biophysical research communications》1975,67(2):782-789
Cytokinin binding protein from tobacco leaves was isolated and purified to a single protein by means of affinity chromatography on benzyladenine-linked Sepharose column combined with polyacrylamide gel electrophoresis. In vitro binding of this protein to [14C] benzyladenine was inhibited remarkably by cold benzyladenine and kinetin and slightly by adenine, but not adenosine. The molecular weight of the protein was determined to be about 4,000 daltons by gel filtration and SDS polyacrylamide gel electrophoresis. 相似文献
9.
First we have confirmed the previous observation that the macrophage migration inhibitory factor (MIF) was adsorbed on normal peritoneal macrophages when they were incubated at 4 C for 60 min. It was found that macrophages fixed with 2% glutaraldehyde gave more reproducible results than viable cells in terms of "adsorption" of guinea pig MIF. The adsorption was achieved more completely at 37 C than at 4 C, indicating that this reaction is a temperature-dependent phenomenon. Using these glutaraldehyde-fixed macrophages, a kind of cell-affinity column was successfully developed. The guinea pig MIF preparation lost its activity when it was passed through this affinity column, and MIF adsorbed on the column was recovered by elution with 0.1 M (L)-fucose of 0.1 M (D)-glucose. Such MIF active eluate was found to be at least 30--40 fold more pure than the original MIF preparation which had been previously fractionated according to its molecular weight. Therefore, this type of macrophage-affinity column may be useful for the purification of MIF. 相似文献
10.
The gene encoding proline dehydrogenase (ProDH) from Pseudomonas fluorescens was isolated using PCR amplification and cloned into pET23a expression vector. The expression of the recombinant target enzyme
was induced by addition of IPTG. The produced His-fusion enzyme was purified and its kinetic properties were studied. The
3D structure modeling was also performed to identify key amino acids involved in FAD-binding and catalysis. The PCR product
contained a 1033 bp open reading frame encoding 345 amino acid residue polypeptide chain. SDS-PAGE analysis revealed a MW
of 40 kDa, whereas the native enzyme exhibited a MW of 40 kDa suggesting a monomeric protein. The K
m and V
max values of the P. fluorescens ProDH were estimated to be 35 mM and 116 μmol/min, respectively. ProDH activity was stable at alkaline pH and the highest
activity was observed at 30°C and pH 8.5. The modeling analysis of the three dimensional structure elucidated that Lys-173
and Asp-202, which were oriented near the hydroxyl group of the substrate, were essential residues for the ProDH activity.
This study, to our knowledge, is the first data on the cloning and biochemical and structural properties of P. fluorescens ProDH. 相似文献
11.
Several iron-binding pigments (siderochromes) produced by Pseudomonas fluorescens have been isolated and partially characterized. They include ferribactin and various forms of pyoverdine, as well as some previously unreported compounds. In particular, the existence of ferribactin has been independently confirmed for the first time. Column and thin layer chromatographic procedures have been developed to fractionate, purify, and identify the siderochromes. We find ferribactin to contain nine amino acids, one residue each of glutamine, tyrosine, and glycine, and two each of serine, lysine, and N-hydroxyornithine, rather than 10 as earlier reported. Pyoverdine is a peptide with the same composition as ferribactin except for the absence of glutamine and the substitution of a fluorescent chromophore for tyrosine. Paper electrophoresis reveals an extra ionizable group in ferric pyoverdine relative to pyoverdine or ferribactin which provides that complex a definite cathodic mobility at pH 3. Optical spectra of the pyoverdine fluorescent component indicate that, in conjunction with the two hydroxamate groups, it is involved in the metal ion coordination, conferring on pyoverdine a dramatically increased affinity for Fe(III) relative to ferribactin. 相似文献
12.
Isolation and partial characterization of Bromelia hemisphaerica protease by affinity chromatography
Hemisphaericin, the protease from Bromelia hemisphaerica fruit juice was isolated by affinity chromatography in one step, using a mercurial sepharose derivative. The enzyme behaves as a single component in immunodifussion, immunoelectrophoresis and polyacrylamide electrophoresis in the presence of SDS and 2-mercaptoethanol. Association and dissociation of active components were evidenced in electrophoresis at pH 3.6 and at pH 8.6. Immunoelectrophoresis analyses also disclosed a certain degree of internal immunological heterogeneity. The results are explained by the presence of an enzyme subunit, of about 8000 daltons, endowed with polymeric properties induced by the pH and oxidative environment. 相似文献
13.
Partial purification and characterization of manganese-oxidizing factors of Pseudomonas fluorescens GB-1. 总被引:1,自引:0,他引:1
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M Okazaki T Sugita M Shimizu Y Ohode K Iwamoto E W de Vrind-de Jong J P de Vrind P L Corstjens 《Applied microbiology》1997,63(12):4793-4799
The Mn(2+)-oxidizing bacterium Pseudomonas fluorescens GB-1 deposits Mn oxide around the cell. During growth of a culture, the Mn(2+)-oxidizing activity of the cells first appeared in the early stationary growth phase. It depended on the O2 concentration in the culture during the late logarithmic growth phase. Maximal activity was observed at an oxygen concentration of 26% saturation. The activity could be recovered in cell extracts and was proportional to the protein concentration in the cell extracts. The specific activity was increased 125-fold by ammonium sulfate precipitation followed by reversed-phase and gel filtration column chromatographies. The activity of the partly purified Mn(2+)-oxidizing preparation had a pH optimum of circa 7 and a temperature optimum of 35 degrees C and was lost by heating. The Mn(2+)-oxidizing activity was sensitive to NaN3 and HgCl2. It was inhibited by KCN, EDTA, Tris, and o-phenanthroline. Although most data indicated the involvement of protein in Mn2+ oxidation, the activity was slightly stimulated by sodium dodecyl sulfate at a low concentration and by treatment with pronase and V8 protease. By polyacrylamide gel electrophoresis, two Mn(2+)-oxidizing factors with estimated molecular weights of 180,000 and 250,000 were detected. 相似文献
14.
The lysosomal enzyme alpha-galactosidase A (alpha-Gal A) metabolizes neutral glycosphingolipids that possess alpha-galactoside residues at the non-reducing terminus, and inherited defects in the activity of alpha-Gal A lead to Fabry disease. We describe here an efficient and rapid purification procedure for recombinant alpha-Gal A by sequential Concanavalin A (Con A)-Sepharose and immobilized thio-alpha-galactoside (thio-Gal) agarose column chromatography. Optimal elution conditions for both columns were obtained using overexpressed human alpha-Gal A. We recommend the use of a mixture of 0.9 M methyl alpha-mannoside and 0.9 M methyl alpha-glucoside in 0.1 M acetate buffer (pH 6.0) with 0.1 M NaCl for the maximum recovery of glycoproteins with multiple high-mannose type sugar chains from Con A column chromatography, and that the Con A column should not be reused for the purification of glycoproteins that are used for structural studies. Binding of the enzyme to the thio-Gal column requires acidic condition at pH 4.8. A galactose-containing buffer (25 mM citrate-phosphate buffer, pH 5.5, with 0.1 M galactose, and 0.1 M NaCl) was used to elute alpha-Gal A. This procedure is especially useful for the purification of mutant forms of alpha-Gal A, which are not stable under conventional purification techniques. A protocol that purifies an intracellular mutant alpha-Gal A (M279I) expressed in COS-7 cells within 6h at 62% overall yield is presented. 相似文献
15.
16.
J C Lee 《The International journal of biochemistry》1984,16(11):1143-1147
Affinity chromatography on beta,gamma-methylene guanosine 5'-triphosphate-Sepharose was used to purify protein synthesis initiation factor eIF-2 from chicken reticulocytes. Gel filtration of the purified factor gave a molecular weight of 150,000, whereas electrophoresis of the purified factor on polyacrylamide gel containing sodium dodecyl sulfate revealed three non-identical subunits with apparent molecular weight of 57,000, 41,000 and 33,000. With Met-tRNA and GTP, the factor formed a ternary complex which would bind the 40S ribosomal subunits. Treatment of the factor with N-ethylmaleimide resulted in a loss of activity. Two sulfhydryl groups per eIF-2 molecule were essential for activity. 相似文献
17.
Aerobic purification of hydrogenase from Rhizobium japonicum by affinity chromatography. 总被引:18,自引:9,他引:9
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We purified active hydrogenase from free-living Rhizobium japonicum by affinity chromatography. The uptake hydrogenase of R. japonicum has been treated previously as an oxygen-sensitive protein. In this purification, however, reducing agents were not added nor was there any attempt to exclude oxygen. In fact, the addition of sodium dithionite to aerobically purified protein resulted in the rapid loss of activity. Purified hydrogenase was more stable when stored under O2 than when stored under Ar. Sodium-chloride-washed hydrogen-oxidizing membranes were solubilized in Triton X-100 and deoxycholate and loaded onto a reactive red 120-agarose column. Purified hydrogenase elutes at 0.36 M NaCl, contains a nickel, and has a pH optimum of 6.0. There was 452-fold purification resulting in a specific activity of 76.9 mumol of H2 oxidized per min per mg of protein and a yield of 17%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed subunits with estimated molecular weights of 65,000 and 33,000. Hydrogenase prepared in this manner was used to raise and affinity purify antibodies against both subunits. 相似文献
18.
19.
Specific gonadotropin receptors of the rat testis were purified 15,000-fold after detergent extraction by a single affinity chromatography step on agarose coupled to human chorionic gonadotropin. Receptors were eluted in the free form at pH 3.2 and did not aggregate in the absence of detergent. The purified receptors displayed about 50% of the maximum theoretical binding activity, and retained the high binding affinity and hormonal specificity of the original gonadotropin receptors of the cell membrane. 相似文献
20.
Two Sepharose-bound substrate analogs, 6'-aminohexanoyl-(2-N-sphingosyl-O-beta-D-glucoside) and 6'-aminohexyl-dodecanedioyl-1-(2-N-sphingosyl-1-O-beta-D-glu coside), were synthesized and used sequentially for the affinity purification of lysosomal beta-glucosidase (N-acyl-sphingosyl-1-O-beta-D-glucoside:glucohydrolase, EC 3.2.1.45). The capacities of these nondegradable affinity supports were 0.1 and 0.15 mg enzyme/ml settled gel, respectively. The purified enzyme had a specific activity of 75 mumol min-1 mg-1. The preparation had a single protein band with a molecular weight of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, evidencing its apparent homogeneity. Isoelectric focusing on granular gels revealed four molecular forms of the enzyme with pI values of 4.0, 4.5, 4.7, and 5.8 to 6.2. The purified enzyme hydrolyzed glucosyl ceramide and 4-methylumbelliferyl-beta-D-glucoside with Km and Vmax values of 0.6 and 2.5 mM, and 101 and 26.1 mumol min-1 mg-1, respectively. The enzyme also hydrolyzed octyl beta-glucoside, a linear mixed-type inhibitor of the enzyme. Binding constants (Ki) were determined for the inhibitors, sphingosyl-1-O-beta-D-glucoside (Ki = 20 microM) and its N-hexyl derivative (Ki = 0.3 microM). The enzyme had a half-life of 65 and 30 min at 50 degrees C and pH 5.0 or 6.0, respectively. In addition, two other classes of ligands were used for the purification of lysosomal beta-glucosidase, and their capacities and specificities were compared to those of the substrate analog affinity supports. These included (i) the alkyl amine inhibitors octylamine, decylamine, and tetradecylamine; and (ii) the inhibitors, 6-aminohexanoyl-beta-glucosylamine and aminododecanoyl-1-(2-N-sphingosyl-1-O-beta-D-glucoside). Compared to these other ligand columns, the substrate analog affinity supports had about 100- to 1000-fold greater capacities or afforded 8- to 40-fold greater purification of human lysosomal beta-glucosidase. 相似文献