scholarly journals Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities

1987 ◽  
Vol 245 (3) ◽  
pp. 831-841 ◽  
Author(s):  
J E Baldwin ◽  
R M Adlington ◽  
J B Coates ◽  
M J C Crabbe ◽  
N P Crouch ◽  
...  
Keyword(s):  
Metal Ion ◽  
Cyclopropane Ring ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ring Expansion ◽  
Maximum Activity ◽  
Reversible Inhibitor ◽  
Synthetase Activity ◽  
Synthetic Analogue ◽  
Ph Optimum ◽  
Deacetoxycephalosporin C Synthetase

Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at −70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.

Guanosine 3’:5’-Cyclic Monophosphate -Dependent Particulate Protein Kinase Activity from Yeast (Saccharomyces cerevisiae)

1999 ◽  
Vol 54 (1-2) ◽  
pp. 84-93 ◽  
Author(s):  
Hans Eckstein ◽  
Birgit Flügge
Keyword(s):  
Protein Kinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Temperature Tolerance ◽  
Maximum Activity ◽  
Partial Purification ◽  
Protein Kinase Activity ◽  
Ph Optimum ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Concentrations

Continuing our studies on cGMP in growing yeast we detected a particulate cGMPdependent protein kinase (Pk-G), which was solubilized by detergents and NaCl. It achieves maximum activity at 25 °C and pH = 6.8, high concentrations of substrate proteins or cGMP produce saturation. Casein and histones are appropriate substrates, phosphatase-pretreated histone H-2a provokes outstandingly high activity. Pk-G differs from cAMP-dependent protein kinase (Pk-A) with respect to pH optimum, temperature tolerance above 50 °C, and stability. Partial purification is achieved by chromatography with DEAE-cellulose, Sepharose, and cGMP-substituted Sepharose. The latter step also markedly removes Pk-A. At least three proteins with Pk-G-activity and high cGMP-affinity are separated by polyacrylamide-gel-electrophoresis. Their apparent molecular masses, as deduced from comigrating marker proteins, differ considerably from those of other Pk-G’s, but also of Pk-A’s

scholarly journals Purification of 2-oxoaldehyde dehydrogenase and its dependence on unusual amines

1975 ◽  
Vol 149 (3) ◽  
pp. 609-617 ◽  
Author(s):  
J Dunkerton ◽  
S P James
Keyword(s):  
Enzyme Activity ◽  
Gel Electrophoresis ◽  
General Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Ph Values ◽  
Sheep Liver ◽  
Living Tissues

1. 2-Oxoaldehyde dehydrogenase was purified from sheep liver and gave one band on polyacrylamide-gel electrophoresis. 2. The enzyme was completely dependent for its activity on the presence of Tris or one of a number of related amines, all of general structure: (See article). When more than one R group was hydrogen no enzyme activity was observed. 3. Only one of these amines is known to exist in living tissues and large concentrations of all amines were required for maximum activity. L-2-Aminopropan-1-ol was the most effective amine on the basis of substrate Km and Vmax. values and the amine Km values. 4. The enzyme was activated by phosphate which lowered the Km values for methylglyoxal, amine and NAD+. 5. The pH optimum of the enzyme was 9.3 and there was no activity at pH values below 7.8. A search for activators that might produce activity at pH 7.4 proved unsuccessful. 6. The enzyme was inhibited by rather large concentrations of barbiturates (6-46 mM) and nitro-alcohol analogues of the activating amines (66-139 mM).

scholarly journals Purification and Characterization of 1-Naphthol-2-Hydroxylase from Carbaryl-Degrading Pseudomonas Strain C4

2007 ◽  
Vol 189 (7) ◽  
pp. 2660-2666 ◽  
Author(s):  
Vandana P. Swetha ◽  
Aditya Basu ◽  
Prashant S. Phale
Keyword(s):  
Molecular Mass ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Substrate Inhibition ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sole Source ◽  
Maximum Activity ◽  
Ph Optimum ◽  
Phenol Derivatives

ABSTRACT Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source of carbon and energy via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. 1-Naphthol-2-hydroxylase (1-NH) was purified 9.1-fold to homogeneity from Pseudomonas sp. strain C4. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme is a homodimer with a native molecular mass of 130 kDa and a subunit molecular mass of 66 kDa. The enzyme was yellow, with absorption maxima at 274, 375, and 445 nm, indicating a flavoprotein. High-performance liquid chromatography analysis of the flavin moiety extracted from 1-NH suggested the presence of flavin adenine dinucleotide (FAD). Based on the spectral properties and the molar extinction coefficient, it was determined that the enzyme contained 1.07 mol of FAD per mol of enzyme. Although the enzyme accepts electrons from NADH, it showed maximum activity with NADPH and had a pH optimum of 8.0. The kinetic constants Km and V max for 1-naphthol and NADPH were determined to be 9.6 and 34.2 μM and 9.5 and 5.1 μmol min−1 mg−1, respectively. At a higher concentration of 1-naphthol, the enzyme showed less activity, indicating substrate inhibition. The Ki for 1-naphthol was determined to be 79.8 μM. The enzyme showed maximum activity with 1-naphthol compared to 4-chloro-1-naphthol (62%) and 5-amino-1-naphthol (54%). However, it failed to act on 2-naphthol, substituted naphthalenes, and phenol derivatives. The enzyme utilized one mole of oxygen per mole of NADPH. Thin-layer chromatographic analysis showed the conversion of 1-naphthol to 1,2-dihydroxynaphthalene under aerobic conditions, but under anaerobic conditions, the enzyme failed to hydroxylate 1-naphthol. These results suggest that 1-NH belongs to the FAD-containing external flavin mono-oxygenase group of the oxidoreductase class of proteins.

scholarly journals Purification and properties of a cellulase from Aspergillus niger

1977 ◽  
Vol 165 (1) ◽  
pp. 33-41 ◽  
Author(s):  
P L Hurst ◽  
J Nielsen ◽  
P A Sullivan ◽  
M G Shepherd
Keyword(s):  
Amino Acid ◽  
Aspergillus Niger ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Cellulolytic Enzyme ◽  
Protein Amino Acid ◽  
Ph Optimum

A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.

scholarly journals Purification and some properties of a novel heat-stable cis-toluene dihydrodiol dehydrogenase

1987 ◽  
Vol 244 (3) ◽  
pp. 585-590 ◽  
Author(s):  
H D Simpson ◽  
J Green ◽  
H Dalton
Keyword(s):  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sole Source ◽  
Product Inhibition ◽  
Enzyme Mechanism ◽  
Bacillus Species ◽  
Ph Optimum ◽  
Heat Stable ◽  
Dihydrodiol Dehydrogenase

cis-Toluene dihydrodiol dehydrogenase was purified 200-fold from cells of a thermotolerant Bacillus species grown with toluene as the sole source of carbon and energy. The purified enzyme preparation was remarkably heat-stable and exhibited a half-life of 100 min at 80 degrees C, the temperature optimum. The activation energy of the reaction was 36 kJ.mol-1. Isoelectric focusing indicated that the pI of the native enzyme was 6.4 and that of the denatured enzyme 6.5. Although the pH optimum was 9.8, the enzyme was most stable at pH 8. The Mr of the enzyme was approx. 172,000 as determined by gel filtration and 166,000 by polyacrylamide-gel electrophoresis. The enzyme was composed of six apparently identical subunits with Mr values of 29,500. Kinetic analysis revealed that the Km for cis-toluene dihydrodiol was 92 microM and for NAD+ was 80 microM. The apparent Km values for cis-benzene dihydrodiol and cis-naphthalene dihydrodiol were 330 microM and 51 microM respectively. The enzyme was inhibited by mercurials but was unaffected by metal-ion chelators. Steady-state kinetics and product-inhibition patterns suggested that the enzyme mechanism was ordered Bi Bi.

scholarly journals Purification and properties of a manganese-stimulated deoxyribonuclease produced during sporulation of Bacillus subtilis

1978 ◽  
Vol 172 (1) ◽  
pp. 69-76 ◽  
Author(s):  
A Akrigg
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Duplex Dna ◽  
Ph Optimum ◽  
Endonucleolytic Cleavage ◽  
Single Strand Breaks ◽  
Purification And Properties

A DNAase (deoxyribonuclease) was isolated from culture supernatants of sporulating Bacillus subtilis 168. The purified enzyme migrated as a single band during polyacrylamide-gel electrophoresis. The enzyme differs from other DNAases of B. subtilis in molecular weight, metal-ion requirement and mode of action. The enzyme was inactive in the absence of metal ions, and exhibited optimum activity with 10 mM-Mn2+, although Mg2+, Cd2+ and Co2+ could also permit some activity. The pH optimum for the enzyme was pH 7.5, and it degraded linear-duplex DNA or closed-circular-duplex DNA to acid-soluble material. There was little or no activity on single-stranded DNA or rRNA. Sucrose-gradient analysis of the products of DNAase action on bacteriophage T7 DNA showed that endonucleolytic cleavage had occurred by the introduction of single-strand breaks in both strands of the duplex. The molecular weight of the enzyme was determined, by gel filtration on Sephadex G-75, to be 12000.

Purification and some properties of a thermostable DNA polymerase from a Thermotoga species

1990 ◽  
Vol 68 (11) ◽  
pp. 1292-1296 ◽  
Author(s):  
H. D. Simpson ◽  
T. Coolbear ◽  
M. Vermue ◽  
R. M. Daniel
Keyword(s):  
Dna Polymerase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Thermostable Enzyme ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Size Exclusion ◽  
Small Scale ◽  
Ph Optimum

A stable DNA polymerase (EC 2.7.7.7) has been purified from the extremely thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1 by a five-step purification procedure. First, the crude extract was treated with polyethylenimine to precipitate nucleic acids. The endonuclease activity coprecipitated. DEAE-Sepharose, CM-Sephrarose, and hydroxylapatite column chromatography were used to purify the preparation. As a final step on a small scale, preparative sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis was used. The purified DNA polymerase exhibited a molecular weight of 85 000, as determined by both SDS–polyacrylamide gel electrophoresis and size-exclusion chromatography. Its pH optimum was in the range pH 7.5–8. When assayed over the temperature range 30–80 °C, the maximum activity in a 30-min assay was at 80 °C. The enzyme was moderately thermostable and exhibited half-lives of 3 min at 95 °C and 60 min at 50 °C in the absence of substrate. Several additives such as Triton X-100 enhanced thermostability. During storage at 4 °C and −70 °C, the stability of the enzyme was improved by the addition of gelatin.Key words: DNA polymerase, thermostable enzyme, Thermotoga.

scholarly journals Purification and properties of S-adenosylmethionine: aldoxime O-methyltransferase from Pseudomonas sp. N.C.I.B. 11652

1985 ◽  
Vol 226 (1) ◽  
pp. 147-153 ◽  
Author(s):  
D B Harper ◽  
J T Kennedy
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Pseudomonas Sp ◽  
Ph Optimum

An enzyme catalysing the O-methylation of isobutyraldoxime by S-adenosyl-L-methionine was isolated from Pseudomonas sp. N.C.I.B. 11652. The enzyme was purified 220-fold by DEAE-cellulose chromatography, (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and chromatography on calcium phosphate gel. Homogeneity of the enzyme preparation was confirmed by isoelectric focusing on polyacrylamide gel and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme showed a narrow pH optimum at 10.25, required thiol-protecting agents for activity and was rapidly denatured at temperatures above 35 degrees C. The Km values for isobutyraldoxime and S-adenosyl-L-methionine were respectively 0.24 mM and 0.15 mM. Studies on substrate specificity indicated that attack was mainly restricted to oximes of C4-C6 aldehydes, with preference being shown for those with branching in the 2- or 3-position. Ketoximes were not substrates for the enzyme. Gel filtration on Sephadex G-100 gave an Mr of 84 000 for the intact enzyme, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated an Mr of 37 500, suggesting the presence of two subunits in the intact enzyme. S-Adenosylhomocysteine was a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 0.027 mM. The enzyme was also susceptible to inhibition by thiol-blocking reagents and heavy-metal ions. Mg2+ was not required for maximum activity.

Purification and characterization of an endo-β-1,3-glucanase from Trichoderma harzianum

1996 ◽  
Vol 42 (10) ◽  
pp. 1039-1044 ◽  
Author(s):  
Eliane Ferreira Noronha ◽  
Cirano José Ulhoa
Keyword(s):  
Trichoderma Harzianum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Maximum Activity ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Isolated Cell

β-1,3-Glucanases are produced by Trichoderma harzianum when it is grown in the presence of chitin or isolated cell wall from fungi. An endo-β-1,3-glucanase from the culture filtrate of T. harzianum was purified by gel filtration on Sephacryl S-200, followed by hydrophobic interaction chromatography on phenyl-Sepharose. A typical procedure provided 134-fold purification with a 3.6% yield. The molecular mass of the purified endo-β-1,3-glucanase was found to be approximately 36 kDa, as estimated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis on a 10% w/v slab gel. The enzyme was active toward glucans containing β-1,3-linkages and hydrolysed laminarin to form oligosaccharides. The Km and Vmax values for β-1,3-ghicanases, using laminarin as substrate, was 1.18 mg∙mL−1 and 1.26 U∙mL−1, respectively. The pH optimum for the enzyme was pH 4.4 and maximum activity was obtained at 45–50 °C. Enzyme activity was strongly inhibited in the presence of HgCl2 and stimulated by cations such as Zn2+ and Ca2+.Key words: endo-β-1,3-glucanase, Trichoderma harzianum, purification, characterization.

scholarly journals Purification and characterization of the major aminopeptidase from human skeletal muscle

1983 ◽  
Vol 211 (3) ◽  
pp. 567-573 ◽  
Author(s):  
D Mantle ◽  
M F Hardy ◽  
B Lauffart ◽  
J R McDermott ◽  
A I Smith ◽  
...  
Keyword(s):  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Quadriceps Muscle ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Naturally Occurring ◽  
Thiol Proteinases

The major aminopeptidase from human quadriceps muscle was purified (as judged by polyacrylamide-gel electrophoresis) by anion-exchange chromatography (two steps) and gel filtration (two steps). The enzyme showed maximum activity at pH 7.3, in the presence of 1 mM-2-mercaptoethanol and 0.5 mM-Ca2+ ions; activation of the enzyme occurred in the presence of several other bivalent cations. Inhibition of activity was obtained in the presence of metal-ion-chelating agents and inhibitors of aminopeptidases and thiol proteinases. The molecular weight of the enzyme was 102 000 (by gel filtration). The enzyme hydrolysed several amino acyl-7-amido-4-methylcoumarin derivatives; highest activity was obtained with alanyl-7-amido-4-methylcoumarin. The enzyme also degraded a series of dipeptides, alanine oligopeptides and some naturally occurring peptides. Of particular interest was the high activity of the enzyme towards the enkephalins.

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