Purification of isopenicillin N synthetase from Streptomyces clavuligerus

1986 ◽  
Vol 32 (12) ◽  
pp. 953-958 ◽  
Author(s):  
Susan E. Jensen ◽  
Brenda K. Leskiw ◽  
Leo. C. Vining ◽  
Yair Aharonowitz ◽  
Donald W. S. Westlake ◽  
...  
Keyword(s):  
High Performance ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Streptomyces Clavuligerus ◽  
High Performance Liquid Chromatographic ◽  
Inhibitory Effect ◽  
Isopenicillin N Synthetase ◽  
Isopenicillin N

Isopenicillin N synthetase was purified from Streptomyces clavuligerus by sequential salt precipitation, ion-exchange and gel-filtration chromatography using both conventional open column and high-performance liquid chromatographic techniques. Material from the final purification step had a specific activity of 204.1 × 10−3 units/mg of protein which represented a 130-fold purification over the cell-free extract. The purified isopenicillin N synthetase was determined to have a molecular weight of 33 000 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and to have a Km of 0.32 mM with respect to its substrate δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine. The enzyme showed a sensitivity to thiol-specific inhibitors with N-ethylmaleimide giving the strongest inhibitory effect.

Partial purification and characterization of isopenicillin N epimerase activity from Streptomyces clavuligerus

1983 ◽  
Vol 29 (11) ◽  
pp. 1526-1531 ◽  
Author(s):  
Susan E. Jensen ◽  
Donald W. S. Westlake ◽  
Saul Wolfe
Keyword(s):  
Pyridoxal Phosphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Streptomyces Clavuligerus ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Major Protein ◽  
Major Protein Band ◽  
Isopenicillin N

Epimerase activity, which converts isopenicillin N to penicillin N, has been partially purified from cell-free extracts of Streptomyces clavuligerus. No stimulating cofactors of this activity were found, and neither EDTA nor anaerobic incubation caused significant inhibition of activity. Although pyridoxal phosphate did not stimulate epimerase activity, the presence of this cofactor was necessary for the stabilization of enzymic activity during the purification process. Epimerase activity was purified 35.5-fold by a combination of salt precipitation, gel filtration, and ion exchange chromatography. Gel filtration indicated that the epimerase has a molecular weight of 60 000 and sodium dodecyl sulphate – polyacrylamide gel electrophoresis of the 35.5-fold purified epimerase showed a major protein band running near that location. Pyridoxal phosphate antagonists did not uniformly inhibit epimerase activity, but the inhibitory effect of hydroxylamine could be partially reversed by pyridoxal phosphate.

Purification and characterization of a β-glucosidase from solid-state cultures ofHumicola griseavar.thermoidea

1996 ◽  
Vol 42 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Edivaldo Ximenes Ferreira Filho
Keyword(s):  
Solid State ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Performance Liquid Chromatographic ◽  
Exchange Chromatography ◽  
Glucosidase Activity ◽  
Apparent Homogeneity ◽  
Sds Page

The thermophilic fungus Humicola grisea var. thermoidea produced β-glucosidase activity when grown in a solid-state culture on wheat bran as carbon source. A β-glucosidase was purified to apparent homogeneity by ultrafiltration, gel filtration chromatography on Sephacryl S-100, and ion-exchange chromatography on S-Sepharose, as judged by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) on a 12.5% (w/v) slab gel. The enzyme had a molecular mass of 82 and 156 kDa, as estimated by SDS–PAGE and gel filtration on a high performance liquid chromatographic column, respectively, suggesting that the native enzyme may consist of two identical subunits. The purified enzyme was thermostable at 60 °C for 1 h with a half-life of 15 min at 65 °C, and displayed optimum activity at 60 °C and a pH range of 4.0–4.5. The Kmand Vmaxvalues for p-nitrophenyl β-D-glucopyranoside were determined to be 0.316 mM and 0.459 IU∙mL−1, respectively. D-Glucose, D-gluconic acid lactone, Hg2+, Cu2+, and Mn2+inhibited β-glucosidase activity. The enzyme activity was competitively inhibited by D-glucose (Ki = 0.6 mM). The purified enzyme was very active against cellobiose and p-nitrophenyl β-D-glucopyranoside.Key words: Humicola, β-glucosidase, purification, characterization.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

scholarly journals Molecular Characterization of a Recombinant Manganese Superoxide Dismutase fromLactococcus lactisM4

2014 ◽  
Vol 2014 ◽  
pp. 1-9
Author(s):  
Boon Hooi Tan ◽  
Thean Chor Leow ◽  
Hooi Ling Foo ◽  
Raha Abdul Rahim
Keyword(s):  
Superoxide Dismutase ◽  
Active Sites ◽  
Manganese Superoxide Dismutase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Gel Filtration Chromatography ◽  
T7 Promoter

A superoxide dismutase (SOD) gene ofLactococcus lactisM4 was cloned and expressed in a prokaryotic system. Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues. Expression ofsodAunder T7 promoter exhibited a specific activity of 4967 U/mg when induced with 1 mM of isopropyl-β-D-thiogalactopyranoside. The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27 kDa. However, the SOD was in dimer form as revealed by gel filtration chromatography. The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25°C and pH 7.2. It was stable up to 45°C. The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD. Although it has 98% homology to SOD ofL. lactisIL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172).

scholarly journals Purification, characterization and inhibition of human skin collagenase

1978 ◽  
Vol 169 (2) ◽  
pp. 265-276 ◽  
Author(s):  
David E. Woolley ◽  
Robert W. Glanville ◽  
Dennis R. Roberts ◽  
John M. Evanson
Keyword(s):  
Human Skin ◽  
Culture Medium ◽  
Serum Proteins ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Collagen Fibrils ◽  
Ph Optimum

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32μg of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5–8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25°C, producing the two characteristic products TCA(¾) and TCB(¼). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25°C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37°C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the α-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37°C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins α2-macroglobulin and β1-anti-collagenase both inhibited the enzyme, but α1-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

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.

Purification and preliminary characterization of 2-monoacylglycerol acyltransferase from rat intestinal villus cells

1985 ◽  
Vol 63 (5) ◽  
pp. 341-347 ◽  
Author(s):  
F. Manganaro ◽  
A. Kuksis
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cell Protein ◽  
Racemic Mixture ◽  
Small Intestinal Mucosa ◽  
Small Intestinal ◽  
Monoacylglycerol Acyltransferase

We have purified the monoacylglycerol acyltransferase from rat small intestinal mucosa to homogeneity by a combination of hydrophobic absorption, guanidine dissociation, and gel filtration. The purified enzyme gives a single band of 37 000 daltons on sodium dodecyl sulphate – polyacrylamide gel electrophoresis. The enzyme has a specific activity of about 5900 nmol/mg per hour and represents 0.12% of total cell protein, corresponding to about a 600-fold purification. The enzyme does not acylate diacylglycerols to triacylglycerols, which is consistent with the separate physical existence of the mono- and di-acylglycerol acyltransferases. The enzyme acylates the 2-monoacylglycerols to yield an essentially racemic mixture of diacylglycerols. It does not acylate glycerol 3-phosphate.

Purification and Characterization of Kallikrein from Plasma of Patients with Acute Pancreatitis

1981 ◽  
Vol 60 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Naotika Toki ◽  
Hiroyuki Sumi ◽  
Sumiyoshi Takasugi
Keyword(s):  
Acute Pancreatitis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Purification And Characterization ◽  
Α2 Macroglobulin ◽  
Sepharose 4B

1. A kallikrein-like enzyme in plasma of patients with acute pancreatitis was further purified by successive hydroxyapatite/cellulose and Sepharose-4B column chromatography. 2. By these procedures 0.26 mg of purified enzyme with a specific activity of 215 S-2266 chromozyme units/mg of protein was obtained from 10 ml of original plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent molecular weight of 31 000 as measured by gel filtration on Sephadex G-200. 4. It was confirmed immunologically that this enzyme was pancreatic kallikrein, which is distinct from plasma kallikrein, and that it could combine with α2-macroglobulin only in the presence of trypsin.

scholarly journals O-Acetylserine Sulfhydrylase fromMethanosarcina thermophila

2000 ◽  
Vol 182 (1) ◽  
pp. 45-50 ◽  
Author(s):  
Birthe Borup ◽  
James G. Ferry
Keyword(s):  
Specific Activity ◽  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Substrate Inhibition ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cysteine Biosynthesis ◽  
High Sequence Similarity ◽  
Methanosarcina Thermophila ◽  
Moderately Thermophilic

ABSTRACT Cysteine is the major source of fixed sulfur for the synthesis of sulfur-containing compounds in organisms of the Bacteriaand Eucarya domains. Though pathways for cysteine biosynthesis have been established for both of these domains, it is unknown how the Archaea fix sulfur or synthesize cysteine. None of the four archaeal genomes sequenced to date contain open reading frames with identities to eitherO-acetyl-l-serine sulfhydrylase (OASS) or homocysteine synthase, the only sulfur-fixing enzymes known in nature. We report the purification and characterization of OASS from acetate-grown Methanosarcina thermophila, a moderately thermophilic methanoarchaeon. The purified OASS contained pyridoxal 5′-phosphate and catalyzed the formation of l-cysteine and acetate from O-acetyl-l-serine and sulfide. The N-terminal amino acid sequence has high sequence similarity with other known OASS enzymes from the Eucarya andBacteria domains. The purified OASS had a specific activity of 129 μmol of cysteine/min/mg, with a Km of 500 ± 80 μM for sulfide, and exhibited positive cooperativity and substrate inhibition withO-acetyl-l-serine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at 36 kDa, and native gel filtration chromatography indicated a molecular mass of 93 kDa, suggesting that the purified OASS is either a homodimer or a homotrimer. The optimum temperature for activity was between 40 and 60°C, consistent with the optimum growth temperature for M. thermophila. The results of this study provide the first evidence for a sulfur-fixing enzyme in the Archaea domain. The results also provide the first biochemical evidence for an enzyme with the potential for involvement in cysteine biosynthesis in theArchaea.

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