A Fibrinolytic Agent from a Saturnid Caterpillar Partial Purification and Characterization

1973 ◽  
Vol 29 (01) ◽  
pp. 135-142 ◽  
Author(s):  
C. L Arocha-Pinango ◽  
N. A Marsh ◽  
D Robinson
Keyword(s):  
Clinical Syndrome ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Heat Stable ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Human Plasminogen ◽  
The Relationship ◽  
Stable Material

SummaryPurification and characterization studies were performed on a proteolytic agent obtained from a Saturnid moth caterpillar. Starting material possessed caseinolytic, fibrinolytic and plasminogen-activator activities. The fibrinolytic activity was stable over a wide range of pH and temperature. Purification was performed by molecular exclusion chromatography and ion exchange chromatography. A pH- and heat-stable material was obtained having a molecular weight in the range of 16,000-18,000. This material did not possess the ability to activate human plasminogen but retained direct caseinolytic and fibrinolytic activities. It had no effect on thrombin- and arvin- clotting times of human plasma, on partial thromboplastin time or on the whole blood thrombin generation test. Electrophoresis on cellulose acetate agar gel and polyacrylamide showed the material to be very basic and moving away from any detectable protein. The isoelectric point was found to be greater than pH 10. The relationship between the characterized material and the clinical syndrome caused by contact with the caterpillar remains to be determined.

scholarly journals Partial purification and characterization of xylanase produced by Penicillium expansum

2006 ◽  
Vol 49 (3) ◽  
pp. 475-480 ◽  
Author(s):  
André Luiz de Souza Querido ◽  
Jorge Luiz Cavalcante Coelho ◽  
Elza Fernandes de Araújo ◽  
Virgínia Maria Chaves-Alves
Keyword(s):  
Enzymatic Activity ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Penicillium Expansum ◽  
Partial Purification ◽  
Major Protein ◽  
Purification And Characterization ◽  
Exclusion Chromatography ◽  
Molecular Exclusion Chromatography

An extracellular xylanase was found to be the major protein in the filtrate culture of Penicillium expansum when grown on 0.3 % wheat bran, which showed no xylanase multiplicity. The enzime was partial purified by.ammonium sulfate fractioning, molecular exclusion chromatography, ultrafiltration and anion exchange chromatography. The protein eluation profile showed only one form of xylanase that was partially characterized. The activity of purified xylanase was optimal at pH 5.5 and 40 ºC. The enzyme was stable at pH between 5.5 and 6.5 and temperatures between 20-40 ºC. The enzyme showed a Km of 3.03 mM and Vmax of 0.027 mumol min-1 mug -1 of protein. The enzymatic activity was increased 31 % by Mg2+ and 28 % by Al3+.

scholarly journals Purification and characterization of the heat-stable serine proteinase from Thermomonospora fusca YX

1987 ◽  
Vol 246 (2) ◽  
pp. 511-517 ◽  
Author(s):  
T W Gusek ◽  
J E Kinsella
Keyword(s):  
Thermal Stability ◽  
Ionic Strength ◽  
Serine Proteinase ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Purification And Characterization ◽  
Thermomonospora Fusca ◽  
Heat Stable

The proteinase secreted from Thermomonospora fusca YX grown on cellulose was purified by (NH4)2SO4 fractionation and cation-exchange chromatography. The isolated proteinase readily hydrolysed several proteins and demonstrated activity towards casein from 35 to 95 degrees C (at pH 8.0) with maximum activity at 80 degrees C. It exhibited broad pH and ionic-strength optima centered at pH 9.0 and 0.2 M-NaCl respectively, and it retained high activity in the presence of 2% (w/v) SDS, 20 mM-dithiothreitol and 1.0 M-NaCl. The proteinase, which was fully inhibited by phenylmethanesulphonyl fluoride, had an Mr of 14,500 and an isoelectric point at 9.21. A measurement of proteinase thermal stability demonstrated a T50% (15 min) of 85 degrees C at pH 4.5.

Partial Purification and Some Properties of Brassica napus Lipase

1994 ◽  
Vol 49 (5-6) ◽  
pp. 293-301 ◽  
Author(s):  
Cornelia Fuchs ◽  
Gerd Hansen
Keyword(s):  
Brassica Napus ◽  
Size Exclusion Chromatography ◽  
Lipase Activity ◽  
Sodium Deoxycholate ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Partial Purification ◽  
Exclusion Chromatography ◽  
Molecular Masses

Abstract Lipase (triacylglycerol acylhydrolase EC 3.1.1.3) from rape (Brassica napus cv. Ceres) was isolated from cotyledons of dark-grown seedlings. The enzyme was partially purified by poly­ ethylene glycol precipitation. Delipidation of the lipase with n-hexane was required prior to further purification by anion exchange chromatography and size exclusion chromatography. A purification factor of 337 was ultimately achieved and the purification process was moni­tored by SDS-PAGE. Here, at least two protein bands with molecular masses of 62 and 64 kD a respectively were found in the active fraction obtained by size exclusion chromatography. Sodium deoxycholate was found to stimulate the lipase activity, but appeared to cause aggregation of the enzyme. It was not possible to estimate the isoelectric point of the dialyzed rape lipase due to the high molecular mass of the aggregates. Two simple methods to detect lipase activity directly on polyacrylamide gel were applied. No esterase activity was found by using p-nitrophenyl acetate as substrate.

scholarly journals The deoxyribonucleic acid polymerases from the diatom Cylindrotheca fusiformis. Partial purification and characterization of four distinct activities

1977 ◽  
Vol 167 (3) ◽  
pp. 601-610 ◽  
Author(s):  
T W Okita ◽  
B E Volcani
Keyword(s):  
Deoxyribonucleic Acid ◽  
Maximum Activity ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Cylindrotheca Fusiformis ◽  
High K ◽  
Zinc Chelator ◽  
Relationship Of ◽  
The Relationship

Four extramitochondrial DNA polymerases from the marine photosynthetic diatom Cylindrotheca fusiformis were isolated and purified more than 1200-fold by chromatography on DNA-cellulose and DEAE-Sephadex. The enzymes were equally susceptible to inhibition by the thiol-blocking agents N-ethylmaleimide and p-chloromercuribenzoate, the zinc chelator o-phenathroline, and the nucleic acid interchelators ethidium bromide and acriflavin; they displayed similar pH optima, preferred activated DNA, and had strict dependence on high K+ for maximum activity. They were differentiated on the basis of their kinetic parameters, template-primer utilization and salt requirements. The four activities varied with growth stage of C. fusiformis. Activities of polymerases A and D doubled in exponential-phase cells as compared with those in stationary-phase cells, and the increase in polymerase B and chloroplast activity C was 20-40%. The relationship of the diatom polymerases to the complements in other organisms is discussed.

Post-proline endopeptidase. Partial purification and characterization of the enzyme from pig kidneys

1982 ◽  
Vol 47 (4) ◽  
pp. 1139-1148 ◽  
Author(s):  
Karel Hauzer ◽  
Linda Servítová ◽  
Tomislav Barth ◽  
Karel Jošt
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Prolyl Leucyl Glycinamide

Post-proline endopeptidase was isolated from pig kidneys and partially purified. The procedure consisted of fractionation with ammonium sulphate, ion exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-200 and rechromatography on DEAE-Sephadex A-50. The preparation had 55 times higher specific activity than the crude extract and did not contain any contaminating enzymic activities. The enzyme cleaved a number of proline-containing peptides and was strictly specific in catalyzing the hydrolysis of the peptide bond on the carboxyl side of the proline residue. The optimum pH for the hydrolysis of the synthetic peptides benzyl-oxycarbonylglycyl-prolyl-leucyl-glycinamide and benzyloxycarbonyl-glycyl-proline β-naphtylamide was 7.8-8.0 and, in the case of benzyloxycarbonylglycyl-proline p-nitroanilide, 7.2 to 7.5. For the hydrolysis of the tetrapeptide benzyloxycarbonylglycyl-prolyl-leucyl-glycinamide, the Km value of 75 μ mol l-1 was obtained.

scholarly journals Purification and Characterization of Transglutaminase from Streptomyces sp. TTA 02 SDS 14

2016 ◽  
Vol 11 (3) ◽  
Author(s):  
Lia Siti Nur'amaliyah ◽  
Dewi Seswita Zilda ◽  
Nisa Rachmania Mubarik
Keyword(s):  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Phenacyl Bromide ◽  
Optimum Temperature ◽  
Purification And Characterization ◽  
Streptomyces Sp ◽  
Exclusion Chromatography ◽  
Sulfonyl Fluoride

Streptomyces sp. TTA 02 SDS 14 is a transglutaminase producing bacteria which previously had been  screened along with more than one hundred isolates. This research aimed to purify and characterize transglutaminase from this strain. Transglutaminase was purified from crude enzyme by ultrafiltration, Q-Sepharose ion exchange chromatography and Sepacryl S200 size exclusion chromatography sequentially, obtaining yield and purification fold of  1.36%  and 27 folds, respectively. The molecular weight of the purified transglutaminase was 72 kDa detected by zymogram gel electrophoresis. The optimum temperature and pH were 50°C and 6. The transglutaminase was stable at 45°C and could be activated in the presence of 5 mM and 10 mM of Na+, K+, Li+,Ca2+, Mg2+, BPB (4-bromo-phenacyl bromide), and IAA (iodo acetamide acid), but the activity was inhibited by  the presence of Cu+, Zn2+, and PMSF (phenyl methyl sulfonyl fluoride).

Partial Purification and Characterization of Polyphenol Oxidase Extracted from Agaricus bisporus (J.E.Lange) Imbach

2007 ◽  
Vol 5 (1) ◽  
Author(s):  
Hicham Gouzi ◽  
Abdelhafid Benmansour
Keyword(s):  
Polyphenol Oxidase ◽  
Agaricus Bisporus ◽  
Mixed Type ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Heat Stable ◽  
Monophenolase Activity ◽  
Specificity Constant ◽  
Diphenolase Activity

Polyphenol oxidase (PPO) from mushrooms (Agaricus bisporus (J.E.Lange) Imbach) was partially purified and characterized. The enzyme exhibited both monophenolase and diphenolase activities that were measured spectrophotometrically using L-tyrosine and pyrogallol as substrates. A two-fold purification in both activities was achieved by ammonium sulfate fractionation. The monophenolase activity was 3.35 EU/ml, and the diphenolase activity was 189.3 EU/ml. PPO was relatively stable at -15°C for 44 days. The enzyme was not very heat stable, and its activity decreased when incubated at the temperatures higher than 35°C. PPO activity showed two pH optima, at 5.3 and 7.0 at 25°C when pyrogallol was used as the substrate.Mono-, di- and triphenols were substrates for PPO. Using Vmax/Km as a specificity constant, pyrocatechol was the better substrate followed by pyrogallol. The kinetic parameters of the enzyme were: Vmax = 78 EU/min/ml, Km = 1.4 mM and KS = 250 mM for pyrogallol and Vmax = 168 EU/min/ml, Km = 0.40 mM and KS = 270 mM for the pyrocatechol. Of the inhibitors tested, competitive-type inhibition was observed with benzoic acid and sodium azide. A mixed-type inhibition was observed with L-cysteine and sodium fluoride.

Purification and characterization of the extracellular proteinase ofPseudomonas fluorescensNCDO 2085

1986 ◽  
Vol 53 (3) ◽  
pp. 457-466 ◽  
Author(s):  
David J. Fairbairn ◽  
Barry A. Law
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Extracellular Proteinase ◽  
Original Activity ◽  
Heat Stable ◽  
D Values

SUMMAEYPseudomonas fluorescensNCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 °C and pH 7·0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3·5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5·40±0·05 and a mol. wt of 40200±2100. It is heat-stable having D-values at 74 and 140 °C of 1·6 and 1·0 min respectively; 40 and 70% of the original activity remained after HTST (74 °C/17 s) and ultra high temperature (140°C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from otherPseudomonasspp.

N-Acetyl-ß-ᴅ-hexosaminidases of the Brine Shrimp Artemia: Partial Purification and Characterization

1991 ◽  
Vol 46 (9-10) ◽  
pp. 781-788 ◽  
Author(s):  
Klaus-Dieter Spindler ◽  
Brigitte Funke-Höpfner
Keyword(s):  
Brine Shrimp ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Strong Inhibitor ◽  
Molecular Masses ◽  
Dose Dependent ◽  
Enzyme I

Abstract N-Acetyl-β-ᴅ-hexosaminidases (EC 3.2.1.52) from Artemia nauplii were isolated and char­acterized. Three different enzymes I, II1 and II2 were separated according to their behaviour on anion exchange chromatography and gel filtration columns. Their apparent molecular masses were 83,000 ± 7000, 110,000 ± 10,000 and 56,000 ± 5000 Da with corresponding S-values of 8.6, 11.9 and 7.9. All three enzymes also differ in their apparent pH-optima (5.1, 4.5 and 6.1) and they all bind to concanavalin A. The three enzymes have about the same affinities (app. Km between 0.16 and 0.72 mmol/1) for the three substrates (p-nitrophenyl-N-acetyl-β-ᴅ-glucosamine or p-nitrophenyl-N-acetyl-β-ᴅ-galactosamine and N ,N′-diacetyl-chitobiose) and are therefore N-acetyl-β-ᴅ-hexosaminidases. In contrast, the three enzymes behave quite differently, both in terms of their inhibitor constants and the type of inhibition. The substrates inhibit both enzymes II1 and II2 but not enzyme I. On the other hand, N-acetyl-β-ᴅ-galactosamine inhibits enzyme I in a non-competi­tive way but not enzymes II1 and II2. All three enzymes are inhibited by the end product N-acetyl-β-ᴅ-glucosamine, enzyme I in a competitive manner, both enzymes II1 and II2 in a non-competitive way. 2-Acetamido-2-deoxy-ᴅ-galactonolactone is a strong inhibitor for en­zyme I (Ki = 13 μtmol/l) with much lower affinities towards enzymes II1 and II2 (Ki = 0.63 and 1.03 mmol/l). All three enzymes are inhibited in a dose-dependent way and completely reversi­ble by α-methyl-mannoside.

Purification and Characterization of an Alkaline Protease from Bacillus alcalophilus

2012 ◽  
Vol 192 ◽  
pp. 285-288 ◽  
Author(s):  
Shan Shan Liu ◽  
Li Li Wang ◽  
Lin Yuan
Keyword(s):  
Alkaline Protease ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization ◽  
Highly Active ◽  
Wide Range ◽  
Commercial Applications ◽  
Ph Range

The purification and characterization of an alkaline protease produced by Bacillus alcalophilus were investigated. The enzyme was purified in two steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by cation-exchange chromatography. The purified protease had a molecular mass of approximately 28 kDa, was highly active over an alkaline pH range of 10.0 to 11.0, and remained stable over a pH range of 7.0 to 12.0. The optimum temperature for the enzyme activity was found to be 40~60°C, while the thermotolerance of the enzyme was poor. Therefore, these characteristics of the protease indicate its potential for a wide range of commercial applications.

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