Purification and characterization of rhodanese from Acinetobacter calcoaceticus

1980 ◽  
Vol 26 (3) ◽  
pp. 281-286 ◽  
Author(s):  
Peter A. Vandenbergh ◽  
Richard S. Berk
Keyword(s):  
Reduced Glutathione ◽  
Acinetobacter Calcoaceticus ◽  
Sucrose Density Gradient ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Soluble Enzyme ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Intracellular Enzyme

Rhodanese (thiosulfate: cyanide sulfur transferase, EC 2.8.1.1) was found to be constitutively present as an intracellular enzyme in Acinetobacter calcoaceticus. The soluble enzyme was purified 40.9-fold by a procedure which included ultracentrifugation, ethanol precipitation, CM-Sephadex batchwise separation, QAE-50 ion exchange chromatography, and sucrose density gradient ultracentrifugation. The enzyme had a molecular weight of approximately 35 000 with a pH optimum of 8–8.5. Activity was substantially enhanced by supplements of 2-mercaptoethanol and to a lesser extent by cysteine–HCl or reduced glutathione. No degradation of the enzyme into smaller subunits was observed when treated with 2-mercaptoethanol.

scholarly journals Purification and properties of l-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus

1987 ◽  
Vol 248 (3) ◽  
pp. 871-876 ◽  
Author(s):  
M E Hoey ◽  
N Allison ◽  
A J Scott ◽  
C A Fewson
Keyword(s):  
Lactate Dehydrogenase ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Potential Inhibitors ◽  
Triton X

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a ‘wall + membrane’ fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.

scholarly journals Purification and characterization of human platelet glutathione-S- transferase

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1595-1599
Author(s):  
J Loscalzo ◽  
J Freedman
Keyword(s):  
Specific Activity ◽  
Human Platelet ◽  
Reduced Glutathione ◽  
Human Platelets ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Complete Purification ◽  
High Concentrations

A glutathione-S-transferase was isolated and purified to homogeneity from human platelets. With a combination of ammonium sulfate fractionation and chromatographic methods, 0.2 mg of pure enzyme was obtained from 9 X 10(11) platelets with a 12% recovery. The purified enzyme had a specific activity of 7.5 U per milligram, representing an approximately 1,100-fold purification. The enzyme was found to be anionic, with an isoelectric point of 4.6. With reduced glutathione as a co-substrate, platelet glutathione-S-transferase was most active with the synthetic substrate, 1-chloro-2,4-dinitrobenzene, less active with 1,2-dichloro-4-nitrobenzene, and essentially inactive with nitroglycerin and 1,2-epoxy-3-(p-nitrophenoxy)-propane. The pH optimum for activity with glutathione and 1-chloro-2,4-dinitrobenzene was 7.0. Indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyindole-3-acetic acid), a chlorobenzene derivative, noncompetitively inhibited human platelet glutathione-S-transferase with an apparent KI of 0.23 mmol/L. This study represents the first complete purification and characterization of a glutathione-S-transferase from platelets. The presence of this enzyme in the platelet, within which high concentrations of reduced glutathione coexist, suggests the potential importance of the platelet in detoxification reactions and in the synthesis of the glutathione adducts of leukotriene metabolism.

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).

Separation and characterization of an α-glucosidase and maltase from Bacillus amyloliquefaciens

1985 ◽  
Vol 31 (8) ◽  
pp. 670-674 ◽  
Author(s):  
William M. Fogarty ◽  
Catherine T. Kelly ◽  
Sunil K. Kadam
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Growth Medium ◽  
Isoelectric Point ◽  
Bacillus Amyloliquefaciens ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Optimum Temperature ◽  
Ph Optimum

A novel α-glucosidase and a maltase were isolated from Bacillus amyloliquefaciens. The formation of both enzymes was induced by trehalose, sucrose, or lactose in the growth medium. Trehalose is by far the most efficient inducer of both systems. The α-glucosidase and maltase were separated and purified by ion-exchange chromatography on DEAE Bio-Gel A. Purified α-glucosidase hydrolysed p-nitrophenyl-α-D-glucoside, isomaltose, and isomaltotriose but sucrose, maltose, or related saccharides were not attacked. β-Glucosides and polymeric glucosides were not degraded. The optimum temperature for α-glucosidase activity was 40 °C and its pH optimum was 5.3. The molecular weight and isoelectric point (pI) of the enzyme were 27 000 and 4.6, respectively. Purified maltase attacked maltose and sucrose, while maltotriose and melezitose were hydrolysed at slower rates and p-nitrophenyl-α-D-glucoside was not degraded. Other properties of the maltase were as follows: optimum temperature for activity, 30 °C; pH optimum, 6.5; molecular weight, 64 000; and pI, 4.7.

Purification and Characterization of Laccase from Curvularia trifol

2010 ◽  
Vol 113-116 ◽  
pp. 2215-2219
Author(s):  
Jie Zhang ◽  
Jing Ren ◽  
Yang Yu ◽  
Bin Song Wang
Keyword(s):  
Enzyme Activity ◽  
Ion Exchange ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Recycling Rate ◽  
Relative Molecular Mass ◽  
Optimal Temperature ◽  
Purification And Characterization ◽  
Alizarin Red

Electrophoretic pure lacquer enzymes were obtained from the thick enzyme of Gladiolus Curvularia trifol with grading precipitation and DEAE2-Cellulose ion exchange chromatography. The overall recycling rate of enzyme activity was 14.97% and the purification reached 2.96 times. The relative molecular mass of enzyme was 92.3Kda. The optimal temperature and pH were 40°C and 3.5, respectively. The Km of ABTS catalyzed by laccase was 1.11× 10- 5mol L-1. Alizarin red and Congo red could be degradated by purified laccase effectively without the participation of small molecule amboceptor. Alizarin red could be degradated by 80% with its being affected by 1000U/L of enzyme activity for 70h, which revealed the huge potentiality of Gladiolus Curvularia trifol and laccase in the degradation of dye.

scholarly journals Purification and Characterization of Cellulase from Termite Ametermes eveuncifer (Silverstri) Soldiers

2016 ◽  
Vol 9 (1) ◽  
pp. 1 ◽  
Author(s):  
B. S. Fagbohunka ◽  
R. E. Okonji ◽  
Ayinla Zainab Adenike
Keyword(s):  
Ion Exchange ◽  
Specific Activity ◽  
Ion Exchange Chromatography ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Ammonium Sulphate Precipitation ◽  
Percentage Yield ◽  
Termite Soldiers

Cellulase enzyme was purified and characterized from termite soldiers (Ametermes eveuncifer) using 70% ammonium sulphate precipitation, ion exchange chromatography and affinity chromatography. The enzyme isolated had a specific activity of 5.04 U/mg with a percentage yield of 11.7%. The enzyme showed maximum activity at 500C and pH 8. The enzyme was not inhibited by Ba2+ at a concentration of 1mM and Pb2+ at 10 mM concentration but was inhibited by other metal ions at 1 mM and 10 mM concentrations of their salts (NaCl, KCl, MnCl2, and NiCl2),

scholarly journals Purification and characterization of hyaluronic acid from chicken combs

2012 ◽  
Vol 42 (9) ◽  
pp. 1682-1687 ◽  
Author(s):  
Claudia Severo da Rosa ◽  
Ana Freire Tovar ◽  
Paulo Mourão ◽  
Ricardo Pereira ◽  
Pedro Barreto ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Gel Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Purification And Characterization ◽  
Tissue Fractionation ◽  
Subsequent Identification

Hyaluronic acid (HA) is an important macromolecule in medical and pharmaceutical fields. The umbilical cord and the chicken comb are some of the tissues richest in this polysaccharide. The profit from obtaining HA from the combs of slaughtered animals is particularly attractive. This work aimed to extract, purify, and characterize HA. The glycosaminoglycan concentration in the chicken comb was found to be about 15µg of hexuronic acid mg-1 of dry tissue. Fractionation using ion exchange chromatography and subsequent identification of the fractions by agarose gel electrophoresis showed that HA corresponded to 90% of the total amount of extracted glycosaminoglycans.

scholarly journals Purification and characterization of human platelet glutathione-S- transferase

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1595-1599 ◽  
Author(s):  
J Loscalzo ◽  
J Freedman
Keyword(s):  
Specific Activity ◽  
Human Platelet ◽  
Reduced Glutathione ◽  
Human Platelets ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Complete Purification ◽  
High Concentrations

Abstract A glutathione-S-transferase was isolated and purified to homogeneity from human platelets. With a combination of ammonium sulfate fractionation and chromatographic methods, 0.2 mg of pure enzyme was obtained from 9 X 10(11) platelets with a 12% recovery. The purified enzyme had a specific activity of 7.5 U per milligram, representing an approximately 1,100-fold purification. The enzyme was found to be anionic, with an isoelectric point of 4.6. With reduced glutathione as a co-substrate, platelet glutathione-S-transferase was most active with the synthetic substrate, 1-chloro-2,4-dinitrobenzene, less active with 1,2-dichloro-4-nitrobenzene, and essentially inactive with nitroglycerin and 1,2-epoxy-3-(p-nitrophenoxy)-propane. The pH optimum for activity with glutathione and 1-chloro-2,4-dinitrobenzene was 7.0. Indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyindole-3-acetic acid), a chlorobenzene derivative, noncompetitively inhibited human platelet glutathione-S-transferase with an apparent KI of 0.23 mmol/L. This study represents the first complete purification and characterization of a glutathione-S-transferase from platelets. The presence of this enzyme in the platelet, within which high concentrations of reduced glutathione coexist, suggests the potential importance of the platelet in detoxification reactions and in the synthesis of the glutathione adducts of leukotriene metabolism.

scholarly journals Purification and characterization of phosphatidylinositol synthase from human placenta

1994 ◽  
Vol 297 (3) ◽  
pp. 517-522 ◽  
Author(s):  
B E Antonsson
Keyword(s):  
Human Placenta ◽  
Microsomal Fraction ◽  
Enzyme Reaction ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Sds Page ◽  
Phosphatidylinositol Synthase ◽  
Triton X

Phosphatidylinositol synthase (CDP-1,2-diacyl-sn-glycerol:myoinositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified from the microsomal fraction of human placenta. The Triton X-100-extracted enzyme was purified 8300-fold over the microsomal fraction by affinity chromatography on CDP-diacylglycerol-Sepharose followed by ion-exchange chromatography on Mono Q. The purified enzyme had a molecular mass of 24,000 Da on SDS/PAGE. The enzyme had a pH optimum at 9.0, required Mn2+ or Mg2+, and was inhibited by Ca2+ and Zn2+. The Km for myo-inositol was determined to be 0.28 mM. Optimal activity was obtained at 0.2-0.4 mM CDP-diacylglycerol; higher concentrations of the lipid substrate inhibited the enzyme reaction. The enzyme was inhibited by nucleoside di- and tri-phosphates, Pi and PPi. CDP competitively inhibited the enzyme reaction with a Kis of 4 mM. The optimal temperature for the PtdIns synthase reaction was 50 degrees C.

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