Purification and partial characterization of glutamate synthase from Rhodospirillum rubrum grown under nitrogen-fixing conditions

Glutamate synthase, a key enzyme in ammonia assimilation, has been purified from the photosynthetic bacterium Rhodospirillum rubrum. The purification procedure involves ion-exchange chromatography, affinity chromatography and gel filtration. The recovery in the procedure is high (62%) and the specific activity is 21 mumol of NADPH oxidized/min per mg. The enzyme is specific for its substrates, and no activity was demonstrated with NADH or NH4+ ions substituting for NADPH and glutamine respectively. The enzyme is composed of two dissimilar subunits with molecular masses of 53 and 152 kDa, and it is shown that Cl- ions have an effect on the aggregation of the enzyme. Km values for the substrates are: NADPH, 16 microM; 2-oxoglutarate, 10 microM; and glutamine, 65 microM. The enzyme is inhibited by amidotransferase inhibitors at micromolar concentrations. The role of the enzyme in the metabolic regulation of nitrogenase is discussed.

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Purification and Characterization of Melanogenic Enzyme Tyrosinase from Button Mushroom

Enzyme Research ◽

10.1155/2014/120739 ◽

2014 ◽

Vol 2014 ◽

pp. 1-6 ◽

Cited By ~ 18

Author(s):

Kamal Uddin Zaidi ◽

Ayesha S. Ali ◽

Sharique A. Ali

Keyword(s):

Agaricus Bisporus ◽

Specific Activity ◽

Gel Filtration ◽

Deae Cellulose ◽

Edible Mushroom ◽

Total Activity ◽

Protein Band ◽

Exchange Chromatography ◽

Ammonium Sulphate Precipitation ◽

Key Enzyme

Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it’s taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

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Kinetic and Thermodynamic Characterization of Lignin Peroxidase Isolated from Agaricus bitorqus A66

International Journal of Chemical Reactor Engineering ◽

10.1515/1542-6580.2837 ◽

2012 ◽

Vol 10 (1) ◽

Author(s):

Ismat Bibi ◽

Haq Nawaz Bhatti

Keyword(s):

Lignin Peroxidase ◽

Thermal Inactivation ◽

Specific Activity ◽

Gel Filtration ◽

Veratryl Alcohol ◽

Exchange Chromatography ◽

Different Temperatures ◽

Scale Experiment ◽

Menten Kinetic

This study deals with purification and characterization of lignin peroxidase (LiP) isolated from Agaricus bitorqus A66 during decolorization of NOVASOL Direct Black dye. A laboratory scale experiment was conducted for maximum LiP production under optimal conditions. Purification & fractionation of LiP was performed on DEAE-Sepharose ion exchange chromatography followed by Sephadex G-50 gel filtration. The purified LiP has a specific activity of 519 U/mg with 6.73% activity recover. The optimum pH and temperature of purified LiP for the oxidation of veratryl alcohol were 6.8 and 45 °C, respectively. Michaelis-Menten kinetic constants (Vmax and Km) were determined using different concentrations of veratryl alcohol (1-35 mM). The Km and Vmax were 16.67 mM and 179.2 U/mL respectively, for veratryl alcohol oxidation as determined from the Lineweaver-Burk plot. Thermal inactivation studies were carried out at different temperatures to check the thermal stability of the enzyme. Enthalpy of activation decreased where Free energy of activation for thermal denaturation increased at higher temperatures. A possible explanation for the thermal inactivation of LiP at higher temperatures is also discussed.

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Evaluation of dialysis treatment in uremic patients by gel filtration of serum

Clinical Chemistry ◽

10.1093/clinchem/36.11.1906 ◽

1990 ◽

Vol 36 (11) ◽

pp. 1906-1910 ◽

Cited By ~ 4

Author(s):

J Osada ◽

T Gea ◽

C Sanz ◽

I Millan ◽

J Botella

Keyword(s):

Ion Exchange ◽

Gel Filtration ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

The Other ◽

Control Group ◽

Dialysis Treatment ◽

Additional Information ◽

Molecular Masses ◽

Two Peaks

Abstract A group of substances of molecular masses between 300 and 1500 Da have been found to be toxic metabolites in patients with uremia. We determined the concentration in serum of these molecules in the following groups of patients: two hemodialyzed groups (one with cuprophane and the other with polyacrylonitrile dialyzers), one group treated with continuous ambulatory peritoneal dialysis, one group of nondialyzed azotemic patients, and one control group of healthy persons. Ultrafiltrates of the subjects' sera were fractionated on Sephadex G-15 followed by ion-exchange chromatography. Eluates were monitored by absorbance at 254 and 206 nm. Partially characterized peaks P1 and P2, obtained by gel filtration, correlated with the concentration of creatinine in serum; their concentrations were significantly (P less than 0.01) larger in hemodialyzed groups than in peritoneal dialyzed or in nondialyzed azotemic patients. After ion-exchange chromatography, two peaks (P'5 and P'6) correlated with serum creatinine and also were larger in hemodialyzed patients than in the other groups. Apparently, adequate discrimination is obtained by gel-filtration analysis and further analysis by ion-exchange chromatography does not provide additional information in most of the affected patients.

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

Bayero Journal of Pure and Applied Sciences ◽

10.4314/bajopas.v13i2.14 ◽

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.

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Optimization of Metal Ions in Sugarcane Bagasse Fermenting Medium for the Production of Streptokinase by Streptococcus equisimilis

Science Letters ◽

10.47262/sl/9.2.132021003 ◽

2021 ◽

Vol 9 (2) ◽

pp. 24-30

Keyword(s):

Metal Ions ◽

Sugarcane Bagasse ◽

Specific Activity ◽

Gel Filtration ◽

Value Added ◽

Cost Effective ◽

Fermentation Medium ◽

Exchange Chromatography ◽

Sds Page ◽

The Cost

Streptokinase is a fibrinolytic enzyme and a product of β-hemolytic Streptococci strains. This enzyme is used as a medication to break down clots in some cases of heart disease. Streptococcus equisimilis, a species of group C Streptococci, is widely used for the production of streptokinase by fermentation technology. In this study, the sugarcane bagasse fermentation medium was optimized for metal ions (KH2PO4, MgSO4.7H2O, CaCO3 and NaHCO3) at various levels to attain the maximal production of streptokinase. Sugarcane bagasse was used due to its profuse availability and as an ideal substrate for microbial processes for the manufacturing of value-added products. The results showed that maximal streptokinase production was found at 0.04% KH2PO4, 0.04% MgSO4.7H2O, 0.15% NaHCO3 and 0.04% CaCO3. Finally, the optimized medium resulted in 84.75 U/mg specific activity and 74.5% recovery. The purification process was carried out simultaneously using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Finally, a purified sample of streptokinase was run on SDS-PAGE and resolute 47 kDa molecular weight. The use of β-hemolytic Streptococci to obtain streptokinase is not free from health risks and is related to anaphylaxis. This study provides a way forward for the cost-effective ways to obtain streptokinase for the treatment of thrombosis.

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PURIFICATION AND PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

IIUM Engineering Journal ◽

10.31436/iiumej.v18i2.654 ◽

2017 ◽

Vol 18 (2) ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Dzun Noraini Jimat ◽

Intan Baizura Firda Mohamed ◽

Azlin Suhaida Azmi ◽

Parveen Jamal

Keyword(s):

Specific Activity ◽

Hot Spring ◽

Gel Filtration ◽

Maximum Activity ◽

Exchange Chromatography ◽

Bacillus Sp ◽

Sds Page ◽

Fast Flow ◽

Microbial Strain

A newly bacterial producing L-asparaginase was successful isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60Â°C.

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Purification and Characterization of Endoglucanase from local isolate of Aspergillus flavus

Baghdad Science Journal ◽

10.21123/bsj.10.3.844-853 ◽

2013 ◽

Vol 10 (3) ◽

pp. 844-853

Author(s):

Baghdad Science Journal

Keyword(s):

Aspergillus Flavus ◽

Specific Activity ◽

Gel Filtration ◽

Temperature Stability ◽

Exchange Chromatography ◽

Purification And Characterization ◽

Original Activity ◽

A Value ◽

Optimum Ph

Endoglucanase produced from Aspergillus flavus was purified by several steps including precipitation with 25 % ammonium sulphate followed by Ion –exchange chromatography, the obtained specific activity was 377.35 U/ mg protein, with a yield of 51.32 % .This step was followed by gel filtration chromatography (Sepharose -6B), when a value of specific activity was 400 U/ mg protein, with a yield of 48 %. Certain properties of this purified enzyme were investigated, the optimum pH of activity was 7 and the pH of its stability was 4.5, while the temperature stability was 40 °C for 60 min. The enzyme retained 100% of its original activity after incubation at 40 °C for 60 min; the optimum temperature for enzyme activity was 40 °C.

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Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants (Camellia sinensis (L.) O. Kuntze)

Acta Biochimica et Biophysica Sinica ◽

10.1111/j.1745-7270.2005.00053.x ◽

2005 ◽

Vol 37 (6) ◽

pp. 363-370 ◽

Cited By ~ 16

Author(s):

Ye-Yun Li ◽

Chang-Jun Jiang ◽

Xiao-Chun Wan ◽

Zheng-Zhu Zhang ◽

Da-Xiang Li

Keyword(s):

Specific Activity ◽

Gel Filtration ◽

Maximum Activity ◽

Exchange Chromatography ◽

Optimum Activity ◽

Development Of Resistance ◽

Sds Page ◽

C 50 ◽

Tea Plants

Abstractβ-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity, with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50 °C and was stable at temperatures lower than 40 °C. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.

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Hyaluronidase in ram semen. Quantitative determination, and isolation of multiple forms

Biochemical Journal ◽

10.1042/bj2520865 ◽

1988 ◽

Vol 252 (3) ◽

pp. 865-874 ◽

Cited By ~ 16

Author(s):

R A Harrison

Keyword(s):

Ionic Strength ◽

Quantitative Determination ◽

Specific Activity ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

High Yield ◽

Poly Vinyl Alcohol ◽

The Media ◽

Low Ionic Strength

A study was made of hyaluronidase in ram semen. The end-group assay conditions used to determine activity quantitatively were chosen to ensure reliability as well as sensitivity [Gacesa, Savitsky, Dodgson & Olavesen (1981) Anal. Biochem. 118, 76-84]; they led to 1 W.H.O. Standard International Hyaluronidase Unit displaying 0.1263 EC munit (1 EC unit of activity releases 1 mumol equivalent of N-acetylglucosamine end groups/min at 37 degrees C). All the activity in the semen was shown to be sperm-derived, and intact spermatozoa were estimated to contain 1.23 EC units per 10(9) cells. In a low-ionic-strength medium, only some 20% of the hyaluronidase was extractable, although up to 80% of the activity could be extracted as the ionic strength was increased; further addition of detergent extracted the remainder. During purification of the enzyme, it was found that inclusion of poly(vinyl alcohol) in the media stabilized the activity; detergent inclusion also improved the yield, especially during early stages. As a consequence both of reliable quantitative determination and of stabilization, a number of forms of hyaluronidase could be isolated in high yield, by using anion-exchange chromatography, cation-exchange chromatography, affinity chromatography and gel filtration. The existence of all these forms was confirmed by electrophoresis and immunoblotting with the use of a monoclonal anti-(ram hyaluronidase) antibody, and their presence in very freshly prepared sperm extracts was demonstrated. The specific activity of the isolated major hyaluronidase form was 15.0 EC units/mg; this was equivalent to 119,000 W.H.O. units/mg, higher than any other previously reported values.

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Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Purification and properties

Biochemical Journal ◽

10.1042/bj2310407 ◽

1985 ◽

Vol 231 (2) ◽

pp. 407-416 ◽

Cited By ~ 11

Author(s):

N Allison ◽

M J O'Donnell ◽

C A Fewson

Keyword(s):

Lactate Dehydrogenase ◽

Ion Exchange ◽

Gel Filtration ◽

Acinetobacter Calcoaceticus ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Purification And Properties ◽

Molecular Masses ◽

Membrane Bound ◽

Lactate Dehydrogenases

Procedures were developed for the optimal solubilization of D-lactate dehydrogenase, D-mandelate dehydrogenase, L-lactate dehydrogenase and L-mandelate dehydrogenase from wall + membrane fractions of Acinetobacter calcoaceticus. D-Lactate dehydrogenase and D-mandelate dehydrogenase were co-eluted on gel filtration, as were L-lactate dehydrogenase and L-mandelate dehydrogenase. All four enzymes could be separated by ion-exchange chromatography. D-Lactate dehydrogenase and D-mandelate dehydrogenase were purified by cholate extraction, (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and chromatofocusing. The properties of D-lactate dehydrogenase and D-mandelate dehydrogenase were similar in several respects: they had relative molecular masses of 62 800 and 59 700 respectively, pI values of 5.8 and 5.5, considerable sensitivity to p-chloromercuribenzoate, little or no inhibition by chelating agents, and similar responses to pH. Both enzymes appeared to contain non-covalently bound FAD as cofactor.

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