The use of Sephadex for the electrophoretic resolution and partial purification of β-hydroxybutyric and isocitric dehydrogenases of Azotobacter vinelandii

1969 ◽  
Vol 15 (4) ◽  
pp. 321-325 ◽  
Author(s):  
Peter Jurtshuk ◽  
C. R. Barrera ◽  
S. Manning
Keyword(s):  
Ammonium Sulfate ◽  
Azotobacter Vinelandii ◽  
Partial Purification ◽  
Marker Enzymes ◽  
Electrophoretic Method ◽  
Enzyme Preparations ◽  
Highly Active ◽  
Combined Use ◽  
Soluble Enzymes ◽  
Electrophoretic Resolution

A relatively quick and convenient electrophoretic method, capable of separating and partially purifying soluble enzymes from cell-free extracts, is described. The method presented has the feature of analytical applicability and is ideally used with preparatory amounts of material. This zonal technique was developed with the Svensson-Porath preparatory electrophoresis column by using Sephadex G-25 as the anticonvectant. The D(−)β-hydroxybutyric and isocitric dehydrogenases of Azotobacter vinelandii strain O were used as the marker enzymes, and both were resolved as sharply defined peaks. The combined use of ammonium sulfate fractionation and electrophoresis yielded highly active and partially purified enzyme preparations of the β-hydroxybutyric and isocitric dehydrogenases.

Extraction and Partial Purification of Protease from Bilimbi (Averrhoa bilimbi L.)

2013 ◽  
Vol 10 (2) ◽  
pp. 29
Author(s):  
Normah Ismail ◽  
Nur' Ain Mohamad Kharoe
Keyword(s):  
Molecular Weight ◽  
Protein Content ◽  
Ammonium Sulfate ◽  
Molecular Weight Distribution ◽  
Weight Distribution ◽  
Protease Activity ◽  
Protein Concentration ◽  
Temperature Stability ◽  
Partial Purification ◽  
Ammonium Sulfate Precipitation

Unripe and ripe bilimbi (Averrhoa bilimbi L.) were ground and the extracted juices were partially purified by ammonium sulfate precipitation at the concentrations of 40 and 60% (w/v). The collected proteases were analysed for pH, temperature stability, storage stability, molecular weight distribution, protein concentration and protein content. Protein content of bilimbi fruit was 0.89 g. Protease activity of both the unripe and ripe fruit were optimum at pH 4 and 40°C when the juice were purified at 40 and 60% ammonium sulfate precipitation. A decreased in protease activity was observed during the seven days of storage at 4°C. Molecular weight distribution indicated that the proteases protein bands fall between IO to 220 kDa. Protein bands were observed at 25, 50 and 160 kDa in both the unripe and ripe bilimbi proteases purified with 40% ammonium sulfate, however, the bands were more intense in those from unripe bilimbi. No protein bands were seen in proteases purified with 60% ammonium sulfate. Protein concentration was higher for proteases extracted with 40% ammonium sulfate at both ripening stages. Thus, purification using 40% ammonium sulfate precipitation could be a successful method to partially purify proteases from bilimbi especially from the unripe stage. 

Pengaruh Penambahan Molases Pada Kulit Pisang Kapendis Untuk Media Produksi Xilanase B.Stearothermophillus DSM 22

2019 ◽  
Vol 15 (3) ◽  
Author(s):  
Trismillah
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Banana Peel ◽  
Partial Purification ◽  
Temperature Conditions ◽  
Cavendish Banana ◽  
Working Volume ◽  
Initial Ph ◽  
Xylanase Enzyme

Cavendish banana peel can be used as a substitute for the expensive xylan, while molasses than as a source of carbon as well as nitrogen, minerals and nutrients needed for the growth of microbes that can produce the enzyme. Xylanase produced from Bacillus stearothermopillus DSM 22, using media cavendish banana peels with the addition of molasses 1%, 2%, and 3%. Fermentation is done in a shaker incubator at 550C temperature conditions, initial pH 8, and 250 rpm agitation. The result showed the highest enzyme activity of 4,14 ± 0,16 U/mL min., on the addition 2% molasses after 24 hours. Further fermentation carried out in the fermenter working volume of 3.5 liters, with the condition of temperature 550C, pH 8, aeration 1 vvm, agitation 250 rpm, the highest spesific enzyme of activity of 51,62 ± 0,16 U/mg after 24 hours. Partial purification of xylanase enzyme fermentation is done with the results of microfiltration, ultrafiltration, ammonium sulfate (0-80%) and dialysis. There is an increase in the purity of the enzyme at each stage of purification, the highest purity on dialysis 3.23 times of crude enzymes.Kulit buah pisang kapendis dapat digunakan sebagai pengganti xilan yang harganya mahal, sementara molases selain sebagai sumber karbon serta nitrogen, mineral dan nutrisi dibutuhkan untuk pertumbuhan mikroba yang dapat menghasilkan enzim. Xilanase yang dihasilkan dari Bacillus stearothermopillus DSM 22, menggunakan media kulit pisang kapendis dengan penambahan molase 1%, 2%, dan 3%. Fermentasi dilakukan dalam shaker inkubator pada temperatur 550C, pH awal 8, dan agitasi 250 rpm. Hasilnya menunjukkan aktivitas enzim tertinggi 4,14 ± 0,16 U/mL min., pada penambahan 2% molases setelah 24 jam. Selanjutnya fermentasi dilakukan di dalam fermentor, volume kerja dari 3,5 liter, dengan kondisi temperatur 550C, pH 8, aeration 1 vvm, agitasi 250 rpm, aktivitas spesifik tertinggi 51,62 ± 0,16 U/mg setelah 24 jam. Pemurnian parsial fermentasi enzim xilanase dilakukan dengan hasil mikrofiltrasi, ultrafiltrasi, amonium sulfat (0-80%) dan dialisis. Ada peningkatan kemurnian enzim pada setiap tahap pemurnian, kemurnian tertinggi pada dialisis 3,23 kali dari enzim kasar.Keywords: Xylanase, B. stearothermophillus DSM 22, Cavendish banana peel, molasses, enzyme activity

Highly Active Modified Variants of Recombinant Phospholipase А2 from Streptomyces violaceoruber for Effective Biosynthesis in Yeasts

2019 ◽  
pp. 30-41 ◽  
Author(s):  
E.P. Sannikova ◽  
A.V. Malysheva ◽  
F.A. Klebanov ◽  
D.G. Kozlov
Keyword(s):  
New Technologies ◽  
Specific Activity ◽  
Point Mutations ◽  
Egg Yolk ◽  
Enzyme Preparations ◽  
Highly Active ◽  
High Calcium ◽  
Pla2 Enzyme ◽  
Streptomyces Violaceoruber ◽  
The Cost

The capacity of yeast to produce the highly active variants of PLA2 has been confirmed. The high-active variants were based on the original enzyme from the strain А-2688 of Streptomyces violaceoruber. To reduce the enzyme toxicity and to increase its expression, various approaches were tested including point mutations, construction of artificial N- and/or C-end pro-regions, hybridization with other proteins and engineering or inactivation of glycosylation sites. As a main result, the modified PLA2 enzymes were obtained which have the same secretion level as their low-active predecessors, but specific activity of which was at least tenfold higher. As the main feature, the selected mutants were characterized by a lower affinity for Ca2+ that probably accounts for their low toxicity (and high expression capacity) at the stage of biosynthesis and their ability to activate under special conditions, e.g. during the egg yolk fermentation. The data obtained can provide a basis for the cost reduction of highly active PLA2 enzyme preparations in industries where the application of high calcium concentrations is allowed. recombinant phospholipase А2, Streptomyces violaceoruber, yeasts, secretion, producer strain The work was initiated by the Innovation Center Biriuch - New Technologies, Ltd., and was supported within the framework of the State Assignment no. 595-00004-18 PR.

scholarly journals Characterization of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatases

1986 ◽  
Vol 233 (3) ◽  
pp. 839-844 ◽  
Author(s):  
P P J Dunn ◽  
A R Slabas ◽  
A L Moore
Keyword(s):  
Adenosine Triphosphatase ◽  
Kinetic Properties ◽  
Ph Profile ◽  
Enzyme Preparations ◽  
Adenosine Triphosphatases ◽  
Membrane Bound ◽  
Cold Stability ◽  
Arum Maculatum ◽  
Guanosine Triphosphatase ◽  
Soluble Enzymes

The catalytic properties of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatase have been analysed. The pH profile, effect of inhibitors, cold-stability and substrate specificity are characteristic of mitochondrial adenosine triphosphatases, although a high guanosine triphosphatase activity does appear to be restricted to plant mitochondrial adenosine triphosphatases. The kinetic properties of nucleoside 5′-triphosphate hydrolysis by membrane-bound and soluble enzymes have been studied by means of double-reciprocal plots. These plots were linear in the absence of an activating anion, which may indicate that the catalytic and/or regulatory mechanism of Arum maculatum adenosine triphosphatase is different from that of other enzyme preparations. It is suggested that the differences in subunit composition of plant and mammalian adenosine triphosphatases reported previously [Dunn, Slabas & Moore (1985) Biochem. J. 225, 821-824] are structurally, rather than functionally, significant.

scholarly journals Combined use of antibiotics and enzyme preparations for sinusitis. Basic study.

1986 ◽  
Vol 79 (11) ◽  
pp. 1745-1749
Author(s):  
TAKUO MAEYAMA
Keyword(s):  
Basic Study ◽  
Enzyme Preparations ◽  
Combined Use

scholarly journals ASPERMATOGENESIS, ANAPHYLAXIS, AND CUTANEOUS SENSITIZATION INDUCED IN THE GUINEA PIG BY HOMOLOGOUS TESTICULAR EXTRACT

1955 ◽  
Vol 101 (6) ◽  
pp. 591-604 ◽  
Author(s):  
Jules Freund ◽  
George E. Thompson ◽  
Murray M. Lipton
Keyword(s):  
Acetic Acid ◽  
Guinea Pig ◽  
Ammonium Sulfate ◽  
Guinea Pigs ◽  
Anaphylactic Shock ◽  
Proteolytic Enzymes ◽  
Trichloracetic Acid ◽  
Oil Emulsion ◽  
Highly Active ◽  
Water In Oil Emulsion

Guinea pig testicles were extracted with acetic acid; the extract was purified by removing material in consecutive precipitations with 30 per cent saturated ammonium-sulfate, trichloracetic acid, and chloroform. The solution so purified, when administered with complete adjuvants, was highly active in inducing impairment of spermatogenesis in guinea pigs. The activity resisted autoclaving at 15 pounds' pressure for 20 minutes, proteolytic enzymes, and formamide. Anaphylactic shock and cutaneous reaction to the purified homologous extract occurred in guinea pigs sensitized by the extract combined with adjuvants. For the production of aspermatogenesis it was essential to incorporate killed mycobacteria into the water-in-oil emulsion containing the antigen; but anaphylactic sensitization did not require the presence of mycobacteria.

Highly active enzyme preparations immobilized via matrix-conjugated anti-Fc antibodies

1991 ◽  
Vol 539 (2) ◽  
pp. 335-341 ◽  
Author(s):  
Beka Solomon ◽  
Eran Hadas ◽  
Rela Koppel ◽  
Fidi Schwartz ◽  
Gideon Fleminger
Keyword(s):  
Active Enzyme ◽  
Enzyme Preparations ◽  
Highly Active

AN ANTIVIRAL SUBSTANCE FROM PENICILLIUM CYANEO-FULVUM BIOURGE: I. PRODUCTION AND PARTIAL PURIFICATION

1965 ◽  
Vol 11 (6) ◽  
pp. 913-919 ◽  
Author(s):  
Patricia M. Cooke ◽  
J. W. Stevenson
Keyword(s):  
Ammonium Sulfate ◽  
Ethyl Alcohol ◽  
Antiviral Agent ◽  
Partial Purification ◽  
Fractional Precipitation ◽  
Antiviral Substance

Penicillium cyaneo-fulvum Biourge, which produces a toxin-neutralizing substance, has been shown to produce a separate and distinct antiviral agent. The two products may be separated in a semipurified state by fractional precipitation with ammonium sulfate and ethyl alcohol.

scholarly journals Partial purification of thrombopoietin from the plasma of thrombocytopenic rabbits

Blood ◽  
1979 ◽  
Vol 54 (2) ◽  
pp. 377-388 ◽  
Author(s):  
BL Evatt ◽  
J Levin ◽  
KM Algazy
Keyword(s):  
Ammonium Sulfate ◽  
Phosphate Buffer ◽  
Carboxymethyl Cellulose ◽  
Room Temperature ◽  
Deae Cellulose ◽  
Partial Purification ◽  
Elution Volume ◽  
Ammonium Sulfate Saturation ◽  
Citrate Phosphate ◽  
Citrate Phosphate Buffer

Abstract Partially purified thrombopoiesis-stimulating activity was prepared from the plasma of thrombocytopenic rabbits using ammonium sulfate precipitation and DEAE cellulose, Sephadex, and carboxymethyl cellulose chromatography. The protein fraction precipitated by an ammonium sulfate saturation of 60%-80%, previously shown to contain thrombopoiesis-stimulating activity, was used as starting material. Column chromatography was carried out at room temperature at pH 5.6. Under these conditions, thrombopoiesis-stimulating activity (thrombopoietin) was retained by DEAE cellulose (0/03 M citrate- phosphate buffer) and carboxymethyl cellulose (0/003 M citrate- phosphate buffer), and eluted with 0.4 M NaCl. Thrombopoietin was retarded by Sephadex G-100; the ratio of the elution volume to the void volume was 1.32:1. Immunoelectrophoretic analysis of partially purified thrombopoietin indicated that following removal of most of the albumin by DEAE chromatography, only proteins with the mobilities of beta- globulins and albumin and traces of other anodally migrating proteins were detectable in the fractions that contained thrombopoiesis- stimulating activity. Thrombopoietin was not dialyzable and was stable from at least pH 5.6 to 7.5. It was approximately 1000-fold purified following sequential chromatography with DEAE and carboxymethyl cellulose. Although the three fractions described reproducibly stimulated thrombopoiesis, as measured by increased levels of selenomethionine-75Se (75SeM) in the circulating platelets, platelet counts did not increase.

scholarly journals 185 Effects of Prohexadione-Ca on Gibberellin Levels in Young Apple Shoots

HortScience ◽  
2000 ◽  
Vol 35 (3) ◽  
pp. 422D-422 ◽  
Author(s):  
Steve J. Croker ◽  
Peter Hedden ◽  
Wilhelm Rademacher
Keyword(s):  
Shoot Growth ◽  
Dry Weight ◽  
Apical Part ◽  
Enzyme Preparations ◽  
Highly Active ◽  
Intact Plants ◽  
Run Off ◽  
Typical Experiment ◽  
Inactive Metabolite

Prohexadione-Ca (BAS 125 W) is a new growth retardant for the inhibition of excessive vegetative growth in apple and other plant species. From work with enzyme preparations, it is known that prohexadione-Ca mimics 2-oxoglutaric acid, the co-substrate of dioxygenases, which catalyze late steps in gibberellin (GA) biosynthesis. As a result, the formation of growth-active GAs is reduced. In order to have a better understanding of its effects in intact plants, we have analyzed the GA status of treated and untreated apple plantlets. In a typical experiment, the following results were obtained: Plants (cv. Jonagold on M9 at 19 cm of new shoot growth) were sprayed until run-off with an aqueous preparation containing 25 ppm of active ingredient. After 22 days of cultivation under greenhouse conditions, total new shoot growth of the controls and the treated plants was 55 cm and 44 cm, respectively. In the apical part of this material the following GAs (roughly ordered in biosynthetic sequence) were detected at the following levels (control/treated in microgram per kilogram dry weight): GA19 (31/62), GA29 (24/36), GA20 (11/20), GA1 (4/3), and GA8 (8/3). These results clearly demonstrate that prohexadione-Ca blocks primarily the hydroxylation of GA20 into GA1. This leads to reduced levels of the highly active GA1 and of GA8, its inactive metabolite, whereas GA20 and the other inactive precursors accumulate. The data support older observations obtained in vitro, which indicate that GA20 3β-hydroxylase and related dioxygenases are the primary targets of prohexadione-Ca and similar compounds.

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