Isolation and purification of the D(–)β-hydroxybutyric dehydrogenase of Azotobacter vinelandii

1968 ◽  
Vol 14 (7) ◽  
pp. 775-783 ◽  
Author(s):  
Peter Jurtshuk ◽  
S. Manning ◽  
C. R. Barrera
Keyword(s):  
Ammonium Sulfate ◽  
Metal Ion ◽  
Azotobacter Vinelandii ◽  
Specific Activity ◽  
Alkaline Ph ◽  
Membranous Structure ◽  
Isolation And Purification ◽  
Resting Cell ◽  
Direct Use ◽  
Marked Stability

It has been possible to isolate and purify the D(–)β-hydroxybutyric dehydrogenase from cell-free extracts of Azotobacter vinelandii. Standardized resting cell suspensions were disrupted by sonic oscillation, and purification of the enzyme was achieved by use of protamine sulfate, ammonium sulfate, and hydroxyapatite or by direct use of a Svensson-Porath preparatory column electrophoretic unit. The D(–)β-hydroxybutyric dehydrogenase was found to be a classical, soluble, NAD+-dependent dehydrogenase and neither bound nor associated with any intracellular membranous structure. The highest specific activity achieved was 17 μmoles NAD+ reduced per minute per milligram protein at 37°. Two unusual features were noted with this dehydrogenase: (a) loss of activity occurred when the enzyme was dialyzed in the absence of metal ion; and (b) marked stability was observed when the enzyme was exposed to alkaline pH even at 40 °C for hours.

scholarly journals Pemurnian Parsial dan Karakterisasi Urease dari Biji Kacang Panjang (Vigna unguiculata subsp sesquipedalis L.)

2018 ◽  
Vol 14 (1) ◽  
pp. 72
Author(s):  
Zusfahair Zusfahair ◽  
Dian Riana Ningsih ◽  
Amin Fatoni ◽  
Darul Santri Pertiwi
Keyword(s):  
Vigna Unguiculata ◽  
Crude Extract ◽  
Metal Ion ◽  
Urease Activity ◽  
Specific Activity ◽  
Partial Purification ◽  
Isolation And Purification ◽  
Molecular Weights ◽  
Sds Page ◽  
Analytical Result

<p>Urease merupakam enzim yang digunakan dalam hidrolisis urea menjadi amoniak dan asam bikarbonat dan telah banyak digunakan dalam proses industri. Tujuan penelitian adalah isolasi dan pemurnian urease dari kacang panjang serta karakterisasinya. Penelitian dimulai dengan melakukan perkecambahan biji kacang panjang selama 8 hari. Kecambah biji kacang panjang selanjutnya diekstraksi dengan menggunakan buffer fosfat pH 7 dan dipisahkan menggunakan sentrifugasi sehingga diperoleh ekstrak kasar urease. Ekstrak kasar urease selanjutnya difraksinasi menggunakan aseton pada tingkat konsentrasi 20, 40, 60 dan 80%. Fraksi yang mempunyai aktivitas spesifik paling tinggi selanjutnya dianalisis menggunakan metode SDS-PAGE untuk menentukan berat molekulnya dan dikarakterisasi lanjut meliputi: pengaruh suhu, pH, konsentrasi substrat dan penambahan ion logam terhadap aktivitas urease. Aktivitas urease ditentukan dengan metode Nessler. Hasil penelitian menunjukkan aktivitas spesifik urease dari kacang panjang paling tinggi ditemukan pada fraksi aseton (FA) 20. Hasil analisis berat molekul dengan metode SDS-PAGE diperoleh beberapa pita protein yang diduga berukuran sekitar 25 KDa dan 17 KDa. Kondisi optimum dari aktivitas urease diperoleh pada suhu 30 ºC, pH 7 dan konsentrasi urea 16,6 mM dengan nilai aktivitas 407,62 U/mL. EDTA dan ion logam dalam CaCl<sub>2</sub>, NaCl, NiCl<sub>2</sub> dan CuCl<sub>2 </sub>pada variasi konsentrasi 10<sup>-3</sup>, 10<sup>-4 </sup> dan 10<sup>-5 </sup>M merupakan inhibitor urease FA 20 dari kacang panjang.</p><p><strong><span lang="EN-US">Partial Purification and Characterization of Urease from Asparagus Bean (<em>Vigna unguiculata subsp sesquipedalis </em>L<em>.</em>). </span></strong><span lang="EN-US">Urease is an enzyme used in urea hydrolysis to ammonia and bicarbonate acid and has been widely used in industrial processes. The study focused on isolation and purification of urease from asparagus beans and its characterization. The study was started with germination of asparagus beans for 8 days. Germinated asparagus beans were further extracted using phosphate buffer pH 7 and separated by centrifugation to obtain a crude extract of urease. The crude extract of urease was further fractionated using acetone at concentrations of 20, 40, 60 and 80%. The fraction with highest specific activity was then </span><span>analyzed using SDS-PAGE method to determine its molecule weight and characterized further including the influence of </span><span lang="EN-US">temperature, pH, substrate concentration, and metal ion addition to urease activity. The urease activity was determined by the Nessler̕ s method. The results showed that the specific activity of urease from asparagus beans was found with highest activity in fraction of acetone (FA) 20. Analytical result using SDS-PAGE method was obtained some protein bands having molecular weights about 25 KD and 17 KDa. The optimum conditions of urease activity was obtained at 30 °C, pH 7, incubation time 20 min and urea concentration 16.6 mM with activity value 407.62 U/mL. EDTA and </span><span>metal ions contained in </span><span lang="EN-US">CaCl<sub>2</sub>, NaCl, NiCl<sub>2</sub> and CuCl<sub>2</sub> at concentrations of 10<sup>-3</sup>, 10<sup>-4</sup> and 10<sup>-5</sup> M were FA 20 urease inhibitors.</span></p>

THE ISOLATION AND PURIFICATION OF A TRYPSIN INHIBITOR FROM WHOLEWHEAT FLOUR

1964 ◽  
Vol 42 (12) ◽  
pp. 1825-1832 ◽  
Author(s):  
G. Shyamala ◽  
R. L. Lyman
Keyword(s):  
Ammonium Sulfate ◽  
Trypsin Inhibitor ◽  
Carboxymethyl Cellulose ◽  
Moving Boundary ◽  
Specific Activity ◽  
Trypsin Inhibitors ◽  
Ammonium Sulfate Precipitation ◽  
Commercial Sample ◽  
Isolation And Purification ◽  
Single Protein

An impure concentrate of a trypsin inhibitor was prepared from a commercial sample of wholewheat flour by ammonium sulfate precipitation. The impure inhibitor was heat-labile and did not inhibit pepsin or α-chymotrypsin activity. When the crude inhibitor was chromatographed on carboxymethyl cellulose, a single protein peak that was about 20 times more active than the original impure inhibitor preparation was obtained.Uultracentrifugal sedimentation patterns and moving-boundary electrophoresis indicated that the material isolated on the carboxymethyl cellulose was nearly homogeneous. In comparison with other known trypsin inhibitors that have been isolated in nearly pure form, wholewheat trypsin inhibitor appears to have a low specific activity.

scholarly journals SELECTED PROTEOLYTIC ACTIVITY BY EXTRACTS OF DERMESTID LARVAE

2011 ◽  
Vol 19 (19) ◽  
pp. 79-83
Author(s):  
Lavinel G. IONESCU
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Proteolytic Activity ◽  
Specific Activity ◽  
Proteolytic Enzymes ◽  
High Specific Activity ◽  
Water Extract ◽  
Dermestes Maculatus ◽  
Crude Preparation

The larvae of the Beetle Dermestes maculatus De Geer can subsist on a diet consisting largely of protein. Studies have been undertaken to investigate the nature of proteolytic enzymes. A water extract of the larvae yielded a crude preparation that hydrolyzes gelatin, bide powder, hemoglobin substrate, benzoyl-DL-arginine p-nitroamilide, and glutaryl-L-phenylalanine p-nitroanilide. Enzyme activity was found in a non-dialyzable, heat- and acid0labile portion of the extract yielded two fractions with high specific activity towards gelatin. These are precipitated between 40% to 60% saturation of ammonium sulfate and 60% to 80% saturation. The higher specific activity was observed in the 40%-60% fraction. These results suggest that the larvae of these dermestids contain proteolytic enzymes with actions similar to mammalian trypsin and chymotrypsin. The results also suggest that other proteolytic enzymes may be present as well.

scholarly journals A Study on the Photocatalytic Reduction of Some Metal Ions in Aqueous Solution Using UV- Titanium Dioxide System

2019 ◽  
pp. 1-7
Author(s):  
I. O. Ekwere ◽  
M. Horsfall ◽  
J. O. E. Otaigbe
Keyword(s):  
Aqueous Solution ◽  
Titanium Dioxide ◽  
Metal Ions ◽  
Metal Ion ◽  
Uv Light ◽  
Kinetic Studies ◽  
Photocatalytic Reduction ◽  
Alkaline Ph ◽  
Removal Of Metal ◽  
Removal Of Metal Ions

The photocatalytic reduction of Cu (II), Pb (II), Cd (II) and Cr (VI) ions in aqueous solution has been investigated. The photocatalyst utilized was nano titanium dioxide, composed of 80% anatase and 20% rutile; the UV light source was a 15 W UV bulb with a wavelength of 254 nm. The results obtained indicated a reduction efficiency order as follows; Cr6+ > Cu2+ > Pb2+ > Cd2+. It was observed that these results correlate with the respective reduction potentials of the metal ions. The effect of pH on the photocatalytic reduction of the metal ions was also carried out and results obtained indicated that with the exception of Cr (VI) ions, higher percentage removal of metal ions from their aqueous solution was recorded at alkaline pH than at acidic pH. This was attributed to an extensive formation of precipitate by the metal ions at alkaline pH. Kinetic studies revealed that the removal of metal ions from their solutions largely followed the pseudo- first-order kinetics. Therefore, the results of this study will be useful in metal ion removal from industrial waste water using photocatalytic process.

scholarly journals DEVELOPMENT OF RECOMBINANT PEPTIDE TECHNOLOGY WITH ANTIMICROBIAL PROPERTIES OF A BROAD ACTION SPECTRUM

2017 ◽  
pp. 3-14
Author(s):  
Alexander Prosekov ◽  
Alexander Prosekov ◽  
Olga Babich ◽  
Olga Babich ◽  
Irina Milenteva ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Ammonium Sulfate ◽  
Action Spectrum ◽  
Antimicrobial Properties ◽  
Hplc Method ◽  
Salting Out ◽  
Antimicrobial Spectrum ◽  
Recombinant Peptide ◽  
Isolation And Purification ◽  
Protein Nature

Bacteriocins are antibacterial, mainly complex, substances of protein nature. The promising strains producing bacteriocins used in the food industry are lactic acid microorganisms. This study examines the development of a technology for the production of a recombinant peptide with broad-spectrum antimicrobial properties. An important step is the isolation and purification of the recombinant peptide. It has been proved that the highest antimicrobial activity is manifested by a recombinant peptide isolated by a method based on salting out with ammonium sulfate. During the purification of the recombinant bacteriocin preparation, three kinds of columns were used. In the purification process, the volume of bacteriocin produced decreases 3-fold, while the RU/mL increases 3-fold, and RU/mg increases 6-fold. Purification allows the use of a smaller amount of recombinant bacteriocin in technologies with greater efficacy. Based on the results of determining the molecular weight and purity of the recombinant bacteriocin, it was found that the molecular weight of the recombinant bacteriocin having the amino acid sequence: KYYGNGVTCCKHSCSVDXGKASSCIINNGAMAXATGGH GGNHCCGMSRYIQGIPDFLRGYLHGISSANKHKKGRL, is 13 kDa. A technology for the preparation of a broad-action antimicrobial spectrum peptide has been developed. The process of production of antimicrobial peptide includes such stages as: cultivation of the recombinant strain of Escherichia coli BL21DE3; separation of biomass from the nutrient medium; precipitation of bacteriocins by ammonium sulfate; centrifugation; washing the precipitate; centrifugation at 4200 rpm and separation of the preparation; purification of bacteriocins by HPLC method; packing in bags of polymeric and combined materials; storage at a temperature of 18±2°C for 12 months.

THE PREPARATION OF SUCROSE-C14 WITH HIGH SPECIFIC ACTIVITY AND HIGH TRACER YIELD USING DETACHED SUGAR BEET LEAVES

1959 ◽  
Vol 37 (1) ◽  
pp. 193-197 ◽  
Author(s):  
C. D. Nelson ◽  
D. C. Mortimer
Keyword(s):  
Sugar Beet ◽  
Specific Activity ◽  
High Specific Activity ◽  
Good Source ◽  
Isolation And Purification

Radioactive sucrose, in millicurie amounts, was prepared by feeding a detached sugar beet leaf C14O2 in air for 15 to 20 minutes followed by 5 to 20 minutes additional photosynthesis in air. The low endogenous sucrose in the leaf coupled with the improved tracer yield effected by the displacement technique gave sucrose with high specific activity. This sucrose-C14, hydrolyzed by 1% invertase, was a good source for radioactive glucose and fructose. Paper chromatographic procedures were used for the isolation and purification of the sugars.

scholarly journals Investigation of the first step of biotin biosynthesis in Bacillus sphaericus. Purification and characterization of the pimeloyl-CoA synthase, and uptake of pimelate

1992 ◽  
Vol 287 (3) ◽  
pp. 685-690 ◽  
Author(s):  
O Ploux ◽  
P Soularue ◽  
A Marquet ◽  
R Gloeckler ◽  
Y Lemoine
Keyword(s):  
Metal Ion ◽  
Specific Activity ◽  
Bacillus Sphaericus ◽  
Careful Study ◽  
Metal Chelators ◽  
Ph Profile ◽  
E Coli ◽  
Coa Ligase ◽  
Hydrolysis Of

The pimeloyl-CoA synthase from Bacillus sphaericus has been purified to homogeneity from an overproducing strain of Escherichia coli. The purification yielded milligram quantities of the synthase with a specific activity of 1 unit/mg of protein. Analysis of the products showed that this enzyme catalysed the transformation of pimelate into pimeloyl-CoA with concomitant hydrolysis of ATP to AMP. Using a continuous spectrophotometric assay, we have examined the catalytic properties of the pure enzyme. The pH profile under Vmax. conditions showed a maximum around 8.5. Apparent Km values for pimelate, CoASH, ATP. Mg2- and Mg2+ were respectively 145 microM, 33 microM, 170 microM and 2.3 mM. The enzyme was inhibited by Mg2+ above 10 mM. This acid-CoA ligase exhibited a very sharp substrate specificity, e.g. neither GTP nor pimelate analogues (di- or mono-carboxylic acids) were processed. The bivalent metal ion requirement was also investigated: Mn2+ (73%) and Co2+ (32%) but not Ca2+ could replace Mg2+. The enzyme was inhibited by metal chelators such as 1,10-phenanthroline and EDTA. The synthase was a homodimer with a 28,000-M(r) subunit. N-Terminal sequencing definitely proved that this enzyme was encoded by the bioW gene. A careful study of pimelate uptake by B. sphaericus, E. coli and Pseudomonas dentrificans showed that this metabolite crossed the membrane of these microorganisms by passive diffusion, ruling out the involvement of the bioX gene product as pimelate carrier.

Aerobic purification of N5,N10-methylenetetrahydromethanopterin dehydrogenase, separated from N5,N10-methenyltetrahydromethanopterin cyclohydrolase, from Methanobacterium thermoautotrophicum strain Marburg

1989 ◽  
Vol 35 (4) ◽  
pp. 499-507 ◽  
Author(s):  
Biswarup Mukhopadhyay ◽  
Lacy Daniels
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Dehydrogenase Activity ◽  
Specific Activity ◽  
Methanobacterium Thermoautotrophicum ◽  
Anaerobic Conditions ◽  
Cell Extracts ◽  
First Time ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

The N5,N10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum strain Marburg has been purified with reasonable yield and much higher specific activity than previously reported. For the first time it has been shown that both N5,N10-methylenetetrahydromethanopterin dehydrogenase and N5,N10-methenyltetrahydromethanopterin cyclohydrolase activities were stable under air and could be purified using aerobic operations. The dehydrogenase activity from Methanobacterium thermoautotrophicum Marburg was stable in phosphate buffer with or without glycerol or ammonium sulfate under both aerobic and anaerobic conditions. However, the presence of either 2-mercaptoethanol or dithiothreitol in the enzyme solution destroyed the enzyme activity during both aerobic and anaerobic incubations. Dehydrogenase was purified 62-fold using Phenyl-Sepharose and DEAE-Sephadex chromatography in succession under air. Both of these chromatographic methods separated dehydrogenase activity from N5,N10-methenyltetrahydromethanopterin cyclohydrolase; DEAE-Sephadex provided the best separation. Phenyl-Sepharose chromatography of the supernatant of cell extracts containing ammonium sulfate at 60% of saturation provided a 4.7-fold purification and 98% recovery of cyclohydrolase; this result established the air stability of N5,N10-methenyltetrahydromethanopterin cyclohydrolase from Methanobacterium thermoautotrophicum Marburg.Key words: methylenetetrahydromethanopterin dehydrogenase, methenyltetrahydromethanopterin cyclohydrolase, Methanobacterium, aerobic purification, oxygen stability.

scholarly journals Purification and Characterization ofα -l-Arabinopyranosidase andα -l-Arabinofuranosidase from Bifidobacterium breve K-110, a Human Intestinal Anaerobic Bacterium Metabolizing Ginsenoside Rb2 and Rc

2003 ◽  
Vol 69 (12) ◽  
pp. 7116-7123 ◽  
Author(s):  
Ho-Young Shin ◽  
Sun-Young Park ◽  
Jong Hwan Sung ◽  
Dong-Hyun Kim
Keyword(s):  
Ammonium Sulfate ◽  
Column Chromatography ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Bifidobacterium Breve ◽  
Ammonium Sulfate Fractionation ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Ginsenoside Rb2

ABSTRACT Two arabinosidases, α-l-arabinopyranosidase (no EC number) and α-l-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizing Bifidobacterium breve K-110, which was isolated from human intestinal microflora. α-l-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 μmol/min/mg.α -l-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 μmol/min/mg. The molecular mass ofα -l-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that ofα -l-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. α-l-Arabinopyranosidase and α-l-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40°C and pH 4.5 and 45°C, respectively. Both purified enzymes were potently inhibited by Cu2+ and p-chlormercuryphenylsulfonic acid.α -l-Arabinopyranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinopyranoside, followed by ginsenoside Rb2. α-l-Arabinofuranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted on p-nitrophenyl-β-galactopyranoside or p-nitrophenyl-β-d-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified α-l-arabinosidases. This is the first reported purification ofα -l-arabinopyranosidase from an anaerobic Bifidobacterium sp.

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