Preparation of a highly active form of nisin from Streptococcus lactis

1971 ◽  
Vol 17 (1) ◽  
pp. 61-67 ◽  
Author(s):  
F. J. Bailey ◽  
A. Hurst
Keyword(s):  
Active Form ◽  
Gradient Elution ◽  
Antibiotic Activity ◽  
Complex Medium ◽  
Main Peak ◽  
Acetone Precipitation ◽  
Highly Active ◽  
Streptococcus Lactis ◽  
Constant Ph ◽  
Ph Gradient Elution

Cells of Streptococcus lactis (354/07) synthesized and retained nisin when grown in a complex medium with 2.5% glucose at a constant pH of 6.7. Nisin was extracted from cells by a previously used method with hot 0.05 N HCl but milder methods of extraction from whole and broken cells using a variety of solvents were also tested. In the preferred method broken cells were extracted with 0.05 N HCl at 2 °C. The Cl− ions of the extract were exchanged for acetate on columns of the resin Amberlite CG 4B and the eluate was concentrated by acetone precipitation at −19 °C. The nisin was finally purified by pH gradient elution from CM cellulose columns. Three peaks with antibiotic activity were found, two of the peaks were minor and represented less than 5% of the nisin. The main peak gave a single band on electrophoresis. Electrophoresis of the material from the CM cellulose peaks revealed about 44 bands of basic proteins. Nisin made by the hot or cold HCl extraction behaved similarly in electrophoresis and CM cellulose chromatography but the antibiotic activity of the material isolated from the cold extract was nine times greater than that of the material isolated from the hot extract.

scholarly journals Improved enrichment strategies for phosphorylated peptides on titanium dioxide using methyl esterification and pH gradient elution

2008 ◽  
Vol 377 (2) ◽  
pp. 234-242 ◽  
Author(s):  
Eric S. Simon ◽  
Matthew Young ◽  
Antonia Chan ◽  
Zhao-Qin Bao ◽  
Philip C. Andrews
Keyword(s):  
Titanium Dioxide ◽  
Gradient Elution ◽  
Ph Gradient ◽  
Methyl Esterification ◽  
Phosphorylated Peptides ◽  
Ph Gradient Elution

THE INFLUENCE OF L-LYSINE ON FACTOR VIII ELUTED FROM MATRIX BOUND DEXTRAN SULPHATE

1987 ◽  
Author(s):  
P Harrison ◽  
R H Saundry ◽  
G F Savidge
Keyword(s):  
Factor Viii ◽  
Large Scale ◽  
Gradient Elution ◽  
Complete Separation ◽  
Enzymic Activity ◽  
Major Protein ◽  
Dextran Sulphate ◽  
Separation Principle ◽  
Matrix Bound ◽  
Ph Gradient Elution

Factor VIII/vWF displays high affinity for matrix bound sulphate groups. Linear salt and pH gradient elution from Dextran Sulphate Sepharose at 4°C resulted in complete separation of the complex from major protein contaminants with typical yields of 70-90% vWF:Ag and vWF (RCoF). Elution in physiological calcium concentrations gave VIII and VIII:Ag yields of 35% and 65% respectively. Addition of L-Lysine (1.0M) to all buffers inhibited VIII/vWF binding, although lysine gradients (0-1.0M) gave comparable binding and elution profiles as C-1.0M NaCl, but with impaired resolution between protein moieties. However, in the presence of L-Lysine, yields of VIII and VIII:Ag were significantly improved to 80% and 100% respectively when assay standardizations with appropriate lysine concentrations were performed. Furthermore, the binding of fibrinogen could be inhibited by 0.15M L-Lysine, 0.075M NaCl in the equilibrating buffer.The presence of enzymic activity, as assessed by S-2222 and S-2238 in the eluting fractions could be abolished by the application of recryoprecipitated material pre-adsorbed with Al(OK)3 and celite. Incorporation of lysine to the buffers with the associated increase in yields further supports the potential viability of the separation principle for large scale purification of Factor VIII.

scholarly journals Latent and activeabPPO4 mushroom tyrosinase cocrystallized with hexatungstotellurate(VI) in a single crystal

2014 ◽  
Vol 70 (9) ◽  
pp. 2301-2315 ◽  
Author(s):  
Stephan Gerhard Mauracher ◽  
Christian Molitor ◽  
Rami Al-Oweini ◽  
Ulrich Kortz ◽  
Annette Rompel
Keyword(s):  
Crystal Structure ◽  
Single Crystal ◽  
Agaricus Bisporus ◽  
Crystal Packing ◽  
Active Form ◽  
Activation Mechanism ◽  
Mushroom Tyrosinase ◽  
Polyphenol Oxidases ◽  
Highly Active ◽  
Eukaryotic Organisms

Tyrosinases, bifunctional metalloenzymes, catalyze the oxidation of monophenols ando-diphenols too-quinones, the precursor compounds of the brown-coloured pigment melanin. In eukaryotic organisms, tyrosinases are expressed as latent zymogens that have to be proteolytically cleaved in order to form highly active enzymes. This activation mechanism, known as the tyrosinase maturation process, has scientific and industrial significance with respect to biochemical and technical applications of the enzyme. Here, not only the first crystal structure of the mushroom tyrosinaseabPPO4 is presented in its active form (Ser2–Ser383) and in its 21 kDa heavier latent form (Ser2–Thr545), but furthermore the simultaneous presence of both forms within one single-crystal structure is shown. This allows for a simple approach to investigate the transition between these two forms. IsoformabPPO4 was isolated and extensively purified from the natural source (Agaricus bisporus), which contains a total of six polyphenol oxidases (PPOs). The enzyme formed crystals (diffracting to a resolution of 2.76 Å) owing to the employment of the 6-tungstotellurate(VI) salt (Na6[TeW6O24]·22H2O) as a cocrystallization agent. Two of these disc-shaped Anderson-type polyoxoanions [TeW6O24]6−separate two asymmetric units comprising one crystallographic heterodimer ofabPPO4, thus resulting in very interesting crystal packing.

scholarly journals A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi

1997 ◽  
Vol 324 (2) ◽  
pp. 619-626 ◽  
Author(s):  
Javier PEÑA-DÍAZ ◽  
Andrea MONTALVETTI ◽  
Ana CAMACHO ◽  
Claribel GALLEGO ◽  
Luis M. RUIZ-PEREZ ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Reductase Activity ◽  
Active Form ◽  
Single Copy ◽  
Enzymic Activity ◽  
Highly Active ◽  
Frame Sequence ◽  
Isolation And Characterization ◽  
Cell Extracts ◽  
Coa Reductase

We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts. Further examination of the 5′ flanking region by cDNA analysis and establishment of the splice acceptor sites clearly indicated that the corresponding mRNA apparently lacks sequences encoding a membrane N-terminal domain. The reductase gene is a single copy and is located on a chromosome of 1.36 Mb as determined by contour-clamped homogeneous electric field electrophoresis. The overall cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts. Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly sensitive to proteolytic inactivation. Furthermore the enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. Thus Trypanosoma cruziHMG-CoA reductase is unique in the sense that it totally lacks the membrane-spanning sequences present in all eukaryotic HMG-CoA reductases so far characterized.

Monoamine Oxidase Activity in Brain Microvessels Determined Using Natural and Artificial Substrates: Relevance to the Blood—Brain Barrier

1983 ◽  
Vol 3 (4) ◽  
pp. 521-528 ◽  
Author(s):  
F. Lasbennes ◽  
R. Sercombe ◽  
J. Seylaz
Keyword(s):  
Monoamine Oxidase ◽  
Blood Brain Barrier ◽  
Active Form ◽  
Artificial Substrates ◽  
Brain Barrier ◽  
Facilitated Diffusion ◽  
Cerebral Microvessels ◽  
Highly Active ◽  
Blood Brain ◽  
Brain Uptake Index

The possible contribution of cerebrovascular monoamine oxidase (MAO) to the blood–brain barrier to catecholamines was studied in isolated porcine and rat microvessels by determining its activity with various substrates. Michaelis-Menten kinetic constants, Km and Vmax, were determined using noradrenaline (NA) as substrate in a Tris medium. Km values were 0.25 ± 0.05 m M in control and 0.16 ± 0.09 m M in ultrasonically disintegrated (USD) preparations (difference not significant); Vmax in USD preparations (1.83 ± 0.20 n.atoms O2 min−1 mg protein−1) was slightly higher (p < 0.05) than in control preparations (1.35 ± 0.11 n.atoms O2 min−1 mg protein−1), suggesting a certain restriction by the plasma membrane of substrate access to the enzyme. This phenomenon was confirmed in a more physiological, ionic medium; the activity was then approximately doubled for 1 m M NA, whereas that for 1 m M β-phenylethylamine (β-PEA), a lipid-soluble substrate, tended to decrease with USD treatment. These results show that this highly active form of MAO is unlikely to be saturated by physiological concentrations of catecholamine. It can be estimated that, for a plasma concentration of NA of 1 μM, a facilitated diffusion accelerating the entry of the catecholamine into the cells by at least 15-fold would be necessary in order to exceed the catabolic capacity of MAO. It is concluded that circulating catecholamines are not likely to cross the endothelial barrier of cerebral microvessels intact, and that the small quantities of radioactivity detected in the parenchyma in measurements of the brain uptake index essentially represent metabolites due to MAO activity.

scholarly journals Antiviral Guanosine Analogs as Substrates for Deoxyguanosine Kinase: Implications for Chemotherapy

2001 ◽  
Vol 45 (3) ◽  
pp. 739-742 ◽  
Author(s):  
Anita Herrström Sjöberg ◽  
Liya Wang ◽  
Staffan Eriksson
Keyword(s):  
Human Immunodeficiency Virus ◽  
Enzyme Assay ◽  
Active Form ◽  
Nucleoside Analogs ◽  
Highly Active ◽  
B Virus ◽  
Immunodeficiency Virus ◽  
Deoxyguanosine Kinase ◽  
Low Efficiency ◽  
Different Tissues

ABSTRACT A highly active form of human recombinant deoxyguanosine kinase (dGK) phosphorylated purine nucleoside analogs active against cytomegalovirus, hepatitis B virus, and human immunodeficiency virus, such as penciclovir, 2′,3′-dideoxyguanosine and 3′-fluoro-2′,3′-dideoxyguanosine. The antiherpesvirus drug ganciclovir, which is also used in gene therapy, was a substrate for dGK, but with low efficiency. ATP and UTP were both good phosphate donors, with apparent Km values of 6 and 4 μM andV max values of 34 and 90 nmol of dGMP/mg of dGK/min, respectively. With a mixture of 5 mM ATP and 0.05 mM UTP, which represent physiologically relevant concentrations, the activities of dGK with ganciclovir and penciclovir was 1% and approximately 10%, respectively, of that with dGuo. The levels of dGK in different tissues were determined with a selective enzyme assay and the total activities per gram of tissues were similar in liver, brain, heart, and thymus extracts. The fact that the cellular dGK enzyme can phosphorylate antiviral guanosine analogs may help to explain the efficacies and side effects of several forms of chemotherapy.

Growth of Streptococcus cremoris and Streptococcus lactis in a chemostat. Production of cells and survival of bacteria during frozen storage

1973 ◽  
Vol 19 (10) ◽  
pp. 1285-1295 ◽  
Author(s):  
I. J. McDonald ◽  
B. Reiter ◽  
P. L. Rogers
Keyword(s):  
Yeast Extract ◽  
Frozen Storage ◽  
Skim Milk ◽  
Streptococcus Lactis ◽  
Streptococcus Cremoris ◽  
Constant Ph ◽  
Limiting Nutrient

Streptococcus cremoris HP and Streptococcus lactis 829 were grown in chemostats in tryptone yeast extract broth and in supplemented 2% skim milk medium. In both media, lactose was the limiting nutrient. Cultures were grown at various dilution rates in media poised at constant pH and temperature and also at constant dilution rates in media controlled at different pH levels and temperatures. The effects of the various conditions of growth on production of bacteria, viable counts, and acid-producing activities of cells and on the ability of bacteria to survive subsequent frozen storage were determined. None of the conditions of growth tested had very pronounced effects on the ability of cells to survive or on the inability of cells to retain acid-producing activity after being frozen at −70 °C and stored at −40 °C.

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