scholarly journals Characterization of a cellobiose dehydrogenase in the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile

1982 ◽  
Vol 203 (1) ◽  
pp. 277-284 ◽  
Author(s):  
M R Coudray ◽  
G Canevascini ◽  
H Meier
Keyword(s):  
Methylene Blue ◽  
Extracellular Enzyme ◽  
Electron Acceptors ◽  
Cellobiose Dehydrogenase ◽  
Ph Optimum ◽  
Phenazine Methosulphate ◽  
Reduced Activity ◽  
Cellobionic Acid

An extracellular enzyme from culture filtrates of Sporotrichum (Chrysosporium) thermophile (A.T.C.C. 42 464) after growth on cellulose or cellobiose was shown to oxidize cellobiose to cellobionic acid in vitro. Lactose and cellodextrins were also efficiently oxidized, but the enzyme was not active against most mono- and di-saccharides. Several redox substances could act as electron acceptors, but molecular oxygen, tetrazolium salts and NAD(P) were not reduced. Activity was stimulated up to 2-fold in the presence of 0.05 M-Mg2+. The pH optimum of the enzymic reaction was acidic when the activity was tested with dichlorophenol-indophenol or Methylene Blue, but was neutral to alkaline for 3,5-di-t-butyl-1,2-benzoquinone or phenazine methosulphate as electron acceptors. As the enzyme was formed inductively in parallel with the endocellulase, its possible function in relation to cellulolysis is discussed.

scholarly journals Characterization of a cellobiose dehydrogenase from Humicola insolens

1998 ◽  
Vol 330 (1) ◽  
pp. 565-571 ◽  
Author(s):  
Charlotte SCHOU ◽  
H. Margrethe CHRISTENSEN ◽  
Martin SCHÜLEIN
Keyword(s):  
Methylene Blue ◽  
Phanerochaete Chrysosporium ◽  
Soft Rot ◽  
Electron Acceptors ◽  
Cellobiose Dehydrogenase ◽  
Humicola Insolens ◽  
Organic Electron ◽  
Anomeric Carbon ◽  
Prosthetic Groups

The major cellobiose dehydrogenase (oxidase) (CBDH) secreted by the soft-rot thermophilic fungus Humicola insolens during growth on cellulose has been isolated and purified. It was shown to be a haemoflavoprotein with a molecular weight of 92 kDa and a pI of 4.0, capable of oxidizing the anomeric carbon of cellobiose, soluble cellooligosaccharides, lactose, xylobiose and maltose. Possible electron acceptors are 2,6-dichlorophenol-indophenol (DCPIP), Methylene Blue, 3,5-di-t-butyl-1,2-benzoquinone, potassium ferricyanide, cytochrome c and molecular oxygen. The oxidation of the prosthetic groups by oxygen was monitored at 449 nm for the flavin group and at 562 nm for the haem group. The curves were very similar to those of the cellobiose dehydrogenase from Phanerochaete chrysosporium, suggesting a similar mechanism. The pH-optima for the oxidation varied remarkably depending on the electron acceptor. For the organic electron acceptors, the pH-optima ranged from pH 4 for Methylene Blue to pH 7 for DCPIP and the benzoquinone. In the case of the FeIII-containing electron acceptors, the enzyme displayed alkaline pH-optima, in contrast to the properties of cellobiose dehydrogenases from Phanerochaete chrysosporium and Myceliophthora (Sporotrichum) thermophila. The enzyme has optimal activity at 65 °C.

scholarly journals In vitro conversion of proapoprotein A-I to apoprotein A-I. Partial characterization of an extracellular enzyme activity.

1983 ◽  
Vol 258 (19) ◽  
pp. 11430-11433 ◽  
Author(s):  
C Edelstein ◽  
J I Gordon ◽  
K Toscas ◽  
H F Sims ◽  
A W Strauss ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Extracellular Enzyme ◽  
Extracellular Enzyme Activity ◽  
Partial Characterization

scholarly journals Purification and Characterization of Cellobiose Dehydrogenase from the Plant Pathogen Sclerotium(Athelia) rolfsii

2001 ◽  
Vol 67 (4) ◽  
pp. 1766-1774 ◽  
Author(s):  
Ursula Baminger ◽  
Sai S. Subramaniam ◽  
V. Renganathan ◽  
Dietmar Haltrich
Keyword(s):  
Sclerotium Rolfsii ◽  
Cation Radical ◽  
Molecular Size ◽  
Extracellular Enzyme ◽  
Cellobiose Dehydrogenase ◽  
Phytopathogenic Fungus ◽  
Athelia Rolfsii ◽  
Heme Domain ◽  
Acid Cation

ABSTRACT Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. In the presence of a suitable electron acceptor, e.g., 2,6-dichloro-indophenol (DCIP), cytochromec, or metal ions, CDH oxidizes cellobiose to cellobionolactone. The phytopathogenic fungus Sclerotium rolfsii (teleomorph: Athelia rolfsii) strain CBS 191.62 produces remarkably high levels of CDH activity when grown on a cellulose-containing medium. Of the 7,500 U of extracellular enzyme activity formed per liter, less than 10% can be attributed to the proteolytic product cellobiose:quinone oxidoreductase. As with CDH from wood-rotting fungi, the intact, monomeric enzyme from S. rolfsii contains one heme b and one flavin adenine dinucleotide cofactor per molecule. It has a molecular size of 101 kDa, of which 15% is glycosylation, and a pI value of 4.2. The preferred substrates are cellobiose and cellooligosaccharides; additionally, β-lactose, thiocellobiose, and xylobiose are efficiently oxidized. Cytochrome c (equine) and the azino-di-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical were the best electron acceptors, while DCIP, 1,4-benzoquinone, phenothiazine dyes such as methylene blue, phenoxazine dyes such as Meldola's blue, and ferricyanide were also excellent acceptors. In addition, electrons can be transferred to oxygen. Limited in vitro proteolysis with papain resulted in the formation of several protein fragments that are active with DCIP but not with cytochrome c. Such a flavin-containing fragment, with a mass of 75 kDa and a pI of 5.1 and lacking the heme domain, was isolated and partially characterized.

scholarly journals Characterization of ornithine decarboxylase of tobacco cells and tomato ovaries.

1982 ◽  
Vol 201 (2) ◽  
pp. 373-376 ◽  
Author(s):  
Y M Heimer ◽  
Y Mizrahi
Keyword(s):  
Molecular Weight ◽  
Ornithine Decarboxylase ◽  
Pyridoxal Phosphate ◽  
Apparent Molecular Weight ◽  
Ph Optimum ◽  
Tobacco Cells ◽  
Thiol Reagent ◽  
Mammalian Origin

Some characteristics of L-ornithine decarboxylase of tomato ovaries and tobacco cells are described. The enzyme has a pH optimum of 8.0. It requires pyridoxal phosphate and thiol reagent (dithiothreitol) for activity. It is specific for L-ornithine and has an apparent Km of 1.4 × 10-4 M. It has an apparent molecular weight of 107000. Putrescine inhibited the activity in vitro. Spermidine and spermine also inhibit the enzyme, but less effectively. It is concluded that the enzyme is similar to that of mammalian origin and likewise fulfils a function related to cell proliferation.

scholarly journals Mosquito Acetylcholinesterase as a Target for Novel Phenyl-Substituted Carbamates

2019 ◽  
Vol 16 (9) ◽  
pp. 1500
Author(s):  
James M. Mutunga ◽  
Ming Ma ◽  
Qiao-Hong Chen ◽  
Joshua A. Hartsel ◽  
Dawn M. Wong ◽  
...  
Keyword(s):  
Anopheles Gambiae ◽  
Parent Compound ◽  
World Health ◽  
Contact Toxicity ◽  
Ache Inhibitors ◽  
Mosquitocidal Activity ◽  
Health Organization ◽  
Reduced Activity

New insecticides are needed for control of disease-vectoring mosquitoes and this research evaluates the activity of new carbamate acetylcholinesterase (AChE) inhibitors. Biochemical and toxicological characterization of carbamates based on the parent structure of terbam, 3-tert-butylphenyl methylcarbamate, was performed. In vitro enzyme inhibition selectivity (Anopheles gambiae versus human) was assessed by the Ellman assay, as well as the lethality to whole insects by the World Health Organization (WHO) paper contact assay. Bromination at the phenyl C6 position increased inhibitory potency to both AChEs, whereas a 6-iodo substituent led to loss of potency, and both halogenations caused a significant reduction of mosquitocidal activity. Similarly, installation of a hexyl substituent at C6 drastically reduced inhibition of AgAChE, but showed a smaller reduction in the inhibition of hAChE. A series of 4-carboxamido analogs of the parent compound gave reduced activity against AgAChE and generally showed more activity against hAChE than AgAChE. Replacement of the 3-t-buyl group with CF3 resulted in poor anticholinesterase activity, but this compound did have measurable mosquitocidal activity. A series of methyl- and fluoro- analogs of 3-trialkylsilyl compounds were also synthesized, but unfortunately resulted in disappointing activity. Finally, a series of sulfenylated proinsecticides showed poor paper contact toxicity, but one of them had topical activity against adult female Anopheles gambiae. Overall, the analogs prepared here contributed to a better understanding of carbamate structure–activity relationships (SAR), but no new significant leads were generated.

Identification and partial characterization of an extracellular acid phosphatase activity of Leishmania donovani promastigotes

1982 ◽  
Vol 2 (1) ◽  
pp. 76-81
Author(s):  
M Gottlieb ◽  
D M Dwyer
Keyword(s):  
Acid Phosphatase ◽  
Leishmania Donovani ◽  
Surface Membrane ◽  
Extracellular Enzyme ◽  
Growth Cycle ◽  
Growth Media ◽  
Ph Optimum ◽  
Membrane Bound ◽  
Extracellular Acid Phosphatase

An extracellular acid phosphatase was detected in the growth media of Leishmania donovani promastigotes. The enzyme was released at all stages of the growth cycle and in amounts which accounted for 90% of the total amount of this enzyme in the culture. The exoenzyme exhibited a pH optimum of 4.5 to 5.0 and was active with a variety of organic phosphates. The enzymatic activity was excluded from Sephacryl S-300 and was retained by ultrafilters with nominal molecular weight cutoffs of up to 300,000. The results of comparative studies indicated that the extracellular enzyme was distinct from a surface membrane-bound acid phosphatase of L. donovani promastigotes which has been previously described.

scholarly journals Characterization of a Novel Thermostable O-Acetylserine Sulfhydrylase from Aeropyrum pernix K1

2003 ◽  
Vol 185 (7) ◽  
pp. 2277-2284 ◽  
Author(s):  
Koshiki Mino ◽  
Kazuhiko Ishikawa
Keyword(s):  
Escherichia Coli ◽  
Rate Constant ◽  
Binding Motif ◽  
Phosphate Binding ◽  
Ph Optimum ◽  
Hyperthermophilic Archaeon ◽  
Aeropyrum Pernix ◽  
Synthetic Reaction

ABSTRACT An O-acetylserine sulfhydrylase (OASS) from the hyperthermophilic archaeon Aeropyrum pernix K1, which shares the pyridoxal 5′-phosphate binding motif with both OASS and cystathionine β-synthase (CBS), was cloned and expressed by using Escherichia coli Rosetta(DE3). The purified protein was a dimer and contained pyridoxal 5′-phosphate. It was shown to be an enzyme with CBS activity as well as OASS activity in vitro. The enzyme retained 90% of its activity after a 6-h incubation at 100°C. In the O-acetyl-l-serine sulfhydrylation reaction, it had a pH optimum of 6.7, apparent Km values for O-acetyl-l-serine and sulfide of 28 and below 0.2 mM, respectively, and a rate constant of 202 s−1. In the l-cystathionine synthetic reaction, it showed a broad pH optimum in the range of 8.1 to 8.8, apparent Km values for l-serine and l-homocysteine of 8 and 0.51 mM, respectively, and a rate constant of 0.7 s−1. A. pernix OASS has a high activity in the l-cysteine desulfurization reaction, which produces sulfide and S-(2,3-hydroxy-4-thiobutyl)-l-cysteine from l-cysteine and dithiothreitol.

scholarly journals The Final Step of Hygromycin A Biosynthesis, Oxidation of C-5″-Dihydrohygromycin A, Is Linked to a Putative Proton Gradient-Dependent Efflux

2009 ◽  
Vol 53 (12) ◽  
pp. 5163-5172 ◽  
Author(s):  
Vidya Dhote ◽  
Agata L. Starosta ◽  
Daniel N. Wilson ◽  
Kevin A. Reynolds
Keyword(s):  
Biosynthetic Pathway ◽  
High Titer ◽  
Biosynthetic Gene Cluster ◽  
Proton Gradient ◽  
Purification And Characterization ◽  
E Coli ◽  
Reduced Activity

ABSTRACT Hygromycin A (HA) is an aminocyclitol antibiotic produced and excreted by Streptomyces hygroscopicus. Deletion of hyg26 from the hygromycin A biosynthetic gene cluster has previously been shown to result in a mutant that produces 5″-dihydrohygromycin A (DHHA). We report herein on the purification and characterization of Hyg26 expressed in E scherichia coli. The enzyme catalyzes an NAD(H)-dependent reversible interconversion of HA and DHHA, supporting the role of the reduced HA as the penultimate biosynthetic pathway intermediate and not a shunt product. The equilibrium for the Hyg26-catalyzed reaction heavily favors the DHHA intermediate. The high-titer production of the HA product by S. hygroscopicus must be dependent upon a subsequent energetically favorable enzyme-catalyzed process, such as the selective and efficient export of HA. hyg19 encodes a putative proton gradient-dependent transporter, and a mutant lacking this gene was observed to produce less HA and to produce the DHHA intermediate. The DHHA produced by either the Δhyg19 or the Δhyg26 mutant had slightly reduced activity against E. coli and reduced protein synthesis-inhibitory activity in vitro. The data indicate that Hyg26 and Hyg19 have evolved to produce and export the final potent HA product in a coordinated fashion.

scholarly journals Synthesis & Characterization of Magnetit (Fe3O4) and Its Applications As Adsorbent Methylene Blue

2017 ◽  
Vol 11 (2) ◽  
pp. 61
Author(s):  
Retno Agnestisia
Keyword(s):  
Methylene Blue ◽  
Contact Time ◽  
Coprecipitation Method ◽  
X Ray Diffraction ◽  
Ph Optimum ◽  
Adsorption Study ◽  
Batch System ◽  
Optimum Ph ◽  
Blue Solution

Synthesis, characterization and adsorption study of magnetite have beenconducted. Magnetite was synthesized by coprecipitation method. The characterizations of magnetite were carried out with spectroscopy FTIR (Fourier Transform Infrared) and XRD (X-ray diffraction). The adsorption study was conducted using a batch system with the studied adsorption study including optimum pH, optimum contact time and adsorption equilibrium. The results showed that coprecipitation method has succeeded to form magnetite that has magnetism properties. Magnetite can adsorbed methylene blue from aqueous phase, with the maximum adsorption at pH 5 and contact time of 90 minutes.Adsorption of methylene blue by magnetite follows the adsorption pattern of the Langmuir isotherm with the adsorption energy of 25.59 kJ/mol and adsorption capacity of 43.86 mg/g. The results of magnetite synthesis can accelerate the process of separating the adsorbent particles in a methylene blue solution using an external magnetic field.Keywords : magnetite, coprecipitation, adsorption, and methylene blue.

scholarly journals Purification and characterization of a calcium-independent acidic phospholipase A2 from rat lung

1994 ◽  
Vol 304 (1) ◽  
pp. 131-137 ◽  
Author(s):  
R Wang ◽  
C R Dodia ◽  
M K Jain ◽  
A B Fisher
Keyword(s):  
Phospholipase A2 ◽  
Dependent Manner ◽  
Rat Lung ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
New Class ◽  
Terminal Amino ◽  
Dose Dependent

Several phospholipase A2 (PLA2) activities have been identified in rat lung homogenate and shown to be important in metabolism of lung phospholipids. One PLA2 activity is Ca(2+)-independent, active in vitro at pH 4, and inhibited by a substrate analogue, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33). Purification of this rat lung PLA2 by approx. 550-fold was carried out by sequential column chromatographies using DE-52, Sephacryl-100, heparin-Sepharose, and phenyl-Sepharose columns. The purified activity had an acidic pH optimum, was Ca(2+)-independent, was inhibited by MJ33 in a dose-dependent manner (50% inhibition at 3 mol%), was unaffected by treatment with p-bromophenacyl bromide or mercaptoethanol, and had a unique N-terminal amino acid sequence. The apparent molecular mass was 15 kDa on gel electrophoresis and activity was recovered in part by renaturation of protein from the gel. The properties of this enzyme suggest a new class of PLA2.

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