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Cholesterol affects African swine fever virus infection

Bernardes, C.; António, A.; Pedroso de Lima, Maria C.; Valdeira, M. L.
Fonte: Universidade de Coimbra Publicador: Universidade de Coimbra
Tipo: Artigo de Revista Científica Formato: aplication/PDF
ENG
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African swine fever virus (ASFV) enters cells by receptor mediated endocytosis and requires a fusion event between the viral envelope and the limiting membrane of the endosome at low pH. In order to investigate the role of cholesterol in the early stages of ASFV infection, we have studied the effect of the removal of cell and viral membrane cholesterol by cholesterol oxidase treatment of cells and virions, as well as the effect of some inhibitors of cholesterol synthesis on the infectious pathway. In addition, we have investigated viral infection in cholesterol-depleted Vero cells. Both cholesterol-depleted and cholesterol oxidase-treated Vero cells were unaltered in their ability to bind or internalize the virus, but were blocked in ASFV fusion and subsequent virus replication. Our results indicate that ASFV infection is affected by cholesterol in the target membrane.; http://www.sciencedirect.com/science/article/B6T1X-3V3N06F-2/1/e52e3430df628f470dc4b0b8400b4b20

Immobilization of cholesterol oxidase in LbL films and detection of cholesterol using ac measurements

MORAES, Marli L.; SOUZA, Nara C. de; HAYASAKA, Caio O.; FERREIRA, Marystela; RODRIGUES FILHO, Ubirajara P.; RIUL JR., Antonio; ZUCOLOTTO, Valtencir; OLIVEIRA JR., Osvaldo N.
Fonte: ELSEVIER SCIENCE BV Publicador: ELSEVIER SCIENCE BV
Tipo: Artigo de Revista Científica
ENG
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The preserved activity of immobilized biomolecules in layer-by-layer (LbL) films can be exploited in various applications. including biosensing. In this study, cholesterol oxidase (COX) layers were alternated with layers of poly(allylamine hydrochloride) (PAH) in LbL films whose morphology was investigated with atomic force microscopy (AFM). The adsorption kinetics of COX layers comprised two regimes, a fast, first-order kinetics process followed by a slow process fitted with a Johnson-Mehl-Avrami (JMA) function. with exponent similar to 2 characteristic of aggregates growing as disks. The concept based on the use of sensor arrays to increase sensitivity, widely employed in electronic tongues, was extended to biosensing with impedance spectroscopy measurements. Using three sensing units, made of LbL films of PAH/COX and PAHIPVS (polyvinyl sulfonic acid) and a bare gold interdigitated electrode, we were able to detect cholesterol in aqueous solutions down to the 10(-6) M level. This high sensitivity is attributed to the molecular-recognition interaction between COX and cholesterol, and opens the way for clinical tests to be made with low cost. fast experimental procedures. (C) 2008 Published by Elsevier B.V.; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); FAPESP; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); CNPq; CAPES (Brazil); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Filmes nanoestruturados de materiais de interesse biológico: ênfase na interação com modelos de membrana e aplicações em biossensores; Nanostructured films with materials of biological interest: emphasis on interaction with membrane models and biosensor applications

Moraes, Marli Leite de
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 27/08/2008 PT
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A imobilização de moléculas de interesse biológico em superfícies sólidas é essencial para uma série de aplicações biotecnológicas. Dentre as técnicas de imobilização, a automontagem camada por camada por adsorção física possui inúmeras vantagens, incluindo condições brandas e fisiológicas de preparação, capacidade de incorporar diferentes biomoléculas, e controle molecular. Nesta tese foram explorados filmes nanoestruturados de materiais de interesse biológico, bem como modelos de membranas, em que foram empregadas a técnica de automontagem e a preparação de lipossomos. Os lipossomos, que serviram como modelos de membrana, foram imobilizados em filmes automontados e sua integridade estrutural foi mantida. Também foram utilizados para incorporar e estabilizar melanina, e então imobilizados em filmes automontados, com preservação da propriedade fluorescente da melanina. A automontagem também foi utilizada para imobilização das enzimas uricase, fitase e colesterol oxidase, alternadas com camadas de polieletrólitos. Estes filmes mostraram bom desempenho como biossensores amperométricos para uricase e fitase, e como biossensores usando espectroscopia de impedância para a fitase e colesterol oxidase. Tais biossensores foram usados para detectar baixas quantidades de ácido úrico...

Monomolecular films of cholesterol oxidase and S-Layer proteins

Ferraz, Helen Conceicao; Guimaraes, Juliana Aguilar; Moitinho Alves, Tito Livio; Leopoldo Constantino, Carlos Jose
Fonte: Elsevier B.V. Publicador: Elsevier B.V.
Tipo: Artigo de Revista Científica Formato: 6535-6539
ENG
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Cholesterol oxidase (ChOx) is a flavoenzyme that catalyzes the oxidation of cholesterol to cholest-5-en-3-one and subsequently the isomerization to cholest-4-en-3-one. ChOx has been very commonly studied as the detection element in cholesterol biosensors. In the biosensor development field, a relatively new approach is the use of crystalline bacterial cell surface layers, known as S-Layer proteins. These proteins exhibit the ability of self-assembling at surfaces, opening a vast spectrum of applications, both in basic and applied researches. In our study, monomolecular films of ChOx and mixed films of ChOx/S-Layer proteins and DPPC/S-Layer proteins were produced using the Langmuir technique. Characterization of the films was performed by means of surface pressure-molecular area (pi-A) isotherms. Stable monolayers were obtained, which means that they can be transferred to solid substrates by Langmuir-Blodgett technique. Mixed monolayers showed an ideal like behavior. (C) 2011 Elsevier B. V. All rights reserved.

Use of hemoglobin as alternative to peroxidases in cholesterol amperometric biosensors

Souza, Tâmera T.L.; Moraes, Marli L.; Ferreira, Marystela
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 101-106
ENG
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Sophisticated molecular architectures can be produced with the layer-by-layer (LbL) method, which may combine distinct materials on the same film. In this study, we take advantage of this capability to produce cholesterol amperometric biosensors from LbL films containing hemoglobin (Hb) and cholesterol oxidase in addition to the polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(ethylene imine) (PEI). Following an optimization procedure, we found that an LbL film deposited onto ITO substrates, with the architecture ITO(PEI/Hb)5(PEI/COx)10, yielded a sensitivity of 93.4 μA μmol L-1 cm-2 for cholesterol incorporated into phospholipid liposomes, comparable to state-of-the-art biosensors. Hb acted as efficient electron mediator and did not suffer interference from phospholipids. Significantly, cholesterol could also be detected in real samples from chicken egg yolk, with no effects from potential interferents, including phospholipids. Taken together these results demonstrate the possible fabrication of low cost, easy-to-use cholesterol amperometric biosensors, whose sensitivity can be enhanced by further optimizing the molecular architectures of the LbL films. © 2012 Elsevier B.V.

A multicommuted flow procedure for the determination of total and free cholesterol in eggs and human blood serum by chemiluminescence

Leite,Oldair D; Vieira,Heberth J; Fatibello-Filho,Orlando; Rocha,Fábio R. P
Fonte: Sociedade Brasileira de Química Publicador: Sociedade Brasileira de Química
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2010 EN
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58.09331%
A multicommuted flow-based procedure with detection by chemiluminescence for the determination of total and free cholesterol without changes in the flow manifold is proposed. Cholesterol esterase and cholesterol oxidase were both immobilized on glass beads via glutaraldehyde/(3-aminopropyl)-triethoxysilane and mini-columns containing the enzymes were used for online sample treatment. Cholesterol esters were cleaved to cholesterol and fatty acids at the packed reactor containing cholesterol esterase. The reactor containing cholesterol oxidase converted cholesterol to cholest-4-en-3-one also yielding hydrogen peroxide. Detection was based on the chemiluminescence produced by H2O2 in the hexacyanoferrate(III)-luminol system. Influence of both chemical and hydrodynamic variables on the chemiluminescence signals were investigated. The analytical curves were linear from 250 to 2500 mg L-1 and from 500 to 4000 mg L-1, for free and total cholesterol, respectively. Detection limits for both analytes were estimated as 60 mg L-1 at 99.7% confidence level. The sampling rate was 55 h-1 and reagent consumption was 350 µg of luminol and 2.6 mg of potassium hexacyanoferrate(III) per determination. The procedure developed was successfully applied for determination of cholesterol in eggs and in human blood serum with results in agreement with the reference spectrophotometric method at the 95% confidence level.

Purification of Extracellular Cholesterol Oxidase with High Activity in the Presence of Organic Solvents from Pseudomonas sp. Strain ST-200

Doukyu, Noriyuki; Aono, Rikizo
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/1998 EN
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Extracellular cholesterol oxidase of Pseudomonas sp. strain ST-200 was purified from the culture supernatant. This oxidase contained bound flavin and was categorized as a 3β-hydroxysteroid oxidase, converting 3β-hydroxyl groups to keto groups. The molecular mass was 60 kDa. The enzyme was stable at pH 4 to 11 and active at pH 5.0 to 8.5, showing optimal activity at pH 7 at 60°C. The Michaelis constant of the ST-200 cholesterol oxidase was lower than those of commercially available oxidases. The cholesterol oxidation rate was enhanced 3- to 3.5-fold in the presence of organic solvents, with log Pow values (partition coefficients of the organic solvent between n-octanol and water), in the range of 2.1 to 4.2, compared with that in the absence of organic solvents.

Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

Corbin, David R.; Grebenok, Robert J.; Ohnmeiss, Thomas E.; Greenplate, John T.; Purcell, John P.
Fonte: American Society of Plant Physiologists Publicador: American Society of Plant Physiologists
Tipo: Artigo de Revista Científica
Publicado em /07/2001 EN
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Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism.

Cloning of an insecticidal cholesterol oxidase gene and its expression in bacteria and in plant protoplasts.

Corbin, D R; Greenplate, J T; Wong, E Y; Purcell, J P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1994 EN
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We cloned and sequenced structural gene choM, which encodes an insecticidally active cholesterol oxidase in Streptomyces sp. strain A19249. The primary translation product was predicted to be a 547-amino-acid protein whose first 43 amino acids constitute a secretory signal peptide. Expression of the gene with the signal sequence in Escherichia coli resulted in production of a protein that had enzymatic and insecticidal properties which were indistinguishable from those of the cholesterol oxidase secreted by Streptomyces sp. strain A19249. Expression of the gene with or without the signal sequence in tobacco protoplasts resulted in production of an enzymatically active cholesterol oxidase.

Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702.

Murooka, Y; Ishizaki, T; Nimi, O; Maekawa, N
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1986 EN
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The cholesterol oxidase gene (cho) of Streptomyces sp. was cloned into Streptomyces lividans with the vector pIJ702. Deletion analysis of the recombinant plasmid showed that entire coding sequence of the cho gene was located within a 2.5-kilobase segment of the chromosomal DNA obtained from the cholesterol oxidase-producing strain. When cloned cells of S. lividans were grown in an appropriate medium, the cells produced severalfold more cholesterol oxidase extracellularly than did the producing strain.

Cholesterol aggregation and interaction with cholesterol oxidase in supercritical carbon dioxide.

Randolph, T W; Clark, D S; Blanch, H W; Prausnitz, J M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1988 EN
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High-pressure EPR spectroscopy indicates that cholesterol forms aggregates in supercritical carbon dioxide. In pure carbon dioxide, changes in cholesterol aggregate size or packing structure are observed with changing pressure. Near the critical point of carbon dioxide, cholesterol solubility is too low to permit significant aggregation, and monomeric cholesterol is observed. Addition of small amounts of dopants to supercritical carbon dioxide strongly affects cholesterol aggregation. Branched butanols (2-methyl-1-propanol and 2-methyl-2-propanol) and ethanol (to a lesser degree) promote cholesterol aggregation, while methanol, acetone, and 1-butanol do not. Cosolvents that promote aggregation also increase the rate at which cholesterol oxidase from Gloeocysticum chrysocreas catalyzes the oxidation of cholesterol. In supercritical carbon dioxide solutions, the EPR spectroscopy reveals little or no conformational change in cholesterol oxidase as 2-methyl-2-propanol or methanol is added. Damp cholesterol oxidase binds multiple cholesterol molecules; dry enzyme loses the ability to bind cholesterol. When molecular oxygen is the oxidizing agent, the rate of enzymatic cholesterol oxidation is greatly reduced in bone-dry carbon dioxide compared to that in water-saturated carbon dioxide.

The characterization and interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous.

Cheetham, P S; Dunnill, P; Lilly, M D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/03/1982 EN
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The physical properties and the methods used for interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous by treatment with Triton X-100, trypsin or buffer alone provide evidence that these forms differ chiefly in the possession or absence of a hydrophobic anchor region connected by a trypsin-sensitive region. The hydrophobic domain normally integrates the enzyme into the cell membrane and confers amphipathic properties on the solubilized enzyme, causing adsorption to hydrophobic resins, aggregation when detergent is removed and formation of mixed micelles with detergent and cholesterol resulting in surface-dilution kinetic behaviour and activation by relatively high concentrations of water-miscible solvents. By contrast, only the enzymic fragment is extracted with trypsin and it behaves as a conventional soluble enzyme and does not aggregate or interact with hydrophobic resins, detergents or water-miscible solvents. As no phospholipid could be detected in the enzyme extracts, the detergent appears to act as a substitute for the cell-membrane lipids that would normally interact with the hydrophobic region. This cholesterol oxidase is an example of a prokaryotic enzyme possessing two closely associated catalytic functions...

The substrate specificity and stereochemistry, reversibility and inhibition of the 3-oxo steroid delta 4-delta 5-isomerase component of cholesterol oxidase.

Smith, A G; Brooks, C J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/10/1977 EN
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1. 5-Cholesten-3-one was shown to be an intermediate in the conversion of cholesterol into 4-cholesten-3-one by Nocardia cholesterol oxidase. 2. The absence of a C-17 side chain from 5-androstene-3,17-dione slightly increased the Vmax. of the isomerase activity relative to 5-cholesten-3-one (1.7-fold), but greatly increased the Km. 3. Incubations of [4alpha-2H]-and [4beta-2H]-cholesterol with cholesterol oxidase showed that the 4beta-hydrogen atom can be transferred to the 6beta-position. However, incubations of cholesterol, 5-cholesten-3-one and 4-cholesten-3-one with the enzyme in 2H2O led to some incorporation of 2H into the 4-cholesten-3-one products, mostly at position 6beta. 4. Both the isomerase and the oxidase activities of cholesterol oxidase were inhibited by 5,10-seco-19-nor-5-cholestyne-3,10-dione.

Two moles of O2 consumption and one mole of H2O2 formation during cholesterol peroxidation with cholesterol oxidase from Pseudomonas sp. strain ST-200.

Doukyu, N; Aono, R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/08/1999 EN
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47.998926%
Cholesterol oxidase from Pseudomonas sp. strain ST-200 oxidized cholesterol and cholestanol to 6beta-hydroperoxycholest-4-en-3-one and 5alpha-cholestan-3-one respectively. The former was converted spontaneously to several oxysteroids such as 6-hydroxycholest-4-en-3-one and cholest-4-ene-3,6-dione, with the consumption of 2 mol of O(2) and the formation of 1 mol of H(2)O(2) for each mole of cholesterol oxidized. An oxidized form of the cholesterol oxidase dehydrogenates cholesterol, probably to the 5-en-3-one derivative. A reduced form of the enzyme, yielded from the cholesterol dehydrogenation reaction, dioxygenated cholest-5-en-3-one to 6beta-hydroperoxycholest-4-en-3-one.

Substrate and inhibitor specificity of the cholesterol oxidase in bovine adrenal cortex

Raggatt, P. R.; Whitehouse, M. W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1966 EN
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1. Cholesteryl 3β-sulphate is oxidized in vitro by preparations of bovine adrenal-cortex mitochondria to pregnenolone sulphate and isocaproic acid (4-methyl-pentanoic acid) without hydrolysis of the ester linkage. 2. Free cholesterol is the preferred substrate for adrenal-cortex cholesterol oxidase; the apparent Km for cholesteryl sulphate is 500μm and for free cholesterol 50μm under the same conditions. 3. Cholesteryl 3β-acetate is hydrolysed by bovine adrenal-cortex mitochondria in vitro to free cholesterol, which is subsequently oxidized to more polar steroids and isocaproic acid. Evidence was obtained that other cholesterol esters behave similarly. Cholesterol esters may thus act as precursors of steroid hormones. 4. Cholest-4-en-3-one is only poorly oxidized to isocaproic acid and more polar steroids and thus is probably not a significant precursor of steroid hormones. 5. Cholesteryl esters inhibit the oxidation of cholesterol competitively (Ki for cholesteryl phosphate 28μm, for cholesteryl sulphate 110μm, for cholesteryl acetate 65μm) but pregnenolone esters do not inhibit this system. 6. Pregnenolone and 20α-hydroxycholesterol (both metabolites of cholesterol in this system) inhibit the oxidation of cholesterol non-competitively. Ki for pregnenolone is 130μm and Ki for 20α-hydroxycholesterol is 17μm. 7. 25-Oxo-27-norcholesterol inhibits cholesterol oxidation non-competitively (Ki16μm). A number of other Δ5-3β-hydroxy steroids inhibit cholesterol oxidation and evidence was obtained that the 3β-hydroxyl group was necessary for inhibitory activity. 8. Pregnenolone...

Cholesterol oxidase: physiological functions

Kreit, Joseph; Sampson, Nicole S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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An important aspect of catalysis by cholesterol oxidase (3β-hydroxysteroid oxidase) is the nature of its association with the lipid bilayer that contains the sterol substrate. Efficient catalytic turnover is affected by the association of the protein with the membrane as well as the solubility of the substrate in the lipid bilayer. In this review, the binding of cholesterol oxidase to the lipid bilayer, its turnover of substrates presented in different physical environments, and how these conditions affect substrate specificity are discussed. The physiological functions of the enzyme in bacterial metabolism, pathogenesis, and macrolide biosynthesis are reviewed in this context.

Fabricating an Amperometric Cholesterol Biosensor by a Covalent Linkage between Poly(3-thiopheneacetic acid) and Cholesterol Oxidase

Nien, Po-Chin; Chen, Po-Yen; Ho, Kuo-Chuan
Fonte: Molecular Diversity Preservation International (MDPI) Publicador: Molecular Diversity Preservation International (MDPI)
Tipo: Artigo de Revista Científica
Publicado em 13/03/2009 EN
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47.93251%
In this study, use of the covalent enzyme immobilization method was proposed to attach cholesterol oxidase (ChO) on a conducting polymer, poly(3-thiopheneacetic acid), [poly(3-TPAA)]. Three red-orange poly(3-TPAA) films, named electrodes A, B and C, were electropolymerized on a platinum electrode by applying a constant current of 1.5 mA, for 5, 20 and 100 s, respectively. Further, 1-ethyl-3-(3-dimethylamiopropyl)carbodiimide hydrochloride (EDC · HCl) and N-hydroxysuccinimide (NHS) were used to activate the free carboxylic groups of the conducting polymer. Afterwards, the amino groups of the cholesterol oxidase were linked on the activated groups to form peptide bonds. The best sensitivity obtained for electrode B is 4.49 mA M−1 cm−2, with a linear concentration ranging from 0 to 8 mM, which is suitable for the analysis of cholesterol in humans. The response time (t95) is between 70 and 90 s and the limit of detection is 0.42 mM, based on the signal to noise ratio equal to 3. The interference of species such as ascorbic acid and uric acid increased to 5.2 and 10.3% of the original current response, respectively, based on the current response of cholesterol (100%). With respect to the long-term stability, the sensing response retains 88% of the original current after 13 days.

Reusable and Mediator-Free Cholesterol Biosensor Based on Cholesterol Oxidase Immobilized onto TGA-SAM Modified Smart Bio-Chips

Rahman, Mohammed M.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 20/06/2014 EN
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47.998926%
A reusable and mediator-free cholesterol biosensor based on cholesterol oxidase (ChOx) was fabricated based on self-assembled monolayer (SAM) of thioglycolic acid (TGA) (covalent enzyme immobilization by dropping method) using bio-chips. Cholesterol was detected with modified bio-chip (Gold/Thioglycolic-acid/Cholesterol-oxidase i.e., Au/TGA/ChOx) by reliable cyclic voltammetric (CV) technique at room conditions. The Au/TGA/ChOx modified bio-chip sensor demonstrates good linearity (1.0 nM to 1.0 mM; R = 0.9935), low-detection limit (∼0.42 nM, SNR∼3), and higher sensitivity (∼74.3 µAµM−1cm−2), lowest-small sample volume (50.0 μL), good stability, and reproducibility. To the best of our knowledge, this is the first statement with a very high sensitivity, low-detection limit, and low-sample volumes are required for cholesterol biosensor using Au/TGA/ChOx-chips assembly. The result of this facile approach was investigated for the biomedical applications for real samples at room conditions with significant assembly (Au/TGA/ChOx) towards the development of selected cholesterol biosensors, which can offer analytical access to a large group of enzymes for wide range of biomedical applications in health-care fields.

Some enzymatic properties of cholesterol oxidase produced by Brevibacterium sp

Salva,Terezinha J.G.; Liserre,Alcina M.; Moretto,Aloísia L.; Zullo,Marco A.T.; Ventrucci,Gisleine; Menezes,Tobias J.B.
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/1999 EN
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In this study we determined some properties of the cholesterol oxidase from a Brevibacterium strain isolated from buffalo's milk and identified the cholesterol degradation products by the bacterial cell. A small fraction of the enzyme synthesized by cells cultured in liquid medium for 7days was released into the medium whereas a larger fraction remained bound to the cell membrane. The extraction of this fraction was efficiently accomplished in 1 mM phosphate buffer, pH 7.0, containing 0.7% Triton X-100. The enzyme stability under freezing and at 45oC was improved by addition of 20% glycerol. The optimum temperature and pH for the enzyme activity were 53°C and 7.5, respectively. The only steroidal product from cholesterol oxidation by the microbial cell and by the crude extract of the membrane-bound enzyme was 4-colesten-3-one. Chromatographic analysis showed that minor no steroidal compounds as well as 4-colesten-3-one found in the reaction media arose during fermentation process and were extracted together with the enzyme in the buffer solution. Cholesterol oxidation by the membrane-bound enzyme was a first order reaction type.

Cholesterol oxidase from Bordetella species promotes irreversible cell apoptosis in lung adenocarcinoma by cholesterol oxidation

Liu, J; Xian, G; Li, M; Zhang, Y; Yang, M; Yu, Y; Lv, H; Xuan, S; Lin, Y; Gao, L
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
EN
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Cholesterol oxidase (COD), an enzyme catalyzing the oxidation of cholesterol, has been applied to track the distribution of membrane cholesterol. Little investigations about the effect of COD on tumor cells have been performed. In the present study, we provided evidence that COD from Bordetella species (COD-B), induced apoptosis of lung cancer cells in vitro and in vivo. COD-B treatment inhibited Akt and ERK1/2 phosphorylation in dose- and time-dependent manner, which was not reversed and was even aggravated by cholesterol addition. Further investigation indicated that COD-B treatment promoted the generation of reactive oxygen species (ROS) and that cholesterol addition further elevated ROS levels. Moreover, COD-B treatment resulted in JNK and p38 phosphorylation, downregulation of Bcl-2, upregulation of Bax, activated caspase-3 and cytochrome C release, which likely responded to freshly produced hydrogen peroxide that accompanied cholesterol oxidation. Catalase pretreatment could only partially prevent COD-B-induced events, suggesting that catalase inhibited H2O2-induced signal transduction but had little effect on signal pathways involved in cholesterol depletion. Our results demonstrated that COD-B led to irreversible cell apoptosis by decreasing cholesterol content and increasing ROS level. In addition...