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Cholesterol Oxidase (cholesterol + oxidase)
Selected AbstractsA hydrogen-bonding network is important for oxidation and isomerization in the reaction catalyzed by cholesterol oxidaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2009Artem Y. Lyubimov Cholesterol oxidase is a flavoenzyme that catalyzes the oxidation and isomerization of 3,-hydroxysteroids. Structural and mutagenesis studies have shown that Asn485 plays a key role in substrate oxidation. The side chain makes an NH..., interaction with the reduced form of the flavin cofactor. A N485D mutant was constructed to further test the role of the amide group in catalysis. The mutation resulted in a 1800-fold drop in the overall kcat. Atomic resolution structures were determined for both the N485L and N485D mutants. The structure of the N485D mutant enzyme (at 1.0,Å resolution) reveals significant perturbations in the active site. As predicted, Asp485 is oriented away from the flavin moiety, such that any stabilizing interaction with the reduced flavin is abolished. Met122 and Glu361 form unusual hydrogen bonds to the functional group of Asp485 and are displaced from the positions they occupy in the wild-type active site. The overall effect is to disrupt the stabilization of the reduced FAD cofactor during catalysis. Furthermore, a narrow transient channel that is shown to form when the wild-type Asn485 forms the NH..., interaction with FAD and that has been proposed to function as an access route of molecular oxygen, is not observed in either of the mutant structures, suggesting that the dynamics of the active site are altered. [source] Fabrication of a Sensitive Cholesterol Biosensor Based on Cobalt-oxide Nanostructures Electrodeposited onto Glassy Carbon ElectrodeELECTROANALYSIS, Issue 24 2009Abdollah Salimi Abstract Electrodeposited cobalt oxide (CoOx) nanomaterials are not only used for immobilization of cholesterol oxidase (ChOx) but also as electron transfer mediator for oxidation of H2O2 generated in the enzymatic reaction. Voltammetry and flow injection analysis (FIA) were used for determination of cholesterol. FIA determination of cholesterol with biosensors yielded a calibration curve with the following characteristics: linear range up to 50,,M, sensitivity of 43.5,nA ,M,1 cm,2 and detection limit of 4.2,,M. The apparent Michaelis-Menten constant and the response time of the biosensor are 0.49,mM and 15,s, respectively. This biosensor also exhibits good stability, reproducibility and long life time. [source] Electrochemical Cholesterol Sensor Based on Tin Oxide-Chitosan Nanobiocomposite FilmELECTROANALYSIS, Issue 8 2009Anees Abstract A chitosan (CS)-tin oxide (SnO2) nanobiocomposite film has been deposited onto an indium-tin-oxide glass plate to immobilize cholesterol oxidase (ChOx) for cholesterol detection. The value of the Michaelis,Menten constant (Km) obtained as 3.8,mM for ChOx/CS-SnO2/ITO is lower (8,mM) than that of a ChOx/CS/ITO bioelectrode revealing enhancement in affinity and/or activity of ChOx towards cholesterol and also revealing strong binding of ChOx onto CS-SnO2/ITO electrode. This ChOx/CS-SnO2/ITO cholesterol sensor retains 95% of enzyme activity after 4,6 weeks at 4,°C with response time of 5,s, sensitivity of 34.7,,A/mg dL,1 cm2 and detection limit of 5,mg/dL. [source] Purification and properties of a new Brevibacterium sterolicum cholesterol oxidase produced by E. coli MM294/pnH10FEMS MICROBIOLOGY LETTERS, Issue 2 2002Kinya Fujishiro Abstract A gene encoding a cholesterol oxidase from Brevibacterium sterolicum nov. sp. ATCC21387 was isolated by an expression cloning method and highly expressed by a recombinant strain Escherichia coli MM294/pnH10. The purified cholesterol oxidase was a typical flavoprotein with a molecular mass of 46.5 kDa, absorption peaks at 280, 360, and 450 nm. Optimum pH and temperature were found at pH 6.5 and 55°C, respectively. The enzyme acted on 3,-hydroxysteroids such as cholesterol, pregnenolone, and ,-sitosterol at high rates, but on dehydro- epi -androsterone to a lesser degree. The molecular and catalytic properties were different from those of cholesterol oxidase I, which was initially discovered in B. sterolicum nov. sp. ATCC21387. The new enzyme, designated cholesterol oxidase II, was distinguished by its high affinity toward cholesterol (Km= 30,M). [source] Performance characteristics of cholesterol oxidase for kinetic determination of total cholesterolJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 6 2005Pornpen Srisawasdi Abstract The enzymatic method for cholesterol determination can use either an endpoint or a kinetic method. Not much is known concerning the properties (Km and Vmax) of the commercial enzyme for the kinetic method. We measured the Km and Vmax of Brevibacterium, Streptomyces, Pseudomonas fluorescens, and Cellulomonas cholesterol oxidase. Brevibacterium gave the highest Km value (230.3×10,4,M), followed by Streptomyces (2.17×10,4,M), Cellulomonas (0.84×10,4,M), and Pseudomonas (0.61×10,4,M). The Km values and the linearity obtained from Streptomyces (2.6,mmol/L), Pseudomonas (2.1,mmol/L), or Cellulomonas (2.1,mmol/L) were too low. Dichlorophenol isomers, acting as inhibitors, increased the enzyme's Km. The addition of 3,4-dichlorophenol raised the Km of Streptomyces from 2.17×10,4 to 24.89×10,4,M. The linearity was increased from 2.6 to 13.0,mmol/L. The high Km of Brevibacterium resulted in an insensitive reaction and low cholesterol linearity (7.8,mmol/L). An increase in the sample-to-reagent ratio from 1:100 to 1:10 enhanced the reaction rate and the linearity from 7.8 to 20.7,mmol/L. We suggest that Brevibacterium and Streptomyces cholesterol oxidase (with the addition of 3,4 dichlorophenol) are good sources for serum cholesterol determination by the kinetic method. J. Clin. Lab. Anal. 19:247,252, 2005. © 2005 Wiley-Liss, Inc. [source] A hydrogen-bonding network is important for oxidation and isomerization in the reaction catalyzed by cholesterol oxidaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2009Artem Y. Lyubimov Cholesterol oxidase is a flavoenzyme that catalyzes the oxidation and isomerization of 3,-hydroxysteroids. Structural and mutagenesis studies have shown that Asn485 plays a key role in substrate oxidation. The side chain makes an NH..., interaction with the reduced form of the flavin cofactor. A N485D mutant was constructed to further test the role of the amide group in catalysis. The mutation resulted in a 1800-fold drop in the overall kcat. Atomic resolution structures were determined for both the N485L and N485D mutants. The structure of the N485D mutant enzyme (at 1.0,Å resolution) reveals significant perturbations in the active site. As predicted, Asp485 is oriented away from the flavin moiety, such that any stabilizing interaction with the reduced flavin is abolished. Met122 and Glu361 form unusual hydrogen bonds to the functional group of Asp485 and are displaced from the positions they occupy in the wild-type active site. The overall effect is to disrupt the stabilization of the reduced FAD cofactor during catalysis. Furthermore, a narrow transient channel that is shown to form when the wild-type Asn485 forms the NH..., interaction with FAD and that has been proposed to function as an access route of molecular oxygen, is not observed in either of the mutant structures, suggesting that the dynamics of the active site are altered. [source] | ||||||||||