Cholesterol oxidase

1 Cholesterol Oxidase. - Cholesterol oxidase (3P-hydroxysteroid oxidase) catalyses the oxidation and isomerization of 3 P-hydroxysteroids having a double bond at A -A of the steroid ring backbone. The enzyme from the soil bacterium Brevibacterium sterlicum is a monomeric oxidase containing one FAD molecule per molecule of protein. Its crystal structure has been determined at 1.8 A 

The same authors have also investigated this system with three-pulse ESEEM. A small increase in a hfc assigned to N1 was also observed upon substrate binding, thus supporting the notion of an increase in electron density on the pyrimidine ring at the expense of the benzene part of isoalloxazine ring. 

Martinez and co-workers performed three- and four-pulse ESEEM and HY-SCORE on this species. Hfcs to N1 = 1.9 MHz), N3 = 0.7 MHz) andN10(Tiso = 11.7 MHz with = 2.7 MHz and Tj = 29.7 MHz) could also be determined. The authors did not attempt to say which was N1 or N3, giving N(A) and N(B) we have taken the liberty to assign them tentatively by comparison with our own DFT calculations.  

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Figure C1.5.17.(A) Enzymatic cycle of cholesterol oxidase, which catalyses tire oxidation of cholesterol by molecular oxygen. The enzyme s naturally fluorescent FAD active site is first reduced by a cholesterol substrate,...

Cholesterol The end point for the cholesterol reaction can be determined by following dye formation. Additionally, the amount of oxygen consumed can be measured amperometricaHy by an oxygen-sensing electrode (see Electro analytical techniques). The H2O2 produced by cholesterol oxidase requires phenol to produce dye. 

Parameter Cholesterol oxidase (CO) Cholesterol esterase (CE) Glucose oxidase (GOD) Peroxidase (POD)... 

Cholesterol Cholesterol esterase and cholesterol oxidase Medical care... 

Doukyu N, R Aono (1998) Purification of extracellular cholesterol oxidase with high activity in the presence of organic solvents from Pseudomonas sp. strain ST-200. Appl Environ Microbiol 64 1929-1932. 

Ohta T, K Eujishoro, K Yamaguchi, Y Tamura, K Aisaka, T Uwajima, M Hasegawa (1991) Sequence of gene choB encoding cholesterol oxidase of Brevibacterium sterolicum comparison with choA of Streptomyces sp. SA-COO. Gene 103 93-96. 

Chen, Y.-R., Huang, H.-H., Cheng, Y.-F. et al. (2006) Expression of a cholesterol oxidase gene from Arthrobacter simplex in Escherichia coli and Pichia pastoris. Enzyme and Microbial Technology, 39, 854-860. 

A new cholesterol flow injection analysis biosensor has also been described as an application of the H2O2 ECL sensor56. In that work, the luminol electrochemiluminescence, previously studied in aqueous media, was implemented in Veronal buffer added of 0.3% triton X-100 (v/v), 0.3% PEG and 0.4% cholate to enable the solubilisation of the cholesterol and then its efficient oxidation catalyzed by the immobilized cholesterol oxidase. The ECL reaction occurred thus in a micellar medium and the performances of the H2O2 ECL sensor were investigated. 

J.P. Li, T.Z. Peng, and Y.Q. Peng, A cholesterol biosensor based on entrapment of cholesterol oxidase in a silicic sol-gel matrix at a Prussian blue modified electrode. Electroanalysis 15, 1031—1037 (2003). 

A.L. Crumbliss, J.Z. Stonehuerner, R.W. Henkens, J. Zhao, and J.P. O Daly, A carrageenan hydrogel stabilized colloidal gold multi-enzyme biosensor electrode utilizing immobilized horseradish peroxidase and cholesterol oxidase/cholesterol esterase to detect cholesterol in serum and whole blood. Biosens. Bioelectron. 8, 331-337 (1993). 

Chang YC, Chou CC (2002) Growth and production of cholesterol oxidase by alginate-immobilized cells of Rhodococcus equi No 23. Biotechnol Appl Biochem 35 69-74... 

Glucose, cholesterol Immobilized glucose oxidase or cholesterol oxidase to H202, luminol CL Not reported 43... 

The enzyme cholesterol oxidase is an effective oxidation catalyst when used in a micellular system involving scCC>2 and water [25], The use of a perfluoropoly-ether-based surfactant causes the formation of reverse-micelles in which the... 

Phospholipid concentration was determined using our modification of Bartlett s procedure (49,53). Cholesterol concentration and purity were determined by HPLC or enzymatically by cholesterol oxidase (49,53). Purity of phospholipids as raw materials, and the extent of their hydrolysis during various steps of liposome preparation and liposome storage, were assessed by TLC and enzymatic determination of the increase in level of nonesterified fatty acids (10,38,49-51,53). 

LPDS, LDL (100 tg/mL), and compound 58-035 (2 tg/mL) at 37°C. At various times, cells were harvested, treated with cholesterol oxidase, and lipid extracts analyzed by HPLC. 

Bioelectrocatalysis involves the coupling of redox enzymes with electrochemical reactions [44]. Thus, oxidizing enzymes can be incorporated into redox systems applied in bioreactors, biosensors and biofuel cells. While biosensors and enzyme electrodes are not synthetic systems, they are, essentially, biocatalytic in nature (Scheme 3.5) and are therefore worthy of mention here. Oxidases are frequently used as the biological agent in biosensors, in combinations designed to detect specific target molecules. Enzyme electrodes are possibly one of the more common applications of oxidase biocatalysts. Enzymes such as glucose oxidase or cholesterol oxidase can be combined with a peroxidase such as horseradish peroxidase. 

An experimental set-up such as that depicted in Fig. 5.12.C was also used by Nieman s group for the determination of free and total cholesterol in serum [37]. A solution of the enzyme (cholesterol oxidase) at pH 7 was forced through a microporous membrane. The pH gradient across the membrane (from 7 to 9) facilitated the enzymatic degradation of the analyte and subsequent diffusion of the hydrogen peroxide formed to the PMT in the reagent/sample stream in order to react with luminol in the presence of horseradish peroxidase as the catalyst at pH 9 —the copper chelate commonly used as catalyst for this purpose requires pH 11. The system per-... 

The oxidation reactions of luminol and lucigenin can be used to assay for H Oj. For example, analysis of glucose in biological systems can be achieved using a three-enzyme system of mutarotase, glucose oxidase and horseradish peroxidase by correlation with the amount of HjOj released. Similarly, cholesterol can be measured using cholesterol oxidase. The fact that the rate of luminol oxidation depends on the concentration of the catalyst can be used as a method for determination of Co +, Fe +, Cr + and Mn + and other catalysts.Some examples of the use of luminol, isolumi-nol and their derivatives in immunoassays are shown in Table 3.11. ... 

Cholesterol oxidase Streptomyces iavenduiae NCIM 2421 (Varma and Nene, 2003)... 

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