Display of Microbial Glucose Dehydrogenase and Cholesterol Oxidase on the Yeast Cell Surface for the Detection of Blood Biochemical Parameters.

High levels of blood glucose are always associated with numerous complications including cholesterol abnormalities. Therefore, it is important to simultaneously monitor blood glucose and cholesterol levels in patients with diabetes during the management of chronic diseases. In this study, a glucose dehydrogenase from Aspergillus oryzae TI and a cholesterol oxidase from Chromobacterium sp. DS-1 were displayed on the surface of Saccharomyces cerevisiae, respectively, using the yeast surface display system at a high copy number. In addition, two whole-cell biosensors were constructed through the immobilization of the above yeast cells on electrodes, for electrochemical detection of glucose and cholesterol. The assay time was 8.5 s for the glucose biosensors and 30 s for the cholesterol biosensors. Under optimal conditions, the cholesterol biosensor exhibited a linear range from 2 to 6 mmol·L-1. The glucose biosensor responded efficiently to the presence of glucose at a concentration range of 20-600 mg·dL-1 (1.4-33.3 mmol·L-1) and showed excellent anti-xylose interference properties. Both biosensors exhibited good performance at room temperature and remained stable over a three-week storage period.

A mass spectrometry-based isotope-coded mass tag method to map thiol accessibility in biological systems.

Interactions at biological membranes are important for many cellular functions, but because of the dynamic nature of these interactions, traditional methods of structure investigation are limited. Here, we describe a method that utilizes thiol-maleimide chemistry to monitor the solvent-accessible surface of membrane-protein complexes and membrane dynamics in vitro in real time. This method, called the isotope-coded mass tag (ICMT) method, has been used previously to locate thiols in transmembrane peptides, to elucidate lipid flipping kinetics in bilayers, to determine the oxidation state of disulfide bonds, and to investigate the protein-lipid interface of the peripheral membrane protein cholesterol oxidase. The method requires only microgram quantities of protein or thiolated lipid, uses common laboratory equipment and routine instrumentation, and can be applied to many different systems to investigate macromolecular interactions in the presence or absence of biological membranes.

Development and evaluation of the internal-controlled real-time PCR assay for Rhodococcus equi detection in various clinical specimens.

Rhodococcus equi is the causative agent of rhodococcosis in horses, resulting in significant morbidity and mortality in foals. This bacterium has also been isolated from a variety of animals and is being increasingly reported as a cause of infection in humans, mainly in immunosuppressed individuals. Laboratory diagnostics of R. equi infections based only on conventional microbiological methods shows low accuracy and can lead to misidentification. The objective of the study was to develop and evaluate a real-time PCR assay for direct detection of R. equi in various clinical specimens, including tissue samples. The species-specific region of the gene encoding R. equi cholesterol oxidase, choE, was used as a qPCR-target. The diagnostic applicability of the assay was confirmed by testing various tissue specimens obtained from horses with clinical signs of rhodoccocal infection and swine submaxillary lymph nodes. The rate of R. equi detection in clinical specimens by the developed assay was higher in comparison to the culture method (90% vs. 60.0% of positive samples) and conventional PCR (90.0% vs. 20.0% of positive samples). In case of 13 samples that were negative in the culture-based method, R. equi was detected by the developed assay. Only in one case, it gave negative result for culture-positive sample. The assay may provide a simple and rapid tool to complement the classical methods of R. equi detection based on culture and phenotypic identification of isolates, as the performed evaluation indicated a high specificity and accuracy of the results.

Immobilization of cholesterol oxidase on magnetic fluorescent core-shell-structured nanoparticles.

The magnetic fluorescent core-shell structured nanoparticles, Fe3O4@SiO2(F)@meso-SiO2 nanoparticles, were prepared. Cholesterol oxidase (COD) was immobilized on their surface to form Fe3O4@SiO2(F)@meso-SiO2@COD nanoparticles. Optimal immobilization was achieved with 2.5% (v/v) APTES, 2.0% (v/v) GA, 10mg COD (in 15 mg carrier) and solution pH of 7.0. Fe3O4@SiO2(F)@meso-SiO2@COD nanoparticles showed maximal catalytic activity at pH7.0 and 50°C. The thermal, storage and operational stabilities of COD were improved greatly after its immobilization. After the incubation at 50°C for 5h, the nanoparticles and free COD retained 80% and 46% of its initial activity, respectively. After kept at 4°C for 30 days, the nanoparticles and free COD maintained 86% and 65% of initial activity, respectively. The nanoparticles retained 71% of its initial activity after 7 consecutive operations. Since Fe3O4@SiO2(F)@meso-SiO2@COD nanoparticles contained tris(2,2-bipyridyl)dichloro-ruthenium(II) hexahydrate (Ru(bpy)3Cl2) and were optical sensitive to oxygen in solution, it might be used as the sensing material and has the application potential in multi parameter fiber optic biosensor based on enzyme catalysis and oxygen consumption.

Bioanalytical device based on cholesterol oxidase-bonded SAM-modified electrodes.

A rapid, simple and reproducible two-step method for constructing cholesterol biosensors by covalently bonding cholesterol oxidase (ChOx) to a 3,3'-dithiodipropionic acid di(N-succinimidyl ester) (DTSP)-modified gold electrode is described. Exhaustive characterizations of both the immobilization process and the morphological properties of the resulting ChOx monolayer were performed via a quartz crystal microbalance (QCM) and atomic force microscopy (AFM) operated under liquid conditions, respectively. In addition, scanning electrochemical microscopy (SECM) measurements were performed in order to check that the immobilized enzyme retains its catalytic activity. The replacement of the natural electron acceptor (O(2)) in the enzymatic reaction with an artificial mediator, hydroxymethylferrocene (HMF), was also studied. Finally, cholesterol was amperometrically determined by measuring the hydrogen peroxide produced during the enzymatic reaction at +0.5 V. The optimized cholesterol biosensor exhibited a sensitivity of 54 nA mM(-1) and a detection limit of 22 microM.

Enzymic conversion of 3beta-hydroxy-5-ene-steroids and their sulfates to 3-oxo-4-ene-steroids for increasing sensitivity in LC-APCI-MS.

A method of increasing the sensitivity of 3beta-hydroxy-5-ene (Delta5)-steroids in liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) based on the structural conversion by cholesterol oxidase (ChO) was demonstrated. The Delta5-steroids were rapidly converted to their 3-oxo-4-ene (Delta4)-forms by the treatment with ChO and the obtained Delta4-forms provided 3-14-fold higher sensitivity compared to intact steroids in the positive-APCI-MS. This enzymic conversion method was also applied to the sulfated conjugates of Delta5-steroids after solvolysis. The method enabled the detection of trace levels of dehydroepiandrosterone and androstenediol 3-sulfate in human serum, which could not be detected by the usual LC-MS.

Cholesterol oxidase: sources, physical properties and analytical applications.

Since Flegg (H.M. Flegg, An investigation of the determination of serum cholesterol by an enzymatic method, Ann. Clin. Biochem. 10 (1973) 79-84) and Richmond (W. Richmond, The development of an enzymatic technique for the assay of cholesterol in biological fluids, Scand. J. clin. Lab. Invest. 29 (1972) 25; W. Richmond, Preparation and properties of a bacterial cholesterol oxidase from Nocardia sp. and its application to enzyme assay of total cholesterol in serum, Clinical Chemistry 19 (1973) 1350-1356) first illustrated the suitability of cholesterol oxidase (COD) for the analysis of serum cholesterol, COD has risen to become the most widely used enzyme in clinical laboratories with the exception of glucose oxidase (GOD). The use is widespread because assays incorporating the enzyme are extremely simple, specific, and highly sensitive and thus offer distinct advantages over the Liebermann-Burchard analytical methodologies which employ corrosive reagents and can be prone to unreliable results due to interfering substances such as bilirubin. Individuals can now readily determine their own serum cholesterol levels with a simple disposable test kit. This review discusses COD in some detail and includes the topics: (1) The variety of bacterial sources available; (2) The various extraction/purification protocols utilised in order to obtain protein of sufficient clarification (purity) for use in food/clinical analysis; (3) Significant differences in the properties of the individual enzymes; (4) Substrate specificities of the various enzymes; (5) Examples of biological assays which have employed cholesterol oxidase as an integral part of the analysis, and the various assay protocols; (6) New steroidal products of COD. This review is not a comprehensive description of published work, but is intended to provide an account of recent and current research, and should promote further interest in the application of enzymes to analytical selectivity.

Some enzymatic properties of cholesterol oxidase produced by Brevibacterium sp

In this study we determined some properties of cholesterol oxidase from a "Brevibacterium" strain isolated from buffalo's milk and identified the cholesterol degradation products by bacterial cell. A small fraction of the enzyme synthesized by cells cultured in liquid medium for 7 days was released into the medium whereas a larger fraction remained boud to the cell membrane. The extraction of this fraction was efficiently accomplished in 1 mM phosphate buffer, pH 7.0, containing 0.7(per cent) Triton X-100. The enzyme stability under freezing and at 45ºC was imrproved by addition of 20(per cent) glycerol. The optimum tempereature and pH for the enzyme activity were 53ºC 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 enzyme in the buffer solution. Cholesterol oxidation by the membrane-bound enzyme was a first order reaction type

Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide.

A sensitive peroxyoxalate chemiluminescent (PO-CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4'-oxalyl- bis[(trifluoromethyl-sulphonyl)imino]trimethylene-bis(4- methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4- d]pyrimidine as fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 x 10(-7)-1 x 10(-4) mol/L. Relative standard deviations for oxidase assays were 5.1-12.7% (n = 10). Detection limits were 1 x 10(-3) U/mL for uricase, 5 x 10(-4) U/mL for choline oxidase, 5 x 10(-3) U/mL for cholesterol oxidase and 5 x 10(-4) U/mL xanthine oxidase (sample to blank ratio, 3).

Combination of an enzymatic method and HPLC for the quantitation of cholesterol in cultured cells.

The study of the cellular events that lead to the foam cell formation requires the development of fast, accurate, and sensitive methods to quantify cholesterol in cultured cells. Here we describe a procedure that allows the rapid determination of free and total cholesterol in a reduced number of cells, which makes it very suitable for cholesterol determination in cell cultures. The method consists of the enzymatic conversion of cholesterol to cholest-4-ene-3-one by cholesterol oxidase followed by the analysis of the sample by high performance liquid chromatography (HPLC) to detect this oxidized product. Due to the relatively high wavelength at which cholest-4-ene-3-one has its maximum absorption (240 nm), other cellular components do not interfere with the chromatographic procedure and prior lipid extraction is not required. Moreover, the duration of each chromatogram is about 3 min, contributing to the celerity of the method. All the cholesteryl esters used (oleate, palmitate, stearate and linoleate) were quantitatively hydrolyzed by incubation with cholesterol esterase; this was observed to occur with both pure standards and in cell homogenates. Sensitivity is enough to allow the determination of free and total cholesterol in less than 5 x 10(3) cells. We have applied this method to human monocyte-derived macrophages and the values obtained for free and total cholesterol are in close agreement with published data.

A colorimetric assay for measuring cell-free and cell-bound cholesterol oxidase.

Cholesterol oxidase (cholesterol:oxygen oxidoreductase, EC 1.1.3.6) catalyzes the conversion of sterol delta 5-3 beta-alcohol to the corresponding delta 4-3-ketone with the reduction of oxygen to hydrogen peroxide. Rhodococcus species GK 1, a soil isolated microbe, produces an extracellular and a membrane-bound cholesterol oxidase; the latter is bound to the outer surface of the microbial cell membrane. A simple and sensitive assay is described to measure the two enzyme types; no enzyme extraction is needed for measuring the membrane-bound cholesterol oxidase. In this assay, hydrogen peroxide is reduced by the chromogen 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in the presence of horseradish peroxidase, and the increased absorbance is followed continuously at 600 nm (epsilon m = 1.82 x 10(4) M-1.cm-1 at pH 7.0 and 30 degrees C). The standardized assay medium contained 46.9 mM sodium-potassium phosphate buffer pH 7.0, 0.16% Triton X-100, 312.5 microM ABTS, 50 micrograms peroxidase (12.5 units at 25 degrees C), 6.25% isopropanol, 306.3 microM cholesterol or other sterols (kept in solution with isopropanol), and cholesterol oxidase. Oxidation of one molecule of cholesterol by cholesterol oxidase gives one molecule of hydrogen peroxide which reacts with two molecules of ABTS. The method is reproducible and the results correlate well with those obtained by measuring the absorbance of delta 4-cholest-3-one at 240 nm (epsilon m = 1.40 x 10(4) M-1.cm-1 at pH 7.0 and 30 degrees C) and by the method of Allain et al. (Clin. Chem. 20, 470-475, 1974).(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol pools in rat adrenal mitochondria: use of cholesterol oxidase to infer a complex pool structure.

ACTH stimulates the side-chain cleavage of cholesterol in the adrenal cortex in a cycloheximide-inhibitable manner. Its mechanism involves mobilizing cholesterol to a "steroidogenic pool" where the sterol can be metabolized to pregnenolone. This pool has been proposed to be in the inner mitochondrial membrane where cytochrome P-450scc resides, and regulation may involve transport of cholesterol from the outer to the inner membrane. To investigate the structure of the mitochondrial cholesterol pools, cholesterol oxidase has been used as a membrane-impermeant probe which should have selective access to outer membrane cholesterol. At 37 C, almost all the cholesterol in mitochondria from ether-stressed rats was metabolized by cholesterol oxidase. Depletion of an intermembrane space but not a matrix marker enzyme indicated partial disruption of the outer membrane. However, at 16 C, mitochondria remained largely intact, and cholesterol oxidase identified a unique pool of cholesterol, which was about two-thirds of the total. In experiments using mitochondria from ether-stressed rats, the size of the 16 C cholesterol oxidase accessible and inaccessible pools was compared with that of the steroidogenic pool. The steroidogenic pool was enhanced by pretreatment of some animals with aminoglutethimide (a P-450scc inhibitor) or eliminated with cycloheximide, both of which increased the total mitochondrial cholesterol. This approach reveals that the steroidogenic pool is not equivalent to the cholesterol oxidase-inaccessible pool. Rather, it overlaps both the cholesterol oxidase accessible and inaccessible pools. These results are not consistent with a simple two pool model, but can be explained by assuming a minimum of three cholesterol pools.

Cholesterol oxidase as a structural probe of biological membranes: its application to brush-border membrane.

Cholesterol present in intact brush-border membrane vesicles made from rabbit small intestine is a poor substrate for cholesterol oxidase (EC 1.1.3.6, from Nocardia sp. and Nocardia erythropolis). It becomes susceptible to oxidation by the enzyme only after the addition of detergent, e.g., Triton X-100, in quantities sufficient to disrupt the membrane. This is also true for cholesterol present in bilayers of small unilamellar phosphatidylcholine or phosphatidylserine vesicles made by ultrasonication. The data presented here on intestinal brush-border membrane are in good agreement with results reported on other biological membranes, e.g., from erythrocytes and vesicular stomatitis virus, but are somewhat different from those on rat intestinal brush-border membrane. Our results on phospholipid bilayers agree well with published work on model membranes. From the work presented we conclude that, with our present understanding, cholesterol oxidase can hardly be used to probe the distribution of cholesterol in biological membranes. A prerequisite for using the enzyme successfully as such a probe would be the understanding of the factors controlling the interaction of the enzyme with its substrate cholesterol. The question under which conditions cholesterol oxidase could be useful for probing the distribution and preferred location of cholesterol in biological membranes is discussed.

Colonic bacterial flora and serum cholesterol: alterations induced by dietary citrus pectin.

The influence of dietary pectin on blood lipids and stool bacterial flora, enzyme activity, and physical properties was investigated. Fifteen grams of citrus pectin were added to the normal diets of 10 subjects. A decrease in serum cholesterol was observed after 2 wk of dietary supplementation, but was not statistically significant after the 3rd wk. Fecal bacterial flora was modified by pectin; anaerobic bacteria increased and aerobic bacteria increased. No change in stool bacterial 7 alpha dehydroxylase or cholesterol dehydrogenase activity was observed. The daily stool output, pH, and water content were not influenced by the addition of pectin to the diet.

[Determination of HDL-cholesterol]. / Zur Bestimmung des HDL-Cholesterols.

For the clinical practice methods of the determination of HDL-cholesterol made their way which are based on the precipitation of apolipoprotein-B-containing lipoproteins and a determination of cholesterol following. The expensive methods of the ultracentrifugation serve as reference methods. The most-spread precipitation techniques (heparin/MCl2, dextran sulphate/CaCl2 or MgCl2 photungstic acid/MgCl2) are comparatively observed with regard to their effectiveness, practicability and methodical and technical conditions (influence of the concentration of the precipitation reagents, pH-value, temperature, incubation and centrifugation conditions). Results of own investigations as well as data from literature are presented to the problem of the harmonization of the cholesterol determination with the precipitation technique. According to the opinion of the authors for the enzymatic determination of cholesterol by means of the CHOD-PAP-method the phosphotungstic acid precipitation well stood the test, whereas for the chemical determination of cholesterol after Liebermann-Burchard in manual or automatized works the precipitation by means of dextran sulphate/CaCl2 (40 g/l, 2.0 mol/l) is to be recommended. The superabundant precipitations with phosphotungstic acid and dextran sulphate/MgCl2 (20 g/l, 2.0 mol/l) achieve higher results in Liebermann-Burchard's reaction likely on account of interferences.