Kinetic study of cholesterol oxidation by cholesterol oxidase enzyme as application for cholesterol biosensor

Author(s):  
Meka Saima Perdani ◽  
Muhamad Sahlan ◽  
Siti Farida ◽  
Dwini Normayulisa Putri ◽  
Sri Angky Soekanto ◽  
...  
2013 ◽  
Vol 11 (9) ◽  
pp. 1554-1561 ◽  
Author(s):  
Georgia-Paraskevi Nikoleli ◽  
Zafar Ibupoto ◽  
Dimitrios Nikolelis ◽  
Vlassis Likodimos ◽  
Nikolas Psaroudakis ◽  
...  

AbstractAbstract A novel potentiometric cholesterol biosensor has been fabricated through the immobilization of the stabilized polymeric lipid membrane onto graphene electrode. The stabilized polymeric lipid membrane is composed of cholesterol oxidase enzyme and polymerization mixture; which holds paramount influence on the properties of the cholesterol biosensor. The presented biosensor reveals an appreciable reproducibility, good selectivity and high sensing capability with a linear slope curve of ∼64 mV per decade. The strong biocompatibility among stabilized polymeric lipid membranes and human biofluids provides the possibility to use for real blood samples and other biological applications. Graphical abstract


2010 ◽  
Vol 10 (1) ◽  
pp. 96-101
Author(s):  
Iip Izul Falah ◽  
Ritmaleni Ritmaleni ◽  
M. Utoro Yahya

In view of health, cholesterol is believed as one of many sources can raise several diseases. Hence, both of research in quantification and developing simple, rapid and accurate analysis method of cholesterol in a sample is very important. Aim of this research was to investigate cholesterol oxidation kinetics and its quantification method based on oxidation of cholesterol and formation complex compound of hexathiocyanato ferat(III), {Fe(SCN)6}-3. The kinetics analysis and quantification, involved cholesterol oxidation in 0.1 M and pH 7.0 phosphate buffer solution to produce cholest-4-en-3-one and hydrogen peroxide, in the presence of cholesterol oxidase enzyme. The formed hydrogen peroxide was used to oxidize iron(II) ion, which was reacted furthermore with thiocyanate ion to raise the red-brown complex compound. Results of the study showed that the complex was stable at 10-120 min since the reaction was started, with maximum wavelength of 530-540 nm. While the kinetics analysis gave first order oxidation reaction with a reaction rate constant, kapp = 5.22 x 10-2 min-1. Based on this kinetics data, cholesterol analysis method could be developed i.e. by oxidizing cholesterol within 1.5 h using cholesterol oxidase as a catalyst, and then reacted with Fe2+, in a solution containing thiocyanate ion. Absorbencies of solutions of the complex compound, measured at wavelength of 535 nm, were linearly proportional to their cholesterol concentrations, in the range of 50-450 ppm.   Keywords: cholesterol, quantification, kinetics, hexathiocyanato ferat(III)


Sensors ◽  
2018 ◽  
Vol 18 (10) ◽  
pp. 3368 ◽  
Author(s):  
Kefilwe Mokwebo ◽  
Oluwatobi Oluwafemi ◽  
Omotayo Arotiba

We report the preparation of poly (propylene imine) dendrimer (PPI) and CdTe/CdSe/ZnSe quantum dots (QDs) as a suitable platform for the development of an enzyme-based electrochemical cholesterol biosensor with enhanced analytical performance. The mercaptopropionic acid (MPA)-capped CdTe/CdSe/ZnSe QDs was synthesized in an aqueous phase and characterized using photoluminescence (PL) spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), X-ray power diffraction (XRD), energy dispersive X-ray (EDX) spectroscopy. The absorption and emission maxima of the QDs red shifted as the reaction time and shell growth increased, indicating the formation of CdTe/CdSe/ZnSe QDs. PPI was electrodeposited on a glassy carbon electrode followed by the deposition (by deep coating) attachment of the QDs onto the PPI dendrimer modified electrode using 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) as a coupling agent. The biosensor was prepared by incubating the PPI/QDs modified electrode into a solution of cholesterol oxidase (ChOx) for 6 h. The modified electrodes were characterized by voltammetry and impedance spectroscopy. Since efficient electron transfer process between the enzyme cholesterol oxidase (ChOx) and the PPI/QDs-modified electrode was achieved, the cholesterol biosensor (GCE/PPI/QDs/ChOx) was able to detect cholesterol in the range 0.1–10 mM with a detection limit (LOD) of 0.075 mM and sensitivity of 111.16 μA mM−1 cm−2. The biosensor was stable for over a month and had greater selectivity towards the cholesterol molecule.


2021 ◽  
Vol 9 (12) ◽  
pp. 2554
Author(s):  
Tatyana Lobastova ◽  
Victoria Fokina ◽  
Sergey Tarlachkov ◽  
Andrey Shutov ◽  
Eugeny Bragin ◽  
...  

The application of thermophilic microorganisms opens new prospects in steroid biotechnology, but little is known to date on steroid catabolism by thermophilic strains. The thermophilic strain Saccharopolyspora hirsuta VKM Ac-666T has been shown to convert various steroids and to fully degrade cholesterol. Cholest-4-en-3-one, cholesta-1,4-dien-3-one, 26-hydroxycholest-4-en-3-one, 3-oxo-cholest-4-en-26-oic acid, 3-oxo-cholesta-1,4-dien-26-oic acid, 26-hydroxycholesterol, 3β-hydroxy-cholest-5-en-26-oic acid were identified as intermediates in cholesterol oxidation. The structures were confirmed by 1H and 13C-NMR analyses. Aliphatic side chain hydroxylation at C26 and the A-ring modification at C3, which are putatively catalyzed by cytochrome P450 monooxygenase CYP125 and cholesterol oxidase, respectively, occur simultaneously in the strain and are followed by cascade reactions of aliphatic sidechain degradation and steroid core destruction via the known 9(10)-seco-pathway. The genes putatively related to the sterol and bile acid degradation pathways form three major clusters in the S. hirsuta genome. The sets of the genes include the orthologs of those involved in steroid catabolism in Mycobacterium tuberculosis H37Rv and Rhodococcus jostii RHA1 and related actinobacteria. Bioinformatics analysis of 52 publicly available genomes of thermophilic bacteria revealed only seven candidate strains that possess the key genes related to the 9(10)-seco pathway of steroid degradation, thus demonstrating that the ability to degrade steroids is not widespread among thermophilic bacteria.


Author(s):  
Thabatta L. S. A. Rosa ◽  
Maria Angela M. Marques ◽  
Zachary DeBoard ◽  
Kelly Hutchins ◽  
Carlos Adriano A. Silva ◽  
...  

Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3β-hydroxysteroid dehydrogenase (3β-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3β-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3β-HSD activity with the 17β-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3β-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.


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