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Cholesterol Assay Based on Recombinant Cholesterol Oxidase, ABTS, and Horseradish Peroxidase

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Microbial Steroids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2704))

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Abstract

Cholesterol determination by cholesterol oxidase reaction is a fast, convenient, and highly specific approach with widespread use in clinical diagnostics. Routinely, endpoint measurements with 4-aminophenazone or 4-aminoantipyrine as chromogens and sodium cholate, surfactants, or alcohols as solubilizing agents are used. Here we describe a novel kinetic method to determine cholesterol in 0.05–0.75 mM range in neutral or acidic buffers by use of recombinant cholesterol oxidase from Nocardioides simplex in a coupled reaction with horseradish peroxidase, ABTS as a chromogen, and methyl-β-cyclodextrin as a solubilizing agent.

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References

  1. Owen DM (2015) Cholesterol depletion using methyl-β-cyclodextrin. Chapter 8. In: Methods in membrane lipids. Humana Press, New York, p 91

    Chapter  Google Scholar 

  2. Warnick GR, Remaley AT (2001) Measurement of cholesterol in plasma and other body fluids. Curr Atheroscler Rep 3:404–411

    Article  CAS  PubMed  Google Scholar 

  3. Srisawasdi P, Jearanaikoon P, Kroll MH, Lolekha PH (2005) Performance characteristics of cholesterol oxidase for kinetic determination of total cholesterol. J Clin Lab Anal 19:247–252

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kumari L, Kanwar SS (2012) Cholesterol oxidase and its applications. Adv Microbiol 2:49–65

    Article  Google Scholar 

  5. Richmond W (1973) Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum. Clin Chem 19:1350–1356

    Article  CAS  PubMed  Google Scholar 

  6. Pesce MA, Bodourian SH (1976) Enzymatic rate method for measuring cholesterol in serum. Clin Chem 22:2042–2045

    CAS  PubMed  Google Scholar 

  7. Shao M, Rao Z, Zhang X, Xu M, Yang T, Li H et al (2014) Bioconversion of cholesterol to 4-cholesten-3-one by recombinant Bacillus subtilis expressing choM gene encoding cholesterol oxidase from Mycobacterium neoaurum JC-12. J Chem Technol Biotechnol 90:1811–1920

    Article  Google Scholar 

  8. Ye D, Lei J, Li W, Ge F, Wu K, Xu W et al (2008) Purification and characterization of extracellular cholesterol oxidase from Enterobacter sp. World J Microbiol Biotechnol 24:2227–2233

    Article  CAS  Google Scholar 

  9. Li B, Wang W, Wang F-Q, Wei D-Z (2010) Cholesterol oxidase ChoL is a critical enzyme that catalyzes the conversion of diosgenin to 4-ene-3-keto steroids in Streptomyces virginiae IBL-14. Appl Microbiol Biotechnol 85:1831–1838

    Article  CAS  PubMed  Google Scholar 

  10. Kasabe PJ, Mali GT, Dandgeet PB (2015) Assessment of alkaline cholesterol oxidase purified from Rhodococcus sp. PKPD-CL for its halo tolerance, detergent and organic solvent stability. Protein Expr Purif 116:30–41

    Article  CAS  PubMed  Google Scholar 

  11. Doukyu N, Nihei S (2015) Cholesterol oxidase with high catalytic activity from Pseudomonas aeruginosa: screening, molecular genetic analysis, expression and characterization. J Biosci Bioeng 120:24–30

    Article  CAS  PubMed  Google Scholar 

  12. Qin HM, Zhu Z, Ma Z, Xu P, Guo Q, Li A et al (2017) Rational design of cholesterol oxidase for efficient bioresolution of cholestane skeleton substrates. Sci Rep 7:16375

    Article  PubMed  PubMed Central  Google Scholar 

  13. Ufot UF, Akpanabiatu MI, Cali K, Uffia ID, Udosen I (2015) pH-dependence of manganese (II) oxidation reaction by novel wild-type and mutants recombinant Phlebia radiata manganese peroxidase 3 (rPr-MnP3) enzymes. Am J Mol Biol 12:67–84

    Article  Google Scholar 

  14. Scott SL, Chen W-J, Bakac A, Espenson JH (1993) Spectroscopic parameters, electrode potentials, acid ionization constants, and electron exchange rates of the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) radicals and ions. J Phys Chem 97:6710–6714

    Article  CAS  Google Scholar 

  15. Childs RE, Bardsley WG (1975) The steady-state kinetics of peroxidase with 2,2′-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid) as chromogen. Biochem J 145:93–103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kadnikova EN, Kostić NM (2002) Oxidation of ABTS by hydrogen peroxide catalyzed by horseradish peroxidase encapsulated into sol–gel glass. Effects of glass matrix on reactivity. J Mol Catal B Enzym 18:39–48

    Article  CAS  Google Scholar 

  17. An H, Xiao T, Fan H, Wei D (2015) Molecular characterization of a novel thermostable laccase PPLCC2 from the brown rot fungus Postia placenta MAD-698-R. Electron J Biotechnol 18:451–458

    Article  Google Scholar 

  18. Alshawafi WM, Aldhahri M, Almulaiky YQ, Salah N, Moselhy SS, Ibrahim IH et al (2018) Immobilization of horseradish peroxidase on PMMA nanofibers incorporated with nanodiamond. Artif Cells Nanomed Biotechnol 46:973–981

    Article  Google Scholar 

  19. Hou C, Ghéczy N, Messmer D, Szymańska K, Adamcik J, Mezzenga R et al (2019) Stable immobilization of enzymes in a macro- and mesoporous silica monolith. ACS Omega 4:7795–7806

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Herzog PL, Sützl L, Eisenhut B, Maresch D, Haltrich D, Obinger C et al (2019) Versatile oxidase and dehydrogenase activities of bacterial pyranose 2-oxidase facilitate redox cycling with manganese peroxidase in vitro. Appl Environ Microbiol 85:e00390–e00319

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. López CA, de Vries AH, Marrink SJ (2011) Molecular mechanism of cyclodextrin mediated cholesterol extraction. PLoS Comput Biol 7:e1002020

    Article  PubMed  PubMed Central  Google Scholar 

  22. Charlton SA, Coym JW (2012) The use of methyl-β-cyclodextrin to solubilize cholesterol prior to coating onto a C18 stationary phase. J Chromatogr A 1266:69–75

    Article  CAS  PubMed  Google Scholar 

  23. Britton HTS, Robinson RA (1931) Universal buffer solutions and the dissociation constant of veronal. J Chem Soc 0:1456–1462

    Article  CAS  Google Scholar 

  24. Huang Y, Lin J, Zou J, Xu J, Wang M, Cai H et al (2012) ABTS as an electron shuttle to accelerate the degradation of diclofenac with horseradish peroxidase-catalyzed hydrogen peroxide oxidation. Sci Total Environ 798:149276

    Article  Google Scholar 

  25. Ilyasov IR, Beloborodov VL, Selivanova IA, Terekhov RP (2020) ABTS/PP decolorization assay of antioxidant capacity reaction pathways. Int J Mol Sci 21:1131

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Aliaga C, Lissi EA (1998) Reaction of 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS) derived radicals with hydroperoxides. Kinetics and mechanism. Int J Chem Kinet 30:565–570

    Article  CAS  Google Scholar 

  27. Ferri M, Gianotti A, Tassoni A (2013) Optimisation of assay conditions for the determination of antioxidant capacity and polyphenols in cereal food components. J Food Compos Anal 30:94–101

    Article  CAS  Google Scholar 

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Acknowledgments

This work is supported by the Russian Science Foundation, project No. 21-64-00024.

Author information

Authors and Affiliations

  1. G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Federal Research Center “Pushchino Center for Biological Research of the Russian Academy of Sciences”, Pushchino, Russia

    Alexey V. Sviridov, Mikhail V. Karpov, Victoria V. Fokina & Marina V. Donova

Authors
  1. Alexey V. Sviridov
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  2. Mikhail V. Karpov
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  3. Victoria V. Fokina
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  4. Marina V. Donova
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Editor information

Editors and Affiliations

  1. Área de Bioquímica y Biología Molecular, Departamento de Biología Molecular, Facultad de Veterinaria, Universidad de León, León, Spain

    Carlos Barreiro

  2. Department of Biotechnology, Curia Spain, León, Spain

    José-Luis Barredo

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Sviridov, A.V., Karpov, M.V., Fokina, V.V., Donova, M.V. (2023). Cholesterol Assay Based on Recombinant Cholesterol Oxidase, ABTS, and Horseradish Peroxidase. In: Barreiro, C., Barredo, JL. (eds) Microbial Steroids. Methods in Molecular Biology, vol 2704. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3385-4_9

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  • DOI: https://doi.org/10.1007/978-1-0716-3385-4_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3384-7

  • Online ISBN: 978-1-0716-3385-4

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