Abstract
A new screening method for 6β-hydroperoxycholest-4-en-3-one (HCEO)-forming cholesterol oxidase was devised in this study. As the result of the screening, a novel cholesterol oxidase producer (strain DS-1) was isolated and identified as Chromobacterium sp. Extracellular cholesterol oxidase of strain DS-1 was purified from the culture supernatant. The molecular mass of the purified enzyme was 58 kDa. This enzyme showed a visible adsorption spectrum having peaks at 355 and 450 nm, like a typical flavoprotein. The enzyme oxidized cholesterol to HCEO, with the consumption of 2 mol of O2 and the formation of 1 mol of H2O2 for every 1 mol of cholesterol oxidized. The enzyme oxidized 3β-hydroxysteroids such as cholesterol, β-cholestanol, and pregnenolone at high rates. The K m value for cholesterol was 26 μM. The enzyme was stable at pH 3 to 11 and most active at pH 7.0–7.5, showing optimal activity at pH 7.0 and 65°C. The enzyme retained about 80% of its activity after incubation for 30 min at 85°C. The thermal stability of the enzyme was the highest among the cholesterol oxidases tested. Moreover, the enzyme was more stable in the presence of various organic solvents and detergents than commercially available cholesterol oxidases.
Similar content being viewed by others
References
Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC (1974) Enzymatic determination of total serum cholesterol. Clin Chem 20:470–475
Aono R, Doukyu N (1996) Stereospecific oxidation of 3b-hydroxysteroids by persolvent fermentation with Pseudomonas sp. ST-200. Biosci Biotechnol Biochem 60:1146–1151
Aono R, Doukyu N, Kobayashi H, Nakajima H, Horikoshi K (1994) Oxidative bioconversion of cholesterol by Pseudomonas sp. strain ST-200 in a water-organic solvent two-phase system. Appl Environ Microbiol 60:2518–2523
Arya S, Datta M, Singh SP, Malhotra B (2007) Biosensor for total cholesterol estimation using N-(2-aminoethyl)-3-aminopropyltrimethoxysilane self-assembled monolayer. Anal Bioanal Chem 389:2235–2242
Barrow G, Feltham R (1993) Cowan and Steel’s manual for the identification of medical bacteria, 3rd edn. Cambridge University Press, New York
Basu A, Chattopadhyay P, Roychoudhuri U, Chakraborty R (2007) Development of cholesterol biosensor based on immobilized cholesterol esterase and cholesterol oxidase on oxygen electrode for the determination of total cholesterol in food samples. Bioelectrochemistry 70:375–379
Biellmann J (2001) Resolution of alcohols by cholesterol oxidase from Rhodococcus erythropolis: lack of enantiospecificity for the steroids. Chirality 13:34–39
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Brenner D, Krieg N, Staley J, Garrity G (2005) Bergey’s manual of systematic bacteriology. The Proteobacteria, part C: the Alpha-, Beta-, Delta-, and Epsilonproteobacteria, vol. 2. Springer, New York
Cheetham PSJ, Dunnill P, Lilly MD (1980) Extraction of cholesterol oxidase from Nocardia rhodochrous. Enzyme Microbiol Technol 2:201–205
Cho HJ, Choi KP, Yamashita M (1995) Introduction and expression of the Streptomyces cholesterol oxidase gene (choA), a potent insecticidal protein active against Boll weevil larvae, into tobacco cells. Appl Microbiol 44:133–138
Dang H, Davies A, Schiesser C (1990) Allylic hydroperoxides formed by singlet oxygenation of cholest-5-en-3-one. J Chem Soc Perkin Trans 1:789–794
Dieth S, Tritsch D, Biellmann J-F (1995) Resolution of allylic alcohols by cholesterol oxidase isolated from Rhodococcus erythropolis. Tetrahedron Lett 36:2243–2246
Doukyu N, Aono R (1998) Purification of extracellular cholesterol oxidase with high activity in the presence of organic solvents from Pseudomonas sp. ST-200. Appl Environ Microbiol 64:1929–1932
Doukyu N, Aono R (1999) Two moles of O2 consumption and one mole of H2O2 formation with cholesterol peroxidation by cholesterol oxidase from Pseudomonas sp. ST-200. Biochem J 341:621–627
Doukyu N, Aono R (2001) Cloning, sequence analysis and expression of a gene encoding an organic solvent- and detergent-tolerant cholesterol oxidase of Burkholderia cepacia strain ST-200. Appl Microbiol Biotechnol 57:146–152
Fujishiro K, Uchida H, Shimokawa K, Nakano M, Sano F, Ohta T, Kayahara N, Aisaka K, Uwajima T (2002) Purification and properties of a new Brevibacterium sterolicum cholesterol oxidase produced by E. coli MM294/pnH10. FEMS Microbiol Lett 215:243–248
Fukuyama M, Miyake Y (1979) Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin. J Biochem 85:1183–1193
Hino K, Nakamura M, Nakanishi K, Manabe M (1996) A new method for the homogeneous assay of serum HDL. Clinical Chem 42:296–299
Inouye Y, Tagnchi K, Fujii A, Ishimaru K, Nakamura S, Nomi R (1982) Purification and characterization of extracellular 3b-hydroxysteroid oxidase produced by Streptoverticillium cholesterolieum. Chem Pharm Bull 30:951–958
Isobe K, Shoji K, Nakanishi Y, Yokoe M, Wakao N (2003) Purification and some properties of cholesterol oxidase stable in detergents from gamma-proteobacterium Y-134. J Biosci Bioeng 95:257–263
Johnson TL, Somkuti GA (1991) Isolation of cholesterol oxidase from Rhodococcus equi ATCC33706. Biotechnol Appl Biochem 13:196–204
Kamei T, Takiguchi Y, Suzuki H, Matsuzaki M, Nakamura S (1978) Purification of 3b-hydroxysteroid oxidase of Streptomyces violascens origin by affinity chromatography on cholesterol. Chem Pharm Bull 26:2799–2804
Kazandjian RZ, Durdich JS, Kilbanov AM (1986) Enzymatic analysis in organic solvents. Biotechnol Bioeng 28:417–421
Kreit J, Lefebvre G, Germain P (1994) Membrane-bound cholesterol oxidase from Rhodococcus sp. cells. Production and extraction. J Biotechnol 33:271–282
Laane C, Boeren S, Vos K, Veegar C (1987) Rules for optimization of biocatalysis in organic solvents. Biotechnol Bioeng 30:81–87
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 224:680–685
Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR (1985) Rapid determination of 16S ribosomal RNA sequences for phylogenetic analysis. Proc Natl Acad Sci USA 82:6955–6959
Lee S, Rhee H, Tae W, Shin J, Park B (1989) Purification and characterization of cholesterol oxidase from Pseudomonas sp. and taxonomic study of the strain. Appl Microbiol Biotechnol 31:542–546
Leo A (1993) Calculating log Poct from structures. Chem Rev 93:1281–1306
Liu W-H, Meng M-H, Chen K-S (1988) Purification and some properties of cholesterol oxidases produced by an inducible and a constitutive mutant of Arthrobacter simplex. Agric Biol Chem 52:413–418
Martin PA, Gundersen-Rindal D, Blackburn M, Buyer J (2007) Chromobacterium subtsugae sp. Nov., a betaproteobacterium toxic to Colorado potato beetle and other insect pests. Int J Syst Evol Microbiol 57:993–999
Noma A, Nakayama K (1976) Comparative studies on cholesterol oxidases from different sources. Clin Chim Acta 73:487–496
Okada M, Matsui H, Ito Y, Fujiwara A, Inano K (1998) Low-density lipoprotein cholesterol can be chemically measured: a new superior method. J Lab Clin Med 132:195–201
Purcell JP, Greenplate JT, Jennings MG (1993) Cholesterol oxidase: a potent insecticidal protein active against Boll weevil larvae. Biochem Biophys Res Commun 196:1406–1413
Richmond W (1973) Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of cholesterol in serum. Clin Chem 19:1350–1356
Shirai K, Nema T, Hiroh Y, Itoh Y, Miyashita Y, Watanabe H (1997) Clinical efficacy of the direct assay method using polymers for serum high density lipoprotein cholesterol. J Clin Lab Anal 11:82–86
Shirokane Y, Nakamura K, Mizusawa K (1977) Purification and some properties of an extracellular 3-hydroxysteroid oxidase produced by Corynebacterium cholesterolicum. J Ferment Technol 55:337–345
Smith AJ, Brooks CJW (1974) Application of cholesterol oxidase in the analysis of steroids. J Chromatogr 101:373–378
Srisawasdi P, Chaichanajarernkul U, Teerakranjana N, Kroll M (2008) Implementation of cellulomonas cholesterol oxidase for total serum cholesterol determination by the endpoint method. J Clin Lab Anal 22:50–58
Teng J, Smith L (1996) Sterol peroxidation by Pseudomonas fluorescens cholesterol oxidase. Steroids 61:627–633
Tomioka H, Kagawa M, Nakamura S (1976) Some enzymatic properties of 3b-hydroxysteroid oxidase produced by Streptomyces violascens. J Biochem 79:903–915
Uwajima T, Yagi H, Terada O (1974) Properties of crystalline 3b-hydroxysteroid oxidase of Brevibacterium sterolicum. Agric Biol Chem 38:1149–1156
Vidal J, Espuelas J, Castillo J (2004) Amperometric cholesterol biosensor based on in situ reconstituted cholesterol oxidase on an immobilized monolayer of flavin adenine dinucleotide cofactor. Anal Biochem 333:88–98
Yazdi M, Zahraei M, Aghaepour K, Kamranpour N (2001) Purification and partial characterization of a cholesterol oxidase from Streptomyces fradiae. Enzyme Microb Technol 8:410–414
Acknowledgments
This work was supported in part by the Industrial Technology Research Grant Program in 2005 from New Energy and Industrial Technology Development Organization (NEDO) of Japan, the INOUE ENRYO Memorial Foundation for the Promotion of Sciences, and the Grant for the High Tech Research Center Program organized by Ministry of Education, Culture, Sports, Science and Technology of Japan since 2006. We thank Hiroyuki Ando, Hiromi Kaneko, and Kaori Yoshioka for their technical support.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Doukyu, N., Shibata, K., Ogino, H. et al. Purification and characterization of Chromobacterium sp. DS-1 cholesterol oxidase with thermal, organic solvent, and detergent tolerance. Appl Microbiol Biotechnol 80, 59–70 (2008). https://doi.org/10.1007/s00253-008-1526-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-008-1526-y