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Purification and Characterization of an Extracellular Cholesterol Oxidase of Bacillus subtilis Isolated from Tiger Excreta

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Abstract

A mesophilic Bacillus sp. initially isolated from tiger excreta and later identified as a Bacillus subtilis strain was used to produce an extracellular cholesterol oxidase (COX) in cholesterol-enriched broth. This bacterial isolate was studied for the production of COX by manipulation of various physicochemical parameters. The extracellular COX was successfully purified from the cell-free culture broth of B. subtilis by successive salting out with ammonium sulfate, dialysis, and riboflavin-affinity chromatography. The purified COX was characterized for its molecular mass/structure and stability. The enzyme possessed some interesting properties such as high native Mr (105 kDa), multimeric (pentamer of ∼21 kDa protein) nature, organic solvent compatibility, and a half-life of ∼2 h at 37 °C. The bacterial COX exhibited ∼22 % higher activity in potassium phosphate buffer (pH 7.5) in the presence of a nonionic detergent Triton X-100 at 0.05 % (v/v). The K m and V max value of COX of B. subtilis COX were found to be 3.25 mM and 2.17 μmol min ml−1, respectively. The purified COX showed very little cytotoxicity associated with it.

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References

  1. Ahmad, S., & Goswami, P. (2012). Enhanced production of cell-bound cholesterol oxidase from Rhodococcus sp. NCIM 2891 by the statistical method. Annals of Microbiology, 63, 199–205.

    Article  Google Scholar 

  2. Bholay, A. D., Gadekar, D. J., Sawant, S. K., & Sonawane, S. M. (2013). Bacterial extracellular cholesterol oxidase and its pharmaceutical perspectives. International Journal of Current Microbiology and Applied Sciences, 2, 19–28.

    Google Scholar 

  3. Bradford, M. M. (1976). Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  4. Cheetham, S. J., Dunnill, P., & Lilly, D. (1982). The characterization and interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous. The Biochemical Journal, 201, 515–521.

    Article  CAS  Google Scholar 

  5. Chen, Y., Xin, Y., Yang, H., Zhang, L., Zhang, Y., Xia, X., Tong, Y., & Wang, W. (2013). Immobilization and stabilization of cholesterol oxidase on modified sepharose particles. International Journal of Biological Macromolecules, 8, 6–13.

    Article  Google Scholar 

  6. Doukyu, N., & Aono, R. (1998). Purification of extracellular cholesterol oxidase with high activity in the presence of organic solvents from Pseudomonas sp. strain ST-200. Applied and Environmental Microbiology, 64, 1929–1932.

    CAS  Google Scholar 

  7. Fukuyama, M., & Miyake, Y. (1979). Purification and properties of cholesterol oxidase from Schizophyllum with covalently bound flavin. Journal of Biochemistry, 85, 1183–1193.

    CAS  Google Scholar 

  8. Hao, L., Zhang, Q., Wang, C. L., Chen, M. H., Wang, Y. R., Li, F. J., Yang, H., & Wang, J. (2012). Optimal conditions for cholesterol oxidase production and enzymatic properties study produced by Monascus X-1. Applied Mechanics and Materials, 469, 195–196.

    Google Scholar 

  9. Inouye, Y., Taguchi, K., Fuji, A., Ishimaru, K., Nakamura, S., & Nomi, R. (1982). Purification and characterization of extracellular 3β-hydroxysteroid oxidase produced by Streptoverticillium cholesterolicum. Chemical and Pharmaceutical Bulletin, 30, 951–958.

    Article  CAS  Google Scholar 

  10. Ivashina, T. V., Nikolayeva, V. M., Dovbnya, D. V., & Donova, M. V. (2012). Cholesterol oxidase ChoD is not a critical enzyme accounting for oxidation of sterols to 3-keto-4-ene steroids in fast-growing Mycobacterium sp. VKMAc-1815D. The Journal of Steroid Biochemistry and Molecular Biology, 129, 47–53.

    Article  CAS  Google Scholar 

  11. Jayachitra, A., Krithiga, N., & Bavani C. (2012). Isolation, molecular characterisation and sequencing of cholesterol degrading bacteria. International Journal of Pharmacy & Life Sciences (IJPLS), 3, 1946–1952.

  12. Johnson, T. L., & Somkuti, G. A. (1991). Isolation of cholesterol oxidase from Rhodococcus equi ATCC 33706. Biotechnology and Applied Biochemistry, 13, 196–204.

    CAS  Google Scholar 

  13. Khmelnitsky, Y. L., Hilhorst, R., & Veeger, C. (1988). Detergentless microemulsion as media for enzymatic reactions. Cholesterol oxidation catalyzed by cholesterol oxidase. European Journal of Biochemistry, 176, 265–271.

    Article  CAS  Google Scholar 

  14. Kreit, J., Lefebvre, G., & Germain, P. (1994). Membrane-bound cholesterol oxidase from Rhodococcus sp. cells production and extraction. Journal of Biotechnology, 33, 271–282.

    Article  CAS  Google Scholar 

  15. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  16. Lee, S. Y., Rhee, H. I., Tae, W. C., Shin, J. C., & Park, B. K. (1989). Purification and characterization of cholesterol oxidase from Pseudomonas sp. and taxonomic study of the stain. Applied Microbiology and Biotechnology, 31, 542–546.

    Article  CAS  Google Scholar 

  17. Li, R., Dowd, V. D., Stewart, J., Burton, S. J., & Lowe, C. R. (1998). Design, synthesis and application of an artificial protein a. Nature Biotechnology, 16, 190–195.

    Article  CAS  Google Scholar 

  18. 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. Agricultural and Biological Chemistry, 52, 4130–4138.

    Article  Google Scholar 

  19. Liu, J., Xian, G., Li, M., Zhang, Y., Yang, M., Yu, Y., Lv, H., Xuan, S., Lin, Y., & Gao, L. (2014). Cholesterol oxidase from Bordetella species promotes irreversible cell apoptosis in lung adenocarcinoma by cholesterol oxidation. Cell Death & Disease, 5, e1372. doi:10.1038/cddis.2014.324.

    Article  CAS  Google Scholar 

  20. Mac Lachlan, J., Wotherspoon, A. T. L., Ansell, R. O., & Brooks, C. J. W. (2000). Cholesterol oxidase: sources, physical properties and analytical applications. The Journal of Steroid Biochemistry and Molecular Biology, 72, 169–195.

    Article  CAS  Google Scholar 

  21. Masurekar, P. S., & Goodhue, C. T. (1978). United States Patent No. 4,093,517, Eastman Kodak Company, Rochester, NY.

  22. Mitsuo, Y., Mitsutoshi, T., Hisaya, O., Isao, F., Noriaki, H., & Yoshikatsu, M. (1998). Separation of two reactions, oxidation and isomarization catalyzed by Streptomyces cholesterol oxidase. Protein Engineering, 11, 1075–1081.

    Article  Google Scholar 

  23. Mosmann, T. (1983). Separation of two reactions, oxidation and isomarization catalyzed by Streptomyces cholesterol oxidase. Journal of Immunological Methods, 65, 55–63.

    Article  CAS  Google Scholar 

  24. Niwas, R., Singh, V., Singh, R., Pant, G., Mitra, K., Kant, C., & Tripathi, M. (2014). Cholesterol oxidase production from entrapped cells of Streptomyces sp. Journal of Basic Microbiology, 54, 1–7.

    Article  Google Scholar 

  25. Petrova, Y., Podsukhina, G. M., Dikun, T. A., & Selezneva, A. A. (1979). Conditions of isolation of cholesterol oxidase from Actinomyces lavendulae mycelium. Applied Biochemistry and Microbiology, 15, 125–128.

    Google Scholar 

  26. Pollegioni, L., Piubelli, L., & Molla, G. (2009). Cholesterol oxidase: biotechnological applications. The FEBS Journal, 276, 6857–6870.

    Article  CAS  Google Scholar 

  27. Rhee, C. H., Kim, K. P., & Park, H. D. (2002). Two novel extracellular cholesterol oxidases of Bacillus sp. isolated from fermented flatfish. Biotechnology Letters, 24, 1385–1389.

    Article  CAS  Google Scholar 

  28. Salva, T. J. G., Alcina, M. L., Aloísia, L. M., Marco, A. T. Z., Gisleine, V., & Tobias, J. B. M. (1999). Some enzymatic properties of cholesterol oxidase produced by Brevibacterium sp. Revista de Microbiologia, 30, 315–323.

    Article  CAS  Google Scholar 

  29. Shirokane, Y., Nakamura, K., & Mizusawa, K. (1977). Purification and some properties of an extracellular 3b-hydroxysteroid oxidase produced by Corynebacterium cholesterolicum. Journal of Fermentation Technology, 55, 337–346.

    CAS  Google Scholar 

  30. Srisawasdi, P., Jearanaikoon, P., Kroll, M. H., & Lolekha, P. H. (2005). Performance characteristics of cholesterol oxidase for kinetic determination of total cholesterol. Journal of Clinical Laboratory Analysis, 19, 247–252.

    Article  CAS  Google Scholar 

  31. Tomioka, H., Kagawa, M., & Nakamura, S. (1976). Some enzymatic properties of 3b-hydroxysteroid oxidase produced by Streptomyces violascens. Journal of Biochemistry, 79, 903–905.

    CAS  Google Scholar 

  32. Turfitt, G. E. (1944). The microbiological degradation of steroids 2, oxidation of cholesterol by Proactinomyces sp. The Biochemical Journal, 38, 492–496.

    Article  CAS  Google Scholar 

  33. Uwajima, H., Yabi, H., & Terada, O. (1974). Properties of crystalline 3β-hydroxysteroid oxidase of Brevibacterium sterolicum. Agricultural and Biological Chemistry, 38, 1149–1156.

  34. Uwajima, T., & Terada, O. (1978). On the kinetics of cholesterol oxidase from Brevibacterium sterolicum in the presence of detergent. Agricultural and Biological Chemistry, 38, 1149–1156.

  35. Varma, R., & Nene, S. (2003). Biosynthesis of cholesterol oxidase by Streptomyces lavendulae NCIM 2421. Enzyme and Microbial Technology, 33, 286–291.

  36. Watanabe, K., Aihara, H., Nakagawa, Y., Nakamura, R., & Sasaki, T. (1989). Properties of the purified extracellular cholesterol oxidase from Rhodococus equi no 23. Journal of Agricultural and Food Chemistry, 37, 1178–1182.

    Article  CAS  Google Scholar 

  37. Yang, S., & Zhang, H. (2012). Optimization of cholesterol oxidase production by Brevibacterium sp. employing response surface methodology. African Journal of Biotechnology, 11, 8316–8322.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors are thankful to the University Grants Commission New Delhi, India, for financial assistance to the authors (vide an award letter F. No. F14-2(ST)/2007(SA-III).

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The authors have no conflict of interest at their place of work.

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  1. Department of Biotechnology, Himachal Pradesh University, Shimla, 171 005, India

    Lata Kumari & Shamsher S. Kanwar

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  1. Lata Kumari
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Correspondence to Shamsher S. Kanwar.

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Kumari, L., Kanwar, S.S. Purification and Characterization of an Extracellular Cholesterol Oxidase of Bacillus subtilis Isolated from Tiger Excreta. Appl Biochem Biotechnol 178, 353–367 (2016). https://doi.org/10.1007/s12010-015-1877-7

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