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Production, purification and characterization of cholesterol oxidase from a newly isolated Streptomyces sp.

World Journal of Microbiology and Biotechnology volume 29, pages 2077–2085 (2013)Cite this article

Abstract

Cholesterol oxidase production (COD) by a new isolate characterized as Streptomyces sp. was studied in different production media and fermentation conditions. Individual supplementation of 1 % maltose, lactose, sucrose, peptone, soybean meal and yeast extract enhanced COD production by 80–110 % in comparison to the basal production medium (2.4 U/ml). Supplementation of 0.05 % cholesterol (inducer) enhanced COD production by 150 %. COD was purified 14.3-fold and its molecular weight was found to be 62 kDa. Vmax (21.93 μM/min mg) and substrate affinity Km (101.3 μM) suggested high affinity of the COD for cholesterol. In presence of Ba2+ and Hg2+ the enzyme activity was inhibited while Cu2+ enhanced the activity nearly threefold. Relative activity of the enzyme was found maximum in triton X-100 whereas sodium dodecyl sulfate inactivated the enzyme. The enzyme activity was also inhibited by the thiol-reducing reagents like Dithiothreitol and β-mercaptoethanol. The COD showed moderate stability towards all organic solvents except acetone, benzene and chloroform. The activity increased in presence of isopropanol and ethanol. The enzyme was most active at pH 7 and 37 °C temperature. This organism is not reported to produce COD.

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Acknowledgments

This study was financially supported by Council of Scientific and Industrial Research, India. The authors would like to thank Mr. A.K. Joshi, Technical officer in F.T. Division CDRI Lucknow for his valuable help.

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Affiliations

  1. Division of Fermentation Technology, CSIR-Central Drug Research Institute, Chattar Manzil, PO Box 173, Lucknow, 226001, India

    Ram Niwas, Vineeta Singh, Rajbir Singh & C. K. M. Tripathi

  2. Division of Organic Chemistry, CSIR-National Chemical Laboratory, Pune, 411008, India

    Divya Tripathi

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  1. Ram Niwas
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  2. Vineeta Singh
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  3. Rajbir Singh
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  4. Divya Tripathi
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  5. C. K. M. Tripathi
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Correspondence to C. K. M. Tripathi.

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Niwas, R., Singh, V., Singh, R. et al. Production, purification and characterization of cholesterol oxidase from a newly isolated Streptomyces sp.. World J Microbiol Biotechnol 29, 2077–2085 (2013). https://doi.org/10.1007/s11274-013-1371-8

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  • DOI: https://doi.org/10.1007/s11274-013-1371-8

Keywords

  • Streptomyces sp.
  • Affinity chromatography
  • SDS PAGE
  • Cholesterol oxidase
  • HPLC