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Cloning, sequence analysis, and expression of a gene encoding Chromobacterium sp. DS-1 cholesterol oxidase

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Abstract

Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase. The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid residues. The amino acid sequence of the product showed significant similarity (53–62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1. K m and V max values of the cholesterol oxidase were estimated from Lineweaver–Burk plots. The V max/K m ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures.

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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 the Ministry of Education, Culture, Sports, Science and Technology of Japan, since 2006. We thank Hiromi Kaneko and Kaori Yoshioka for their technical support. We thank Naoki Kajiyama, Kouzou Hirokawa, and Kazuki Shiga (Kikkoman Corp.) for their kind support.

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Authors and Affiliations

  1. Bio-Nano Electronic Research Center, Toyo University, 2100, Kujirai, Kawagoe, Saitama, 350-8585, Japan

    Noriyuki Doukyu

  2. Graduate School of Interdisciplinary New Science, Toyo University, 2100, Kujirai, Kawagoe, Saitama, 350-8585, Japan

    Noriyuki Doukyu

  3. Department of Life Science, Toyo University, 1-1-1 Izumino, Itakura-machi, Gunma, 374-0193, Japan

    Noriyuki Doukyu & Kanpei Shibata

  4. Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka, 599-8531, Japan

    Hiroyasu Ogino

  5. Department of Chemistry and Biochemistry and Program for BioMolecular Science and Engineering, University of California, Santa Barbara, Santa Barbara, CA, 93106, USA

    Martin Sagermann

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  1. Noriyuki Doukyu
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  2. Kanpei Shibata
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  3. Hiroyasu Ogino
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  4. Martin Sagermann
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Correspondence to Noriyuki Doukyu.

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Doukyu, N., Shibata, K., Ogino, H. et al. Cloning, sequence analysis, and expression of a gene encoding Chromobacterium sp. DS-1 cholesterol oxidase. Appl Microbiol Biotechnol 82, 479–490 (2009). https://doi.org/10.1007/s00253-008-1775-9

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  • DOI: https://doi.org/10.1007/s00253-008-1775-9

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