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Preparation and some properties of cholesterol oxidase from Rhodococcus sp. R14-2

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Abstract

Rhodococcus sp. R14-2, isolated from Chinese Jin-hua ham, produces a novel extracellular cholesterol oxidase (COX). The enzyme was extracted from fermentation broth and purified 53.1-fold based on specific activity. The purified enzyme shows a single polypeptide band on SDS-PAGE with an estimated molecular weight of about 60 kDa, and has a pI of 8.5. The first 10 amino acid residues of the NH2-terminal sequence of the enzyme are A-P-P-V-A-S-C-R-Y-C, which differs from other known COXs. The enzyme is stable over a rather wide pH range of 4.0–10.0. The optimum pH and temperature of the COX are pH 7.0 and 50°C, respectively. The COX rapidly oxidizes 3β-hydroxysteroids such as cholesterol and phytosterols, but is inert toward 3α-hydroxysteroids. Thus, the presence of a 3β-hydroxyl group appears to be essential for substrate activity. The Michaelis constant (Km) for cholesterol is estimated at 55 μM; the COX activity was markedly inhibited by metal ions such as Hg2+ and Fe3+ and inhibitors such as p-chloromercuric benzoate, mercaptoethanol and fenpropimorph. Inhibition caused by p-chloromercuric benzoate, mercuric chloride, or silver nitrate was almost completely prevented by the addition of glutathione. These suggests that -SH groups may be involved in the catalytic activity of the present COX.

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Acknowledgements

This work was supported in part by a support program for the training of excellent persons of Beijing (project No. 20061D0500300137) and a grant from the Ministry of Science and Technology of China (National Basic Research Program of China, 2007CB707802) and a support from the Education Department of Heilongjiang Province (No. 10531125).

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Authors and Affiliations

  1. College of Chemical & Environmental Engineering, Beijing Technology & Business University, No.33, Fucheng Road, Haidian District, Beijing, 100037, China

    Chengtao Wang, Yanping Cao, Baoguo Sun & Ji Wang

  2. College of Food Sciences, Heilongjiang August First Land Reclamation University, Daqing, 163319, China

    Chengtao Wang & Yonghuan Zhao

  3. College of Food Science & Nutritional Engineering, China Agricultural University, Beijing, 100083, China

    Baoping Ji

  4. Laboratory of Food Microbiology, Department of Agrotechnology & Food Sciences, Wageningen University, Wageningen, 6703 HD, The Netherlands

    M. J. Robert Nout

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  1. Chengtao Wang
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Correspondence to Baoguo Sun.

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Wang, C., Cao, Y., Sun, B. et al. Preparation and some properties of cholesterol oxidase from Rhodococcus sp. R14-2 . World J Microbiol Biotechnol 24, 2149–2157 (2008). https://doi.org/10.1007/s11274-008-9722-6

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  • DOI: https://doi.org/10.1007/s11274-008-9722-6

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