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Applied and Environmental Microbiology, December 2005, p. 8024-8030, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8024-8030.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Purification of Cytochrome P450 and Ferredoxin, Involved in Bisphenol A Degradation, from Sphingomonas sp. Strain AO1

Miho Sasaki,1 Ayako Akahira,1 Ko-ichi Oshiman,3 Tetsuaki Tsuchido,1,2 and Yoshinobu Matsumura1,2*

Department of Biotechnology, Faculty of Engineering,1 High Technology Research Center, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan,2 Tsukuba Research Institute, Sumitomo Forestry Co., Ltd., 3-2 Midorigahara, Tsukuba 300-2646, Japan3

Received 31 May 2005/ Accepted 31 August 2005

In a previous study (M. Sasaki, J. Maki, K. Oshiman, Y. Matsumura, and T. Tsuchido, Biodegradation 16:449-459, 2005), the cytochrome P450 monooxygenase system was shown to be involved in bisphenol A (BPA) degradation by Sphingomonas sp. strain AO1. In the present investigation, we purified the components of this monooxygenase, cytochrome P450 (P450bisd), ferredoxin (Fdbisd), and ferredoxin reductase (Redbisd). We demonstrated that P450bisd and Fdbisd are homodimeric proteins with molecular masses of 102.3 and 19.1 kDa, respectively, by gel filtration chromatography analysis. Spectroscopic analysis of Fdbisd revealed the presence of a putidaredoxin-type [2Fe-2S] cluster. P450bisd, in the presence of Fdbisd, Redbisd, and NADH, was able to convert BPA. The Km and kcat values for BPA degradation were 85 ± 4.7 µM and 3.9 ± 0.04 min–1, respectively. NADPH, spinach ferredoxin, and spinach ferredoxin reductase resulted in weak monooxygenase activity. These results indicated that the electron transport system of P450bisd might exhibit strict specificity. Two BPA degradation products of the P450bisd system were detected by high-performance liquid chromatography analysis and were thought to be 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol based on mass spectrometry-mass spectrometry analysis. This is the first report demonstrating that the cytochrome P450 monooxygenase system in bacteria is involved in BPA degradation.

* Corresponding author. Mailing address: Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan. Phone: 81-6-6368-0934. Fax: 81-6-6388-8609. E-mail: ymatsu{at}ipcku.kansai-u.ac.jp.

Applied and Environmental Microbiology, December 2005, p. 8024-8030, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8024-8030.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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