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Purification and Characterization of 3-Ketovalidoxylamine A C-N Lyase Produced by Stenotrophomonas maltrophilia

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Abstract

A soluble 3-ketovalidoxylamine A C-N lyase from Stenotrophomonas maltrophilia was purified to 367.5-fold from the crude enzyme, with a yield of 16.4% by column chromatography on High S IEX, Methyl HIC, High Q IEX, and Sephadex G 100. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the enzyme was a neutral protein having an isoelectric point value at pH 7.0. The optimal pH of 3-ketovalidoxylamine A C-N lyase was around 7.0. The enzyme was stable within a pH range of 7.0–10.5. The optimal temperature was found to be near 40 °C, and the enzyme was sensitive to heat. The enzyme was completely inhibited by ethylenediaminetetraacetic acid, and it was reversed by Ca2+. The product, p-nitroaniline, inhibited the enzyme activity significantly at low concentration. The enzyme has C-N lyase activity and C-O lyase activity, and need 3-keto groups. The apparent K m value for p-nitrophenyl-3-ketovalidamine was 0.14 mM.

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Acknowledgements

This work was supported by the National Basic Research Program of China (973 Program; No. 2007CB714306), the Fund of the National High Technology Research and Development Program of China (863 Program; No. 2006AA10A209), the National Natural Sciences Foundation of China (No. 20872132), and the Research and Development Program of Science and Technology Department of Zhejiang Province (No. 2008C32026 and No. 2008C03004-1).

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Authors and Affiliations

  1. College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou, 310015, People’s Republic of China

    Jian-Fen Zhang

  2. Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Jian-Fen Zhang, Yu-Guo Zheng & Yin-Chu Shen

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  1. Jian-Fen Zhang
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  2. Yu-Guo Zheng
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  3. Yin-Chu Shen
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Correspondence to Yu-Guo Zheng.

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Zhang, JF., Zheng, YG. & Shen, YC. Purification and Characterization of 3-Ketovalidoxylamine A C-N Lyase Produced by Stenotrophomonas maltrophilia . Appl Biochem Biotechnol 162, 966–974 (2010). https://doi.org/10.1007/s12010-009-8787-5

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  • DOI: https://doi.org/10.1007/s12010-009-8787-5

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