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Triple fusion of d-amino acid oxidase from Trigonopsis variabilis with polyhistidine and Vitreoscilla hemoglobin

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Abstract

Fusion proteins of d-amino acid oxidase from Trigonopsis variabilis (TvDAAO) with Vitreoscilla Hemoglobin (VHb) and (His)6-tag were constructed and expressed in recombinant Escherichia coli. A fusing-position effect was revealed that (His)6-tag’s N-terminal fusion with TvDAAO (HDAAO) reduced the specific activity by ~29%, while the C-terminal fusion (DAAOH) remained unreduced. The N-terminal fusion of VHb with TvDAAO and DAAOH significantly improved their activity. As in a 5 l fermentor, the activity of the triple fusion VHb-TvDAAO-(His)6 (VDAAOH) reached 2.53 U/mg dry cell at 9 h, ~58% increase than that of DAAOH together with ~40% biomass increase, confirming the positive effect of VHb expression on cell level. After purification, the UV–visible and fluorescence spectrum of DAAOH and VDAAOH were characterized. Enzyme kinetics studies further indicated that VDAAOH behaved a higher K cat, but a weaker substrate affinity of K m relative to DAAOH, revealing two distinct impacts of VHb-coupling with TvDAAO on protein level.

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Acknowledgments

This work was supported by the Foundation for the Author of National Excellent Doctoral Dissertation of P. R. China (No.200345), the National Key Basic Research Project 973 (2007CB714304) and High-tech Project 863 (2008AA02Z207). Many thanks for the kind gift of pBR322-vgb from Prof. Shengli Yang of Shanghai Research Center of Biotechnology, Academic Sinica, Shanghai, China.

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Authors and Affiliations

  1. Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University, 100084, Beijing, China

    Xian-Feng Ma, Hui-Min Yu, Cheng Wen, Qiang Li & Zhong-Yao Shen

  2. Department of Biological Science, University of Science and Technology Beijing, 100083, Beijing, China

    Hui Luo

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  1. Xian-Feng Ma
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Correspondence to Hui-Min Yu.

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Ma, XF., Yu, HM., Wen, C. et al. Triple fusion of d-amino acid oxidase from Trigonopsis variabilis with polyhistidine and Vitreoscilla hemoglobin. World J Microbiol Biotechnol 25, 1353–1361 (2009). https://doi.org/10.1007/s11274-009-0022-6

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