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Expression, purification, and characterization of a membrane-bound d-amino acid dehydrogenase from Proteus mirabilis JN458

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Abstract

Objectives

To characterize a novel membrane-bound d -amino acid dehydrogenase from Proteus mirabilis JN458 (PmDAD).

Results

The recombinant PmDAD protein, encoding a peptide of 434 amino acids with a MW of 47.7 kDa, exhibited broad substrate specificity with d -alanine the most preferred substrate. The K m and V max values for d -alanine were 9 mM and 20 μmol min−1 mg−1, respectively. Optimal activity was at pH 8 and 45 °C. Additionally, this PmDAD generated H2O2 and exhibited 68 and 60% similarity with E. coli K12 DAD and Pseudomonas aeruginosa DAD, respectively, with low degrees of sequence similarity with other bacterial DADs.

Conclusions

d-Amino acid dehydrogenase from Proteus mirabilis JN458 was expressed and characterized for the first time, DAD was confirmed to be an alanine dehydrogenase.

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Acknowledgments

We are grateful for the financial support of this work by the National Key Scientific Instrument and Equipment Development Project of China (2013YQ17052504), the Program for Changjiang Scholars and Innovative Research Team in the University of the Ministry of Education of China (IRT_15R55), and the seventh group of the Hundred-Talent Program of Shanxi Province (2015).

Supporting information

Supplementary Table 1—Purification of P. mirabilis DAD from recombinant Escherichia coli cells.

Supplementary Fig. 1—SDS-PAGE (12%) analysis of recombinant P. mirabilis DAD from E. coli cells.

Author information

Authors and Affiliations

  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China

    Jinjin Xu & Yujie Cai

  2. College of Life Sciences, Northwest University, Xi’an, 710069, Shanxi, China

    Yajun Bai, Taiping Fan & Xiaohui Zheng

  3. Department of Pharmacology, University of Cambridge, Cambridge, CB2 1T, UK

    Taiping Fan

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  1. Jinjin Xu
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  2. Yajun Bai
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  4. Xiaohui Zheng
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Correspondence to Yujie Cai.

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The authors declare that they have no conflict of interest.

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Xu, J., Bai, Y., Fan, T. et al. Expression, purification, and characterization of a membrane-bound d-amino acid dehydrogenase from Proteus mirabilis JN458. Biotechnol Lett 39, 1559–1566 (2017). https://doi.org/10.1007/s10529-017-2388-0

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  • DOI: https://doi.org/10.1007/s10529-017-2388-0

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