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A zinc-containing mannitol-2-dehydrogenase from Leuconostoc pseudomesenteroides ATCC 12291: purification of the enzyme and cloning of the gene

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Abstract

Mannitol-2-dehydrogenase (EC 1.1.1.67) of Leuconostoc pseudomesenteroides ATCC 12291 catalyzing the NADH-dependent reduction of d-fructose to d-mannitol was purified to homogeneity. Native mannitol-2-dehydrogenase has a molecular mass of 155 kDa as determined by gel filtration chromatography. In SDS–PAGE, a single band appeared corresponding to a molecular mass of 43 kDa which indicated that the enzyme was composed of four identical subunits. Enzyme activity was completely inhibited by EDTA and could be restored by zinc ions, but not by Mn2+ or Mg2+ which demonstrated that zinc is a cofactor. Purified mannitol-2-dehydrogenase exhibited a maximal specific activity of 400 μmol fructose reduced min−1(mg protein)−1, using NADH as electron donor. The enzyme showed a high substrate specificity for d-fructose and d-mannitol, however it accepted NADPH as a cofactor with 32% activity (V max ) relative to NADPH (100%). The mdh gene, encoding mannitol-2-dehydrogenase, was identified by hybridization with a degenerate gene probe complementary to the nucleotide sequence encoding the first eight N-terminal amino acids of the enzyme. The mdh gene was cloned on a 4.2-kb DNA fragment, subcloned, and expressed in Escherichia coli. Sequencing of the gene revealed an open reading frame of 1,017 bp, encoding a protein of 338 amino acids with a predicted molecular mass of 36.0 kDa. Plasmid-encoded mdh was functionally expressed, with 70 U/mg of cell-free protein in E. coli. Multiple sequence alignments showed that mannitol-2-dehydrogenase was affiliated with members of the Zn2+-containing medium-chain alcohol/polyol dehydrogenase/reductase protein family (MDR).

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Abbreviations

DTT:

Dithiothreitol

mdh :

Gene encoding NAD+-dependent mannitol-2-dehydrogenase

MDR:

Medium-chain alcohol/polyol dehydrogenase/reductase protein family

IPTG:

Isopropyl−1-thio-β-d-galactopyranoside

SD:

Standard deviation

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Acknowledgements

This work was supported by the Institut für Technologie der Kohlenhydrate-Zuckerinstitut e. V.-Braunschweig, Germany

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Authors and Affiliations

  1. Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany

    Gerald Hahn, Björn Kaup, Stephanie Bringer-Meyer & Hermann Sahm

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  1. Gerald Hahn
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  2. Björn Kaup
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  3. Stephanie Bringer-Meyer
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  4. Hermann Sahm
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Correspondence to Stephanie Bringer-Meyer.

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Hahn, G., Kaup, B., Bringer-Meyer, S. et al. A zinc-containing mannitol-2-dehydrogenase from Leuconostoc pseudomesenteroides ATCC 12291: purification of the enzyme and cloning of the gene. Arch Microbiol 179, 101–107 (2003). https://doi.org/10.1007/s00203-002-0507-2

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  • DOI: https://doi.org/10.1007/s00203-002-0507-2

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