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Cloning and nucleotide sequence of a D,L-haloalkanoic acid dehalogenase encoding gene from Alcaligenes xylosoxidans ssp. denitrificans ABIV

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Abstract

We have cloned DNA fragments of plasmid pFL40 from Alcaligenes xylosoxidans ssp. denitrificans ABIV encoding a D,L-2-haloalkanoic acid halidohydrolase (DhlIV). A 6.5-kb EcoRI/SalI-fragment with inducible expression of the halidohydrolase was cloned in Pseudomonas fluorescens and Escherichia coli. A 1.9-kb HindII-fragment demonstrated expression of the dehalogenase only due to the presence of the promoter from the pUC vector in Escherichia coli. The nucleotide sequence of this DNA-fragment was determined. It had an open reading frame coding for 296 amino acid residues (molecular weight of 32783 D). The dhlIV gene showed sequence homology to a short segment of a D-specific dehalogenase (hadD) from Pseudomonas putida AJ1, but not to any other known DNA sequences. Restriction enzyme patterns indicated similarity between dhlIV and the D,L-isomer specific dehI dehalogenase gene from Pseudomonas putida PP3. There are some indications from restriction enzyme patterns and initial sequencing data, that a gene encoding a σ54 activator protein, similar to the dehR Iregulatory gene from Pseudomonas putida PP3 is located upstream of dhlIV. In contrast to DehI, dehalogenation of D-or L-chloropropionic acid by the DhlIV-protein leads to lactic acid of inverted configuration.

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Authors and Affiliations

  1. Department of Microbiology, University Gesamthochschule Kassel, D-34109 Kassel, Heinrich-Plett-Str. 40, D-34109, Kassel, Germany

    Andre Brokamp & Friedrich R. J. Schmidt

  2. Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124, Braunschweig, Germany

    Birgitta Happe

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  1. Andre Brokamp
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  2. Birgitta Happe
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  3. Friedrich R. J. Schmidt
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Brokamp, A., Happe, B. & Schmidt, F.R.J. Cloning and nucleotide sequence of a D,L-haloalkanoic acid dehalogenase encoding gene from Alcaligenes xylosoxidans ssp. denitrificans ABIV. Biodegradation 7, 383–396 (1996). https://doi.org/10.1007/BF00056422

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