Hall, Gerrit van

Janssen, Dick B.

Ploeg, Jan van der

English

The haloacid dehalogenase of the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10 was purified from a mutant with an eightfold increase in expression of the enzyme. The mutant was obtained by selecting for enhanced resistance to monobromoacetate. The enzyme was purified through (NH4)2SO4 fractionation, DEAE-cellulose chromatography, and hydroxylapatite chromatography. The molecular mass of the protein was 28 kDa as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 36 kDa as determined with gel filtration on Superose 12 fast protein liquid chromatography. The enzyme was active with 2-halogenated carboxylic acids and converted only the L-isomer of 2-chloropropionic acid with inversion of configuration to produce D-lactate. The activity of the enzyme was not readily influenced by thiol reagents. The gene encoding the haloacid dehalogenase (dhlB) was cloned and could be allocated to a 6.5-kb EcoRI-BglII fragment. Part of this fragment was sequenced, and the dhlB open reading frame was identified by comparison with the N-terminal amino acid sequence of the protein. The gene was found to encode a protein of 27,433 Da that showed considerable homology (60.5 and 61.0% similarity) with the two other haloacid dehalogenases sequenced to date but not with the haloalkane dehalogenase from X. autotrophicus GJ10

Dissertations of the University of Groningen

Characterization of the Haloacid Dehalogenase from Xanthobacter autotrophicus GJ10 and Sequencing of the dhlB Gene

https://pure.rug.nl/ws/files/14930615/1991JBacteriolvdPloeg.pdf

Characterization of the Haloacid Dehalogenase from Xanthobacter autotrophicus GJ10 and Sequencing of the dhlB Gene

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Dissertations of the University of Groningen