National
Centre for Biomolecular Research, Masaryk University, Kotlářská 2, 611 37 Brno,
Czech Republic
Haloalkane
dehalogenases are enzymes catalyzing cleavage of a carbon-halogen bond of
halogenated compounds using a hydrolytic mechanism. The products of the
reaction are a halogen ion and a corresponding alcohol. Known sequences of
genes encoding haloalkane dehalogenases dhlA,
dhaA and linB were compared with all sequences accessible in genetic
databases. This comparison revealed a presence of two genes coding for putative
haloalkane dehalogenases in the genome library of Mycobacterium tuberculosis H37Rv. Identified genes were rv2296 and rv2579 [1]. We have used genetically closely related Mycobacterium bovis 5032/66 izolated from the cattle to clone the
genes of putative mycobacterial dehalogenases.
Gene rv2296 was amplified by PCR reaction
using specific primers. PCR amplicon of the gene rv2296 was cloned into expression vector pCR T7/NT- TOPO and the
ligation mixture was transformed to Escherichia
coli OneShot TOP10F competent cells.
The rv2296 gene was sequenced from
both sides to ensure accuracy. Expression of Rv2296 protein was performed in
bacterial cells E. coli BL21(DE3)LysS. Solubility of the
protein was supported using different conditions for preparation of crude
extract. Active purified protein Rv2296 could not be prepared using the
standard purification protocols. The substrate specificity of crude extract
Rv2296 was tested towards thirty-four different halogenated substrates.
Gene rv2579 was amplified by PCR reaction
using specific primers, which introduced hexahistidyl tail to C-terminus of the
protein. PCR amplicon of the gene rv2579
was cloned into pUC18 vector and sequenced. For expression of Rv2579, the gene was re-cloned from pUC18 to
pAQN. E. coli BL21 was used as the host strain for expression of Rv2579
protein. Expressed protein was purified to homogeneity using immobilized metal
affinity chromatography with the yield 5 mg of a protein per 1 L of a bacterial
culture. Protein Rv2579 was tested for temperature- and pH-optimum. The highest activity of protein Rv2579
was observed at 45°C. Protein showed double pH optimum (pH 5.5 and pH 8.5). The
effect of storage temperature and the effect of stabilizing additives on
protein was also determined.
[1] Jesenská
A., Sedláček I., Damborský J. Dehalogenation of Haloalkanes by Mycobacterium
tuberculosis H37Rv and Other Mycobacteria. Applied and Enviromental Microbiology 66(2000): 219-222.
* To whom correspondence may be addressed: Fax:
420-5-41129506; E-mail: andreaj@chemi.muni.cz.