Cloning, expression and characterization of the mycobacterial haloalkane dehalogenases Rv2296 and Rv2579

Iva Těšínská, Marta Monincová, Andrea Jesenská* and Jiří Damborský


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.


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