Cloning and expression of DL-2-haloacid dehalogenase gene from Burkholderia cepacia

Burkholderia cepacia strain KY, which can utilize a herbicide 2,4-D as a sole carbon and energy source, catalyzes the hydrolytic dehalogenation of both D- and L-2-haloalkanoic acids. We have cloned the gene encoding DL-2-haloacid dehalogenase, and obtained a recombinant plasmid (pUCDEXL) containing approximately 4.5 kbp insert. In both of B. cepacia strain KY and this clone E. coli JM109/pUCDEXL, DL-2-haloacid dehalogenase was induced significantly with monohalogenated acetic acids, such as chloroacetate, bromoacetate and iodoacetate. This dehalogenase was also overexpressed in E. coli using three different promoters. In pET vector systems with T7 lac promoter, a large amount of dehalogenase was selectively expressed, but some parts of the protein were accumulated in the form of inclusion bodies. This problem was overcome to carry on growth and induction at 22°C, and at the same time, the maximum specific activity of dehalogenase was reached at 12.6 U/mg, 500-fold higher activity than in wild strain, B. cepacia strain KY grown with 2,4-D.