Abstract

The gene responsible for dechlorination of 4-chlorobenzoate(4CBA) was cloned from chromosomal DNA of Pseudomonas sp. DJ-12 in Escherichia coli XL1-Blue by using pBluescript SK(+) phagemid vector. Two recombinant plasmids of pCJ1 and pCJ2 carrying dechlorinase gene were constructed. The inserted DNAs in the pCJ1 and pCJ2 were found to have the fragment of 9.5 kb carrying dechlorinase gene, but they were oriented in opposite direction. The inserted DNA of 3.4kb in the pCJ101 subclone carrying dechlorinase gene had two restriction sites for AccI and each one site for HincII, KpnI, PstI, and SacIi but the dechlorinase gene in pCJ101 was found to be placed over the HincII site. The dechlorinase fenes in the recombinant cells of E. coli CJ1 and CJ101 were well expressed to show the dechlorination activity of 4CBA. The proteins encoded by dechlorinase genes in E. coli CJ1 and CJ101 were about 49 kDa in molecular weight. But this protein was not produced by E. coli CJ102, CJ103 and E. coli XL1-Blue. Therefore, the proteins produced by Pseudomonas sp. DJ-12 and the cloned cells of E. coli CJ1 and CJ101 were thought to be the dechlorinase enzyme which was the product of dechlorinase gene.

• Keywords: dechlorinase gene; cloning; 4-chlorobenzoate; Pseudomonas sp. DJ-12