Cloning and Expression of Gene encoding meta-Cleavage Enzyme of BTEX Degradation Pathway from Haloalkaliphilic Pseudomonas sp. HA10

The potential degradation of haloalkaliphilic hydrocarbon degrading bacteria is an interesting class of extremophilic organisms that possess special adaptation strategies that make them interesting for the remediation of polluted sites. Haloalkaliphilic pure culture using BTEX as a sole source of carbon and energy was isolated from water samples from Mediterranean Sea, Abu Qir-coastline, Egypt. An analysis of the 16S rRNA gene sequence and morphological and physiological characteristics showed that this strain is a member of the genus Pseudomonas, and it was designated as strain HA10. Strain HA10 could grow at pH from 8 to 11 and salt concentration from 1.5% to 10%.Its optimal conditions for biodegradation of BTEX were pH 10 and 7% salt concentration. Catechol 2,3dioxygenase (C23O) gene as a key catabolic genes in the degradation of BTEX compounds was cloned and sequenced. Analysis of C23O nucleotide sequence revealed a 912bp and encoded a polypeptide of molecular weight of 34.43KDa containing 303 amino acids residues. By comparison, the C23O of Pseudomonas sp. HA10 showed 81-100% identity in amino acids sequence with other C23Os. The C23O gene was overexpressed in E. coli DH5α and the gene product was identified by SDS-PAGE. To our knowledge, this is the first reported C23O gene cloned from haloalkaliphilic bacterium and successfully expressed and such finding is essential for developing in situ bioremediation technologies. [Hamdy A. Hassan, Asmaa A. Aly and Mohamed E. Ebeid. Cloning and Expression of meta-Cleavage Enzyme of BTEX Degradation Pathway from Haloalkaliphilic Pseudomonas sp. HA10. Life Sci J 2014;11(5):403-411] (ISSN:1097-8135). http://www.lifesciencesite.com. 56