Cloning And Expression Of Lipase-Encoding Gene From Burkholderia Sp. GXU56

Lipases, are an important group of biotechnologically relevantenzymes and they find immense applications in food, dairy, detergent andpharmaceutical industries.In this study, the partial genomic library of Burkholderia sp GXU56composed of about 10000 recombinants was constructed. The genesencoding the lipase (lipA) and lipase chaperone (lipB) were isolated fromlibrary screening and complemented by PCR. Nucleotide sequence analysisand alignments of amino acid sequences suggest that lipA is a member ofbacterial lipase family 1.1 and that lipB is a lipase activator of lipA. Thededuced aminoacid sequences for the lipA was found to encode matureproteins of 365 aa (34 kDa). The lipase contained a putative leadersequence, as well as the conserved Ser,His and Asp residues which areknown to function as the catalytic triad in lipases, lipB was found to encodemature proteins of 344aa (37kDa). A possible trans-membranehydrophobic helix was identified in the N-terminal region of the chaperone. High levels of expression of inactive iipase (45ï¼…) were achieved withEscherichia coli Tuner(DE3) harboring pPETBlue-lipA. Hence, for thechaperone lipB, the putative membrane anchor was removed. Thetruncation of the gene led to overexpression of the active chaperone (up to30ï¼…) in E. coli.With this chaperone, it was possible to obtain in a simplein vitro refolding procedure a active lipase with a specific activity of up toa yield of 60,000 U/g of E. coli wet cells.Recently, cell surface engineering has been attracting a great deal ofattention. To production of a recombinant lipase arterially localized on theBacillus subtilis cell surface,lipA gene was fused to the 5'-terminal regionof the cwlb gene which encoding the cell wall-binding domain of the majorcell wall hydrolase (autolysin). SDS-polyacrylamide gel electrophoresisrevealed that the fused protein is localized in the B. subtilis cell wallfraction. However we couldn't detect obvious specific activity of lipase,possibly because lack of chaperone or depredated by cell wall associatedprotease.