The Gene Cloning Of Cold-adapted Lipase From Pseudomonas Sp.lip35 And Expression In Pichia Pastoris

Low-adapted lipases produced by low-temperature microorganisms can catalysis at low temperature. However, low-temperature microorganisms need low temperature grow environments and have low expression level of lipase which have restricted its industrial production and application. Moreover, gene cloning and heterogenous expression give a new approach to potential industrial utilization of low-adapted lipases.However, owing to the low homology observed among different lipase genes, a novel lipase gene is very difficult to be amplified by PCR. The usual strategy for obtaining lipase genes is to construct a DNA library, to anlyze the amino acids sequence or to hybrid due to high G+C value and low similarities of lipase genes. Using these methods several lipase genes had been cloned from lipase-producing strains these years. Especially the Upases from Pseudomonas sp. have been received more and more attention.In this paper, a combination method of usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain Pseudomonas sp.lip35 was described. First, 16SrRNA was amplified from the genomic DNA of the train Pseudomonas sp. lip35. NCBI Blast searches were performed on the DNA sequence to determine the most closely related 16SrRNA species in order to further testify the subfamily of the lipase from the strain lip35. From the 16SrRNA analysis on homology, the strain lip35 has been suggested as a member of Pseudomonas subfamily 1.3 because of high similarity with P. fluorescence and P. chlororaphis whose lipase belonged to subfamily I.3.Several Lipase gene sequences of Pseudomonas subfamily I.3 were downloaded from the Entrez Search and Retrieval System at the National Center for Biotechnology Information. Regions with homology to the lipase gene sequences were obtained using Blastp and Clustal W program. The design of primers was performed carefully by DNAMAN6.0 and Primer premier5.0 softwares. Using this combined method a full-length lipase gene lipA and a partial lipase gene lipB were cloned successfully.Nucleotide sequencing showed lipA gene is 1701bp major open reading frame, and the deduced amino residues encoded a protein of 567 amino acid residues. The deduced amino acid sequence contained a lipase consensus sequence Gly-His-Ser-Leu-Gly-Gly which was most conserved in Pseudomonas family 1.3. The initial codon is ATG, and the stop codon is TAG The amino sequence of LipA showed 74% identical to the lipase from the uncultured bacterium, but did not show any overall homology with lipases from other origins whose properties had been studied. The lipB gene is partial which includes the GHSLGG motif. The active site serine residue was found from residues 205 to 210. Moreover, the SD sequence "GAGG" was also presented at 7bp upstream of initial codon.The expression of cloned cold-adapted lipase gene lipA in Pichia pastoris GS115 with the pPIC9k as the expressive vector was performed according to the methods described in the manual, version 3.0, of the pPIC9K identified the lip gene cloned here was a functional lipase gene. In addition the expression on cold-adapted lipase in Pichia pastoris was also to be studies.The sequences of 16SrRNA, lipA gene and the deduced amino sequence had been disposited in. GenBank, with the accession number EU439251, EU414288 and ABY86751 respectively.