Cloning And Expression Of A Cold-Adapted Lipase Gene From Pseudomonas Sp. PF16

A pychrotrophic bacterial strain producing cold-adapted lipase was isolated from Kunming University of Science and Technoligy. Based on morphological characteristics and 16S rDNA sequence analysis, the isolate strain was identified as one strain of Pseudomonas species, and named as Pseudomonas sp. PF16. The lipase produced by this strain was named as Lip-PF16.Lip-PF16 was an intracellular enzyme. It can hydrolyze olive oil and show activity even at 0℃, so Lip-PF16 can be identified as a cold-adapted lipase. Pseudomonas sp. PF16 was grown at 25℃for 24 h. After centrification, the precipitated cells were resupended and disrupted by sonication. The supernatant resulting from centrification of the cell lysate was patially purified by ultrafiltration and (NE4)2SO4 precipition.The effects of temperature and pH on the stability of the patially purified Lip-PF16 was also completed. The enzyme was stable at temperature of 0℃～25℃and pH of 7.5-10. This lipase show high catalytic activity at lowtemperatures and has potential applications at food processing, cleaning agents, bioremediation et al.The production of the lipase from Pseudomonas sp. PF16 was very limited. To achieve the large-scale production of this lipase, it is worthy to clone and express the lipase gene from Pseudomonas sp. PF16. On the one hand, a genomic library of Pseudomonas sp. PF 16 was constructed. On the other hand, the genomic DNA of this bacterium was used for PCR screening using primers obtained from multiple sequence alignments of lipases belonging to the Pseudomonas species. Lipase gene sequences were downloaded from the EntrezSearch and Retrieval System at the National Center for Biotechnology Information (NCBI). Regions with homology to the lipase gene sequences were obtained using BLASTp and ClustalW program, and the design of primers was carefully performed using DNAMAN 6.0 and Primer Premier5.0 software.This allowed cloning and sequencing of the DNA that partially encodes a novel lipase protein (0-629bp). Subsequently the downstream sequences of this gene were also obtained by PCR using the consensus primers. The gene consisted of 1431 bases, revealing a protein of 637 amino acid residues with a molecular mass of 50 kDa and a pI of 4.58. It was identified as a subfamily 1.3. Its amino acid sequence exhibited high homology to cold-adative lipase (ABI94371) (71%).Lip-PF16 was overexpressed using a T7 RNA polymerase transcription (pET21a) system in the Escherichia coli BL21(DE3). The result indicated that Lip-PF16 was functionally expressed in Escherichia coli BL21(DE3) cells as an inclusion body. The concentration of soluble lipase is not high but with significant activity even at low temperature.The wild type Pseudomonas sp. PF16 strain was used as a source of DNA for the cloning experiment. During the course of these studies, the lipase gene was obtained and fuctionally expressed in Escherichia coli BL21(DE3). The result provides valuable information in further study of this cold-adapted lipase.