Construction Of The Genomic Libaray Of Stenotrophomona.maltophilia 0450 And Cloning And Expression Of Its Lipase Gene

Seventy-one lipase-producing bacterial strains were isolated from oil-stained soil samples by solid plate method, among which one strain was identified as Stenotrophomonas maltophilia by its morphological, biochemical, physiological natures and matching of 16S rRNA gene sequence, and named as S. maltophilia 0450.By further experiments, the optimal conditions for lipase activity are pH9.0 and 45℃. The lipase can be activated by 5mmo1/L Ca2+, but inhibited by Fe2+ and Mn2+. It also exhibits fairly high thermo-stability from pH7.0 to pH 10.0.The genomic libaray of S. maltophilia 0450 was constructed by pHBM803 and E. coli XL10-Gold system. After Rodamin B+olive oil check, the positive clone ps01 was selected and sequenced. The length of inserted fragment is 1.2kb, containing a whole ORF at 1101bps and coding a conserved hypothetical protein of 365 amino acids. The protein has a conservative domain of -G-X1-S-X2-G- as in any other lipase. Ligated the gene with pET28a(+) and expressed in E.coli DH5 by the induction of IPTG, hydrolysis of the obtained protein as a lipase was detected and its molecular mass was measured as 39KDa by SDS-PAGE method. From the above tested natures, we confirm that we have cloned the lipase gene of S. maltophilia 0450 which deposited in GenBank with the accession number DQ647508.