Screening Of Lipase Producing Strains And The Integrative Expression Of Lipase Genes In Bacillus Subtilis

The study was done to obtain a strain which have high lipase production ability and the lipase gene was cloned. We expect to construct an integrative expression vector by technology of genetic engineering to achieve the integration and expressions of lipase gene in Bacillus subtilis pabO2 at the same time, which established the foundation for Constructing a stable expression system and further exploitation of Bacillus subtilis pabO2.1. Seven lipase-yield strains were isolated from oily soil samples through repeating enrichment culture and plate isolation with olive oil as substrate while Victoria blue or Rhodamine as indicator. Among them an isolated bacterial strain number as A05 secreting a large amount of lipase was selected by secondary screening. The lipase activity of strain A05 was assayed 1.07U/ml by alkaline titrimetric method. By testing morphologic and physiological-biochemical characteristics the strain A05 was preliminarily identified as Pseudomonas sp.. The general primers were designed according to conservative sequence of bacteria 16SrDNA. The genomic DNA of Pseudomonas A05 was extracted and amplified the target 16SrDNA sequence. At the same time, this sequence was analyzed by using the NCBI with BLAST database and the phylogenetic tree was established. The Pseudomonas A05 was finally identified as Pseudomonas aeruginosa based on the results above. 2. Alignment of nucleotide sequence of Pseudomonas aeruginosa lipase gene reported from GenBank link was performed by bioinformatics methods. Primers were designed based on the conservative nucleotide sequence and the lipase gene of Pseudomonas aeruginosa A05 was cloned using genomic DNA as template. A05 lipase gene sequence is 1092bp long and encodes a 26-amino-acid signal sequence and a mature sequence of 285 amino acids, the molecule weight of which was estimated 33kDa. The lipase gene sequence blast showed 98% identical to the published sequence. The deduced amino acid sequences showed that an active site of theα/βhydrolase fold enzymes it has by conservative domains analysis which belongs to Esterase-lipase superfamily.3. In order to achieve the integration and expression of lipase gene in Bacillus subtilis, the common integrated expression plasmid pHSG398-16S-lipA was constructed by the following elements:the vector pHSG398 was used as Bacillus backbone; Pseudomonas aeruginosa A05 lipase gene was used as the purpose gene; and the B. subtilis pabO2 16SrDNA was used for expression of homologous gene. The integrated expression plasmid was transformed into host strain B. subtilis pabO2 via chemical transformation, realizing homologous integration under 16SrDNA sequence of the integrated expression plasmid. The molecular mass of the recombinant lipase determined by SDS-PAGE was 37kDa.