Cloning Of A Lipase Gene Fragment From Bacillus And Its Bioinformatics Analysis

Lipase (EC3.1.1.3), Triacylglycerols acyl hydrolase, is an important hydrolytic enzyme which catalyzed the hydrolysis of ester bond of grease, widely exists in animals, plants and micro-organisms. Lipase can catalyzes transesterification, alcoholysis, esterification, acid hydrolysis reaction, and therefore is widely applied in food processing, washing industrial, medical and textile industry. In recent years, the characteristics and applications of microbial lipase has got much attention by the using of genetic engineering techniques to construct recombinant strain. In this paper one lipase gene fragment was cloned from Sphingobacterium sp. And its bioinformatics was also analyzed, which hopes to provide a theoretical foundation for lipase in related fields. The mainly results were: 1. The genomic DNA was extracted from Sphingobacterium sp, one pair of primers was designed by Primer Premier5.0 to amplify the lipase gene fragment and the amplification conditions were also optimized. 2. Extract the genomic DNA of high purity and no degradation. One 800bp DNA fragment was amplified from genomic DNA and named Lip-J. After ligated with the pEASY-Blunt Simple vector and transformed into E.coli JM109 competent cells, the recombinant plasmid was identified by specific amplification that the recombinant plasmid containing the amplified target fragment and then sent to sequencing. 3. The sequencing analysis results and the phylogenetic tree results showed that the amplified lipJ gene fragment was 761bp, inserted into the vector in positive direction. It contained an open reading frame of 639bp. Lipase gene showed 35.89% identity with lipase from Bacillus subtilis and 37.44% with lipase from Bacillus stearothermophilus. so Lip-J was like-lipase gene. 4. The bioinformatics analysis of Lip-J gene fragment showed its putative protein molecular weight is 29416.9Da and its isoelectric point is 5.12 and is an acidic protein.The total number of negatively charged residues (Asp+Glu) is 27, the total number of positively charged residues (Arg+Lys) is 24.The instability coefficient is 37.39, and the total average number of hydrophilic is -0.381, so it is a hydrophilic stabilizing protein. The structure predetermination indicated that the fragment contains 15.1% α-helix,34.0% β-folded,50.1% random coil, and has no transmembrane structure and signal peptide.