Screening Of Lipase Producing Microorganism And Cloning And Expression Of Lipase Gene

Lipase(Lipase, E C 3.1.1.3) was a special kind of ester bond hydrolytic enzymes that can catalytic hydrolysis glyceride generate glycerol and long chain fatty acids. It also was a kind of hydrolase with interface catalytic properties,can catalyze substrate hydrolysis, transesterification, ester synthesis reaction. In animals, plants, seeds and microorganisms are widely exists in all kinds of lipase, the most species of which is derived from microbial lipase. Microbial lipase has been widely used in food processing, detergent production, biodiesel, drug synthesis, wastepaper deinking, tanning, feed production and other industries.The experiment Screening microbial lipase production from yak rumen contents,with olive oil as the sole carbon source, Using the neutral red oil medium to the early screen, and the improved copper soap-photometric method for the determination of enzyme activity were rescreened. Screened the strains(TZ- 2, TZ- 4, TZ- 6, TZ- 7, TZ-8 and TZ – 9), a total of 6 strains lipase producing microorganisms, enzymatic activity were 3.30, 2.96, 5.48, 3.12 and 4.40 U/m L. After morphological observation and physiological and biochemical reaction, found that the TZ- 2, and TZ TZ- 4-6 were bacteria, TZ- 7, TZ- 8 and TZ-9 were fungus. Amplified bacteria strains 16 S r DNA sequence, amplified fungi strains 18 S r DNA or ITS sequence, BLAST the sequence on NCBI, and constructed evolutionary tree. Identification of strains of TZ- 2, TZ- 4 and TZ-6 were Serratia liquefaciens strain, the strain TZ- 8 and TZ- 9 were Geotrichum candidum, strain TZ- 7 was Mucor circinelloides.According to the NCBI Serratia liquefaciens strain lipase gene(gb: EF202840.1) designed primers, Used the Serratia liquefaciens strain genomic DNA as a template, After PCR amplified the target gene,used the p MD19-T as a vector, constructed recombinant plasmid p MD19T-SSL,and imported into the E.coli DH5α.Screened positive clones,identificated with Bam HⅠand Hind Ⅲ digestion,and gene sequencing. Results showed that the gene length was 1848 bp, the gene 100% similarity to the known Serratia liquefaciens lipase gene(gb: EF202840.1).After enzyme-cutting(with Bam HⅠand Hind Ⅲ) the correct sequence of Serratia liquefaciens lipase gene and p ET-28 a vector, constructed recombinant plasmid p ET-28a-SLL, and transformed BL21.Expression induced with a final concentration of 0.8mmol /L IPTG. SDS-PAGE results showed that the protein relative molecular mass was 66.4ku, and same with relative molecular mass of Serratia liquefaciens lipase.Using bioinformatics to predict structure of lipase from Serratia liquefaciens. The results show that the lipase was composed of 615 amino acids, relative molecular mass was 64938.9u, the theoretical isoelectric point was 4.31, which was a hydrophilic protein, containing a coiled-coil structure, no signal peptide and transmembrane region, α-helix content was 26.34%, β angle content was 12.52%,.The amino acid sequence 201 to 210 of ITISGHSLGG was conserved region, And the conserved sequences(GHSLG) was same with the active site conserved sequences of a majority of lipase.