Screen Of A Lipase-Producing Strains, Gene Cloning And Expression Of Lipase

In this thesis, a lipase-producing strains was screened and the enzyme activity of lipase was researched. The strain of lipase-producing was made as microbial additives suspension and the mice were fed by it. The effect of growth, immune function, and intestinal microbial communities of mice were detected.Then the gene of lipase was cloned and expressed. The results were showed as follow:1.8 lipase-producing strains was screened from soil which stained by oil. Through enrichment, plate isolation, flask fermentation and detection of enzyme activity, the lipase-producing strain PZ04 with higher lipase activity was screened. The PZ04 was identified as Bacillus subtilis by using the 16SrDNA sequence analysis method and morphological observation. The optimal temperature and acidity for lipase to decompose lipid was found at 37℃and pH 8.7. Lipase characterization showed that it was highly stable under the pH value ranging from 8.0 to 10.0 and the temperature value ranging from 20-45℃, then the lipase indicated 65% of total activity. The result showed that the lipase-producing PZ04 strain was activated by Zn2+, Mg2+, Ca2, but inhibited by Cu2+, Fe2+.2.96 three weeks mice were selected and randomly divided into 3 groups, feeding with the basic diets, the basic diets with the 0.1% probiotics of B. subtilis PZ04 in the concentration of 5×108CFU/g and 1×109CFU/g. The results indicated that Bacillus subtilis could improve the growth of mice and significantly improved the weight gain in the 4-5week; The thymus index of the probiotics groups had significant differences with the control group in the 3-4week (P<0.05); The changes of intestinal microbial flora in different week were analysed by PCR-DGGE. The results were showed that mice fed with the 109CFU/g of probiotics could make intestinal microbial flora stable. In the fourth week, the increasing of the microbial flora was detected. The results indicated that the immune function and the intestinal microbial flora stability can be improved by Bacillus subtilis.3. The lipase gene from PZ04 was cloned into pET-28a(+) vector by double digest identification with HidIII+Xho I and the recombinant plasmid was transformed into E coli BL21(DE3). The lipase was over expressed in E coli BL21(DE3) after induction by IPTG and revealed a molecular mass of 26kDa. The protein was highly expressed with 2.0mmol/L IPTG at 37℃for 4h. The recombinant pET28a-PZ04 product with 6-His-tag was purified by Ni-NTA Agrose chromatography. The protein was purified 10.5 times. The lipase of the recombinant pET28a-PZ04 characterization showed that it was highly stable under the pH value ranging from 8.0 to 9.5 the the lipase indicated 90% of total activity. At the temperature of 50℃, the lipase indicated 60% of total activity.