Study On Fermentation Conditions And Enzymatic Properties Of Lipase Produced By Pseudomonas Aeruginosa

Lipase is a kind of hydrolase that can decompose triglycerides,and can convert triglycerides into glycerol and fatty acids at the oil-water interface.Studies have shown that many microbial strains have the ability to produce lipase.However,few strains have high lipase activity.In order to enrich the existing resources of high-efficiency lipase-producing strains and meet the needs of industrial application.The screening of lipase-producing microorganisms,optimization of lipase-producing conditions,separation and purification of lipase,investigation of enzymatic properties,cloning and heterologous expression of lipase genes were conclucted in this study.The main results are as follows:（1）Three lipase-producing bacterial strains were preliminarily screened from fermented sesame cake using neutral red oil plates.After re-screening with p-nitrophenol method,K-2was identified as the strain with high activity of lipase.After analysis of colony morphology,physiological and biochemical characteristics,16S r RNA gene cloning,sequence alignment and phylogenetic tree construction,the strain was identified as a strain of Pseudomonas aeruginosa.The single factor experiment was used to investigate the effects of culture time,the amount of inoculum,carbon source,nitrogen source,metal ions and the initial p H of the medium on the production of lipase by strain K-2.Three significant factors including fermentation time,inoculation amount and peptone addition were selected to optimize the enzyme production conditions using Box-Behnken response surface methodology,and the optimal fermentation conditions were obtained as follows:starch 5 g/L,peptone 11.3 g/L,Mg SO₄·7H₂O 1 g/L,KH₂PO₄1 g/L,p H 7.0,culture temperature 37°C,inoculation amount3%and culture time 67.5 h.Under these conditions,the lipase activity produced by the strain K-2 reached the maximum value of 271.28 U/m L.（2）The lipase produced by strain K-2 was isolated and purified by ammonium sulfate salting-out and Sephadex G-100 gel filtration chromatography.The specific activity of the lipase was 282.84 U/mg,the purification multiple was 11.5 times,and the recovery rate was23%.The optimum reaction temperature of that lipase was 40°C.The optimal reaction p H was 9.0,and the lipase was stable at p H 7.0-10.0.After 24 h incubation,the lipase activity remained more than 70%.The enzyme is a basic lipase,and its activity is inhibited by acidic conditions.1 m M Mg²⁺,Cu²⁺and Mn²⁺promoted the lipase activity.Fe³⁺and Zn²⁺inhibited the lipase.Ca²⁺had a significant activation on lipase.Lipase was tolerant to organic solvents to a certain extent.When the volume fraction of solvent is 10%,even strong polar solvents（acetonitrile and methanol）can promote lipase activity,and the relative enzyme activity is115.62%and 118.24%respectively.The optimal substrate of lipase was p-nitrophenol laurate（C12）,which could hydrolyze rapeseed oil,corn oil and peanut oil and produce fatty acids,making the alkaline oil mixture containing phenol red indicator turn yellow.（3）The lipase and molecular chaperone（Lip AH）gene of Pseudomonas aeruginosa K-2was cloned by PCR.The gene sequence was full-length 2008 bp,and encoded 668 amino acids,including two open reading frames,namely lipase gene and molecular chaperone.The cloned gene（Lip AH）was connected to the p ET-28a（+）plasmid vector and transformed into E.coli BL21（DE3）.The lipase activity reached the highest level of 123.16 U/m L when the concentration of IPTG was 0.3 m M,the induction temperature was 20°C,and the induction time was 24 h.According to SDS-PAGE analysis,most of the molecular chaperone of recombinant lipase existed in the form of inclusion bodies,which might make the lipase unable to fold properly,and thus led to the lipase activity produced by the engineering bacteria being lower than the optimal enzyme activity of the optimized wild bacteria.In this paper,a lipase-producing Pseudomonas aeruginosa strain was isolated and screened from fermented sesame cake.It was identified by comparing the colony characteristics,physiological and biochemical characteristics with the sequence of 16S r RNA gene,and the lipase-producing conditions were optimized to investigate the lipase-producing potential of the strain.Then,the enzymatic properties of lipase produced by the strain were studied and the engineering strain was constructed,which can provide strain resources and method reference for developing lipase products with low cost and high enzyme activity.