Study On Overexpression And Regulation Mechanism Of Pseudomonas Lipases

Lipases are an important class of industrial enzymes,which are widely used in chemicals,food,medicine,detergents,textiles,leather,new materials,dairy products,cosmetics,paper making,pollution treatment and bio-energy industries.Among these lipases,bacterial lipases have more types of catalytic reactions,higher reactivity and more stability than other lipases in aqueous and organic phases,especially Pseudomonas lipases possess the best merits.However,although bacterial lipases have many advantages,their yields are generally low.Conventional breeding methods can not meet the large demand of industry,thus their market price is very expensive.By comprehensively analyzing the literatures,there are two strategies which can be used to improve bacterial lipase expression,for lipases which are not dependent on foldase or less dependent on foldase,heterologous expression is usually employed to enhance lipase production?for example,the lipase gene of Pseudomonas fluorescens?;for those strongly dependent on foldase,gene expression regulation mechanism is usually dug out and then using it to improve lipase expression?for example,the lipase gene of Pseudomonas aeruginosa?.Therefore,in this study,Pseudomonas lipase was used to explore the above-mentioned strategies.The main researches and results were summarized below:1.In the heterologous expression strategy,a predictive lipase gene Pflip1 from Pseudomonas fluorescens Pf0-1 was expressed both in Escherichia coli and in Pichia pastoris.The corresponding lipases Pflip1a and Pflip1b were obtained.After confirming they were lipases,the enzymatic properties of Pflip1a were totally investigated.Its optimum pH value was 8.0,optimum substrate was C8,and the optimum temperature was70�C.Then,the effects of various metal ions,EDTA,surfactantsand organic solventson the enzyme activity were studied.It was revealled that it had high tolerance to various metal ions(Na⁺,K⁺,Ca²⁺,Mg²⁺and Ba²⁺),EDTA,nonionic surfactants?NP-40,Triton X-100,Tween 20,Tween 40 and Tween 80?,and organic solvents?N-hexane,acetone,butanol,ethanol,glycerol and methanol?.The conversion of biodiesel was further studied by immobilizing the two lipases Pflip1a and Pflip1b onto magnetic nanoparticles.After 10h treatment,the conversion rates of the immobilized Pflip1a and Pflip1b were respectively80.5%and 68.5%;after 10 batches of running,the two immobilized lipases still retained their initial activities of 70%-82%.The enzymatic properties and biodiesel conversion results suggest that the lipase has great potential in organic synthesis,especially in the production of biodiesel.2.In the strategy of studying regulation mechanism of gene expression,Pseudomonas aeruginosa PAO1 were selected as the research host,the control group and olive oil induction group were employed to analyze the transcriptome and proteomics,respectively.Altogether,24 genes were found to have close relations with the expression of lipA.After further verification,two genes PmrA and PA1397 were the closest ones.Then,their detailed regulation pathways on lipA expression were carefully studied.I.Through deletion mutation of lipC,PmrA,rsmY and rsmA,overexpression of PmrA,rsmY and rsmA,fusion expression of lacZ,qRT-PCR analysis,EMSA and relative lipase activity determination,it has been proved for the first time that PmrA can directly bind to rsmY and then promote the expression of rsmY.RsmY can further inhibit the expression of rsmA,and then inhibit the translation process of lipA through rsmA.II.Through deletion mutation of lipC,PA1397,rsmZ and rsmA,overexpression of PmrA,rsmZ and rsmA,fusion expression of lacZ,qRT-PCR analysis,EMSA and relative lipase activity determination,it was found for the first time that PA1397 can directly bind to rsmZ and then promote the expression of rsmZ.RsmZ can further inhibit the expression of rsmA,and then inhibit the translation process of lipA through rsmA.This study of heterologous expression and regulation mechanism digging laid a solid theoretical foundation for the later construction of highly expressed bacterial lipase engineering strains,especially for the Pseudomonas lipase engineering strains.