Construction Of Mutant Strain Of Pseudomonas Aeruginosa NY3 With High Rhamnolipid Production And Optimization Of Fermentation And Isolation

Rhamnolipid?Rha?is a glycolipid biosurfactant,which has the advantages of high surface activity,low toxicity and biodegradability.Rhamnolipid can be widely used in the remediation of polluted environment,as well as as as as as additives for health care products,cosmetics,pharmaceuticals,food and detergents.At present,rhamnolipid is produced by Pseudomonas aeruginosa fermentation,but its industrial application is limited because of the complex synthesis mechanism and the high cost of isolation and extraction of rhamnolipid crude products.In this paper,Pseudomonas aeruginosa NY3was used as the tested strain.With the help of molecular biological means and instrumental analysis and identification technology,the main purpose was to increase Rha production and reduce the cost of extraction and purification.Relevant research work was carried out,which was of great significance for industrial production..Starting from the biosynthetic pathway,this paper studied the gene regulation mechanism of rhamnolipid production by NY3,constructed a rhamnolipid-producing NY3 mutant strain by DNA recombination technology,and optimized the fermentation conditions of rhamnolipid production by NY3 and the separation and purification of its products.The following results were obtained.?1?The phaG gene of Pseudomonas aeruginosa NY3 was amplified successfully.A suicide recombinant plasmid PEX18-phaG-Sm^R with anti-streptomycin gene and anti-phaG expression was constructed by DNA recombination technology.The recombinant plasmid was integrated into the genome of wild NY3 strain by triploid hybridization,and phaG gene deletion mutant NY3?phaG was successfully constructed.?2?The mutant NY3?phaG can ferment rhamnolipid with rapeseed oil as carbon source.Compared with the wild strain NY3,the yield and purity of rhamnolipid increased by 56%and 6.7%respectively,and the emulsifying performance also improved.?3?Response surface methodology was used to optimize the optimal medium formulation for Rha production by mutant NY3?phaG fermentation:carbon source?rapeseed oil?60g/L,nitrogen source(NaNO₃)5.69g/L,trace element 1.48mL/L,Na₂HPO₄ 0.75g/L,KH₂PO₄ 0.75g/L,MgSO₄·7H₂O 0.06g/L,CaCl₂·2H₂O 0.01 g/L,initial pH 7.5.Under the optimum conditions,the yield of rhamnolipid fermented by mutant increased by 54.5%.?4?From silica gel products with different particle sizes,thin layer silica gel was selected as the best column chromatographic filler for isolation and purification of rhamnolipid.The chromatographic column with this filler can not only separate monosaccharide and disaccharide rhamnolipids,but also remove the pigment impurities in rhamnolipids.Each eluent is 100 mL and the column flow rate is 2 mL/min.The di-rhamnolipids with purity of 87.68%can be obtained by eluting them in the order of chloroform,"chloroform:ethyl acetate=2:1","ethyl acetate:methanol=1:2","ethyl acetate:methanol".