Penicillium Expansum TS414 Lipase in Pichia Pastori: Expression, Purification and Enantioselective Esterification of (R, S)-naproxen
|School||Fujian Normal University|
|Course||Biochemistry and Molecular Biology|
|Keywords||Lipase Fermentation Purification Naproxen Immobilization Esterification Resolution|
This thesis has taken the expression, purification of Penicillium expansum TS414 lipase in Pichia pastori and the recombinant Penicillium expansum TS414 lipase-catalyzed enantioselective esterification of (R,S)-naproxen in the orgnic media as the main lines and carried out the following researches:Firstly,the shake flask fermention conditons for X33-TS414-lip engineering bacterial were optimized.Conditions are as follows:pH:7.5;inoculated quantity:2OD,methanol concentration:0.75%,flask volume:10%,rotation speed:250rpm,temperature:30℃.Based on the conditons, three different methanol feeding strategies were carried out on the fermentation tank. The lipase’s activities of DO based strategy was 104U/ml,enzyme yield was 962U/l·h; chromotropic acid method to maintain a low concentration of methanol was 443U/ml,enzyme production rate was 4614.58U/l·h;Mixed feeding methanol-glycerol fed-batch under chromotropic acid method was 573 U/ml,enzyme production rate was 6821.43U/l·h.This optimized strategy resulted in the highest productivity (6821.43U/l·h),which is 5.51-fold higher than the DO-based strategy.Secondly, through three purification steps which are saturated ammonium sulfate precipitation, Sephacryl S-100 gel filtration chromatography and Phenyl Sepharose hydrophobic interaction chromatography, the recombinant Penicillium expansum TS414 lipase achieved electrophoretically pure,the ratio of enzyme activity from the initial 2988.03U/mg increased to 7909.74U/mg, which was increased 2.65 times, and its activity recovery was 39.69%.The results of the recombinant Penicillium expansum TS414 lipase were similar to the native lipase in kinetic resolution of (R S)-naproxen.Lastly,the esterification reaction for resolution of (R,S)-naproxen by the immobilized recombinant Penicillium expansum TS414 lipase was researched.The optimized conditions were as follows:1.Immobilization conditions:Tianjin Guangfu AB-8 macroporous resin,the resin and the enzyme mass ratio:1:8,pH:9.0,immobilized adsorption time:1.5h, lyophilizing:lh.2.Resolution of esterification reaction:the amount of immobilized enzyme was 26.7g/l,the amount of racemic naproxen was1.48mg, n-propanol concentration was 7.73μl,system’s water content was 0.05%.Under the optimum conditions reacted 120h, the percent conversion and enantiomeric selectivity respectively reached 48.27% and 98.01%. The effect of recombinant Penicillium expansum TS414 lipase-catalyzed enantioselective enantification of (R,S)-naproxen was better.