Molecular Engineering And Catalytic Property Of Lipase From Pseudomonas Aeruginosa

Lipase is an important enzyme used for industry,which is widely used in the field of detergent,food,oil processing,pharmaceutical synthesis,cosmetics,paper manufacture and biodiesel preparation.The lipase from Pseudomonas aeruginosa has some features such as high activity,stability and selectivity,but the formation of active conformation of the lipase is dependent on its specific foldase.The expression of lipase from Pseudomonas aeruginosa and the optimization of induction condition,the directed evolution of lipase from Pseudomonas aeruginosa,site-saturation mutagenesis of lipase from Pseudomonas aeruginosa based on molecular docking,purification of recombinant liapse and catalytic synthsis of castanospermine derivatives were studied.The main contents in present paper include four parts.(1)Lipase and foldase from pseudomonas aeruginosa CS-2 were successfully coexpressed using dual expression plasmid pACYCDuet-1,and the best induction condition was induction temperature(6h),the concentration of IPTG(1.0mM),and induction temperature(37?).(2)The nine lipase mutants were obtained with a proceduce of the first screening and the second screening,using the method of directed evolution.The activities of three lipase mutants(R314Q,S187N/E200G/R314 Q and P104S)were 1.65,1.62 and1.54 times as compared with the wild type.(3)The lipase mutant R314 W was obtained,using a virtual screening based on molecular docking between substrate and enzyme.The activity of lipase mutant(R314 W)was 3.97 folds as compared with the wild type.(4)The lipase mutant(R314W)and the wild type were purifed by affinity chromatography,with a result of one band through electrophoresis.The synthesis of derivatives of castanospermine was performed by the purified wild type lipase,in which conversion rate of castanospermine was 95.3% and yield rate of 6-O-butanoyl castanospermine was 76.4%.The purified lipase mutant(R314W)gave rise to castanospermine conversion of 96.7% and 6-O-butanoyl castanospermine yield of 90.5%.