Optimizing Fermentation Conditions Of Engineering Lipase Strains And Site-directed Mutagenesis Of Penicillum Expansum Lipase

In order to increase the expression level,two,three and four-copy lipase expression vectors were constructed and introduced into the yeast cells.Some conditions related to cell growth and lipase expression such as the medium ingredient,cell density,methanol feeding,medium initial pH and period of the fermentation were investigated by cultivating cells in shaker flasks and a 7 L fermentor,the higher-level expression of lip was achieved in Pichia pastoris.Site-directed mutagenesis of PEL was carried out and one mutant shows that the optimum temperature was lower 5.0℃and thermostability was better than that of wild type.1.Optimize the expression condition of multi-copy engineered strainsSome conditions related to cell growth and lipase expression such as the medium ingredient,cell density,methanol feeding,medium initial pH and period of the fermentation were investigated by cultivating cells in shaker flasks and a 7 L fermentor. The result indicated that the suitable fermentation condition for the recombinant cell and lipase expression is below:the medium initial pH is about 7.0 to 8.0,the temperature is 28 deg C for cell growth and 26 deg C for lipase expression,the methanol inducing should be started at a cell density of 1.0(OD₆₀₀),the methanol feeding is in a volume ratio of 1.5% to the medium per day,and the fermentation time is 120h.In a 7L fermentor cultivate for 60h the lipase yield is 0.92mg/ml,1250U/ml.The activity was about 4.8-folds higher than that of the previous constructed strains.2.Site-directed Mutagenesis of PELQ176V,Y204D of lip were mutanted by overlap extension PCR respectively.The mutant genes were expressed in Pichia pastoris and two mutants were obtained.(1)The optimum temperature of the mutant PEL-Q176V-GS was 5.0℃lower than that of PEL-GS, while the thermostability was 0.7℃higher,the expression field was about 300μg/ml,the secreted activity was about 392U/ml;(2)No lipase activity and yield was detected in supernatant of the mutant PEL-Q176V-GS.