Directed Evolution Of Penicillium Expansum Lipase By Error-prone PCR

In order to improve the thermo-stability of the Penicillum expansum lipase, a library of 13000 mutants was created by error-prone PCR and transformation of the Pichia pastoris.50 mutants with lipase activity were identified through the high throughput olive oil agar plate lipase activity screen and 11 mutants were selected by the further flask shake culture and enzyme activity assay. The activity and thermo-stability of these 11 mutants were measured. The encoding genes of these mutants were amplified with PCR using the genomic DNA as templates and sequencing result indicate that all the 11 mutants has one to three amino acid substitution.(1) The optimum temperature, enzyme activity and Tm of GS-pPIC3.5K-lip07-ER04 were 10℃,22% and 3.28℃respectively higer than that of the wild-type enzyme.(2) The optimum temperature and the Tm of GS-pPIC3.5K- lip07-ER10 were 5℃and 2.15℃respectively higer than that of the wild-type enzyme.its enzyme activity is 950U/mL.(3) The optimum temperature of GS-pPIC3.5K- lip07-ER15 is similar to the wild-type enzyme. Its enzyme activity is 935U/mL.The Tm of this mutant is 2.28℃higer than that of the wild-type enzyme.(4) The optimum temperature and the Tm of GS-pPIC3.5K- lip07-ER17 were 5℃and 1.8℃respectively higer than that of the wild-type enzyme.Its enzyme activity is 1005U/mL.(5) The optimum temperature and the Tm of GS-pPIC3.5K- lip07-ER31 were 5℃and 2.25℃respectively higer than that of the wild-type enzyme.Its enzyme activity is 917U/mL.(6) The optimum temperature of GS-pPIC3.5K- lip07-ER38 is similar to the wild-type enzyme. Its enzyme activity is 1105U/mL.The Tm of this mutant is 2.81℃higer than that of the wild-type enzyme.(7) The optimum temperature of GS-pPIC3.5K- lip07-ER45 is similar to the wild-type enzyme. Its enzyme activity is 1060U/mL.The Tm of this mutant is 2.26℃higer than that of the wild-type enzyme. (8) The optimum temperature of GS-pPIC3.5K- lip07-ER50 is similar to the wild-type enzyme. Its enzyme activity is 425U/mL,about 42% lower than the wild lipase.(9) The optimum temperature of HR1,HR2,HR5 is similar to the wild-type enzyme. Their enzyme activity is 720U/mL,612U/mL,762U/mL respectively.In this study, we succeeded in using error prone PCR generate donzens of PEL mutants.Seven of them are the positive mutants with the thermostability and the enzyme activity improved. Studies are now under way to create more effective substitutions of the PEL that give rise to enhanced thermo-stability. The result indicates that the error-prone PCR is a good approach to improve enzyme's feature。...