The Construction Of Engineered Strains Containing Multiple Lip Copies And Site-directed Mutagenesis Of Psychrophilic Lipase

In this paper, the multi-copy expression vectors containing Penicillum expansum lipase(PEL) gene-lip were constructed in vitro and then the multi-copy engineered strains were obtained after the vectors were transformed into Pichia pastori by electroporation, the higher-level expression of lip was achieved in Pichia pastoris. The expression field and activity of lipase of three copy strain was up to 800μg/ml and 1199U/ml respectively, the activity was about 3.6-folds higher than that of the presious constructed strains. site-directed mutagenesis of PEL was carried out and a psychrophilic mutant was obtained, of which the thermostability was better and the optimum temperature was lower by 10.0℃ than that of wild type.1. Construction and expression of multi-copy engineered strainsLip was cloned into expression vector pAO815, and the multi-copy expression vectors pAO815-xlip(x=1,2,3,4) containing 1-4 lip copies were obtained, which were then transformed into GS115 by electroporation. The multi-copy engineered strains GS-pAO815-xlip (designated xcopy, x=1,2,3,4) were obtained after screening and then expressed by methanol induction in shaker-flasks. The expression protein of lip was analyzed by SDS-PAGE gel and the activity of lipase was assayed, the result indicated that lipase was functionally expressed in 1 -4 copy and up to 95% of the secreted protein; the expression field (800μg/ml) and the activity (1199U/ml) of lipase secreted of 3copy were best, which was about 73.9% and 77.6% higher than that of single copy respectively and the activity was about 3.6-fold higher than that of the presious constructed strains (260 U/ml), so the higher-level expression of lip was achieved in Pichia pastoris. Four different strategies were applied to optimized the cultivation condition for 3copy in shaker flasks, the result indicated that the optimal medium initial pH was 8.0, the optimal cell density before inducing was 1.0 (A600), the optimal methanol concentration was 1.5% and the optimal induction time was 120h.2. Site-directed Mutagenesis of PELPXD,P150S,I164A,PXD/ep8(multiple sites) and P239A of lip were mutanted by overlap extension PCR and megaprimer PCR method respectively. The mutant genes were expressed in Pichia pastoris and five mutants were obtained. The results of the two obvious characterize of mutants showed that: (1) The optimum temperature of the...