Cloning and Expression of A Cold-adapted Lipase Gene from Geotrichum candidum ch-3 in Pichia pastoris

Objective: Lipase is one of primary enzymes applied in industry in the world.To clone and express a cold-adapted lipase gene from Geotrichum candidum ch-3 by gene engineering.Methods: A cold-adapted lipase gene lip was amplified with PCR from the genomic DNA of G.candidum ch3,and it was then linked into pBluescriptⅡSK(+) vector.After it was confirmed by sequencing analysis,it was correctly inserted into the expression vector pPIC9α of Pichia pastoris.Then the recombinant pPIC9α plasmid with target gene was induced and expressed in P.pastoris.Results: The lip gene was expressed successfully by using P.pastoris as host strain and the activity of it was 37U/mL.SDS-PAGE analysis showed that the molecular weight of the lipase protein was about 62kDa,larger than the theoretical value.This result indicated that the expression protein of lip gene might be glycosylated.The optimal temperature for recombinant lipase was 35℃.At 0℃,the enzyme still remained 66% of its activity.It was thermoliable. Conclusion: The successful expression of cold-adapted lipase in recombinant P.pastoris was realized,and it will be the foundation for the study on catalytic reaction mechanism of cold-adapted lipase and its industrialized application.