Improving Thermostability Of The Penicillum Expansum Lipase By Molecular Mutagenesis

Penicillium expansum lipase (PEL) plays an important role in commercial production for its catalyze hydrolysis function. In order to improving the thermostability of the PEL, the lipase encoding gene was mutated by site-directed mutagenesis. Several recombinant vetors which contain double or triple mutant genes were constructed by overlap extension PCR using the cDNA of a random-mutant lipase ep8 (a single site mutant) as the template and two special primers were used to generate mutation site. The recombinant vectors were transformed into Pichia pastoris GS115 by electroporation and corresponding recombinant expression cells PEL-ep8-K56R-GS,PEL-ep8-K56R-GS,PEL-ep8-P90D-GS,PEL-ep8-E207P-GS,PEL-ep8-E221P-GS,PEL-ep8-K226A-GS,PEL-ep8-P82D-GS,PEL-ep8-E113P-GS,PEL-ep8-I164S-GS,PEL-ep8-K115P-GS,PEL-ep8-S139R-GS,PEL-ep8-N157Y-GS,PEL-ep8-K56R-K115R-GS were created. The corresponding mutant lipase PEL-ep8-K56R,PEL-ep8-P90D,PEL-ep8-E207P,PEL-ep8-E221P,PEL-ep8-K226A,PEL-ep8-P82D,PEL-ep8-E113P,PEL-ep8-I164S,PEL-ep8-K115P,PEL-ep8-S139R,PEL-ep8-N157Y,PEL-ep8-K56R-K115R were obtained by flask shaking and the thermostability of these mutant lipases were measured.(1) The Tm of PEL-ep8-K56R is 40.1℃,1.4℃higher than that of wild-type lipase and 0.2℃higher than that of random-mutant lipase. The expression level of the double-mutant lipase is 852U/mL(2) The Tm of PEL-ep8-P90D is 38.8℃, which is close to wild-type lipase. The expession is about 584 U/mL(3) The Tm of triple-mutant PEL-ep8-K56R-K115R is37.5℃,2.5℃lower than that of wild-type lipase. The expression level is 56U/mL.(4) Other double-mutants PEL-ep8-E207P,PEL-ep8-E221P,PEL-ep8-K226A,PEL-ep8-P82D,PEL-ep8-E113P,PEL-ep8-I164S,PEL-ep8-K115P,PEL-ep8-S139R及PEL-ep8 N157Y did not show lipolytic activity when they tested on an olive oil agar plate.In summry,13 mutant vectors were constructed and transformed into the pichia posteris. Three of mutants have lipase activity.