Heterologous Expression Of Penicillium Expansum Lipase Gene And Molecular Mutagenesis

Penicillium expansum lipase(PEL)plays an important role in industry for its catalytic characters.Pichia pastoris is a very good eukaryotic expression system that can stably express heterologous proteins at high level.Expression with the GAP promoter(P_GAP)is methanol-free and sometimes the level of expression can be slightly higher than that obtained with the AOX1 promoter.PEL coding gene was cloned and introduced into yeast integrative plasmid pGAPZA and then transformed into Pichia pastoris GS115 to construct a recombinant which expressed lipase using glyceraldehydes-3-phosphate dehydrogenase gene(GAP)as promoter.The lipase expression level of the recombinant stain was up to 221.5U/ml when glycerol was used as carbon source.For studing thermostability of PEL,nine mutants were created by site-directed mutagenesis based on the structure of the protein.A mutant of PEL-ep8-K202A was obtained which thermostability shows 2.63℃higher than that of the wild-type.1,Overexpression of PEL gene in Pichia pastorisThe Penicillium expansum lipase(PEL)encoding gene was cloned and introduced into the yeast integrative plasmid pGAPZA.By using Zeocin as a selection reagent,the recombinant plasmid pGAPZA-lip07 was selected and transformed into Pichia pastoris GS115 to construct a recombinant stain GS-pGAPZA-lip07 which expressed lipase using GAP as promoter.The expression product—recombinant PEL shows lipolytic activities when it was tested on an olive oil agar plate or measured by titration of NaOH. SDS-PAGE analysis of the recombinant protein shows that the molecular mass of the recombinant protein was about 28KD,which is the same with that of the wild-type PEL-lip07.The lipase expression level of the recombinant stain was up to 221.5U/ml when glycerol was used as carbon source.2,Site-directed Mutagenesis of PELNine mutation genes was created by site-directed mutagenesis using wild type lipase gene(lip07)and the random-mutant gene ep8,which shows higher thermostability,as templates.The mutant genes were subcloned into pAO815 or pPIC3.5K,and then transformed into the Pichia pastoris GS115 for extracelluar expression under the control of the AOX1 promoter.(1)K202A single mutant and double mutantThe PEL-ep8-K202A,a mutant obtained by using ep8 as template,has an optimum temperature 2℃higher than that of the wild-type.The Tm of it is 2.63℃higher than PEL-lip07 and 1.21℃higher than PEL-ep8.The expression yield of the strain is 504.7U/ml,260.0μg/ml,and the specific activity is 1941.0U/mg.while the mutant PEL-lip07-K202A.a mutant abtained by using lip07 as template,has optimum temperature of similar to the wide- type PEL-lip07,while the thermostability is 1.95℃lower than that of wide type.The expression yield is about 125.0μg/ml,the secreted activity is about 273.0U/ml and the catalytic activity is about 2184.0U/mg.(2)N73T double mutantThe Tm of PEL-ep8-N73T is 38.48℃,which is 0.55℃lower than the wild type.The optimum temperature of PEL-ep8-N73T was 5℃lower than the wild type.The expression level is about 71.0U/ml,40.0μg/ml,and the specific activity is 1775.0U/mg.(3)S153P,Q176P single mutantThe Tm of PEL-lip07-Q176P and PEL-lip07-S153P is 37.81℃and 36.36℃,lower than that of the wide-type.But the optimum temperature is similar to the wide type.(4)K202E,S91K,H107R,N157Y single mutantThe four single mutant,PEL-lip07-K202E,PEL-lip07-S91K,PEL-lip07-H107R and PEL-lip07-N157Y did not show lipolytic activities when they were tested on an olive oil agar plate and activity measure.