Improvement Thermostability Of The Penicillum Expansum Lipase By Double-mutagenesis

In order to improve the thermostability of the Penicillium expansum Lipase (PEL),the lipase encoding gene was mutated by site-directed mutagenesis. Several recombinantvectors which contain double mutant genes were constructed by overlap extension PCRusing the cDNA of a random-mutant lipase ep8 (a single site mutant) as the template andtwo special primers were used to generate another mutation site. The recombinant vectorswere transformed into Pichia pastoris GS115 by electroporation and correspondingrecombinant mutant GS-pAO815-ep8-K115R,GS-pAO815-ep8-K55R,GS-pAO815-ep8-E214P,GS-pAO815-ep8-S104V,GS-pAO815-ep8-S139V,GS-pAO815-ep8-N166E,GS-pAO815-ep8-G205P,GS-pAO815-ep8-A170P,GS-pAO815-ep8-A144P,GS-pAO815-ep8-A105P,GS-pAO815-K115R were generated.These recombinant mutants can secret double mutant lipases into the medium whenit was induced by Methanol. Three of these mutant lipases got the thermostabilityimproved.1. PEL-ep8-K115R-GS, generated by introducing a mutation of K115R intoPEL-ep8-GS, had an optimum temperature 5℃lower than that of the wild type lipasePEL-GS and PEL-ep8-GS. While the Tm of it was 3.5℃higher than PEL-GS and 2.0℃higher than PEL-ep8-GS. The expression yield of the strain was 696U/mL, 480ug/mL,and the specific activity was 1449.0U/mg.2. PEL-ep8-E214P-GS, another double mutant lipase generated by introducing aE214P mutation into PEL-ep8-GS, showed the Tm was 4.5℃higher than that ofPEL-GS and 3.2℃higher than that of PEL-ep8-GS. The expression yield of the strainwas about 645U/mL, 220ug/mL, and the specific activity was 2931.8U/mg.3. PEL-ep8-K55R-GS also had the stability improved with the value of Tm 2.3℃higher than that of PEL-GS and 0.8℃higher than PEL-ep8-GS. The expression levelwas about 508U/mL, 220ug/mL, and the specific activity was 2309.1U/mg.Three of the created mutant lipases did not get the thermostability improved but theycan be secreted to the medium properly.1. The Tm of PEL-ep8-N166E-GS was 37.9℃, similar to the wild type. 2. The Tm of PEL-ep8-S104V-GS was 39.1℃,which was 1.5℃lower than thewild type. The expression level is about 455U/mL, 240ug/mL, and the specific activitywas 1897.3U/mg.3. The Tm of PEL-ep8-S139V-GS was 35.7℃, which was 5.4℃lower than thewild type.Four of double mutants did not show any expression level of lipase when checkedby SDS-PAGE and activity measure. They are PEL-ep8-G205P-GS, PEL-ep8-A170P-GS,PEL-ep8-A144P-GS and PEL-ep8-A105P-GS. These mutant lipases were not secreted tothe medium.In summary, there are 10 double mutant lipases were created by site-directedmutagenesis in order to improve the thermostability of the lipase. Three of them got thethermostability improved. Three of them did not get the thermostability improved but canbe secreted to the medium properly.And four of them did not show any expression levelof lipase and were not secreted to the medium.