Directed Evolution Of Penicillium Expansum Lipase And Improvement Of Its Stability

In order to improve the stability of Penicillium expansum lipase(PEL),this study adopted the technology of site-directed mutagenesis to mutate the key points of the enzyme.Several mutants were screened out,and the enzymatic properties were analyzed.The main results were as follows.1?Construction of mutant library and screening of more stable mutantsPEL was mutated by designed degenerateprimers for 83?92 and 151 sites.And then mutant plasmids were transformed into E.coli competent cells,respectively.The mutants E83N?E83P?E83P?D92N and D151Y were screened out with higher stability than the wild-typePEL.2?Construction of multiple-copy engineering strainsThe PEL fragments were amplified by PEL-EcoR I primer and contained the EcoR I cleavage site.They were not connected to linearized and dephosphorylated plasmid pA0815 until they had been digested by EcoRl enzyme,precipitated by isopropyl alcohol and recycled.The recombination plasmids were then transformed into Top 10 competent cells.And the transformants were identified by the newly designed pAO-PEL primers by PCR.One-copy expression vector,pA0815-lip was obtained.The expression cassette containing 5'(Bgl II)-AOX-lip-TT-3'(BamHl)was obtained by digesting pA0815-lip with BglII and BamHI.The expression box was recovered by isopropanol precipitationandligatedwith BamHl digested and dephosphorylated pA0815-lip,then the recombinationplasmids were transformed intoToplO competent cells.The transformants were digestedwith BglIIandBamHI toobtain the correct2-copyexpression vector pA0815-21ip.Similarly,pA0815-31ip,pAO815-4lip expression vectors were obtained.The 4-copy expression vector pA0815-4lip was enzymatically cleaved by SalI and transformated into Pichia pastor is GS115.At last 4-copy engineering strains were identified by YPOM plates.3?Fermentation of and study on its enzymatic properties4-copy engineering strains were fermented.The lipases were collected and theirenzymatic properties were studied as follows.(1)In 37? test,the specific activities on PNBP of the E83V?E83P,?E83N?D92N and D151Y were2.1 times,0.9 times,2.6 times,3.0 times and 1.2 times more than that of the wild-type PEL.(2)The Tm values of wild-type and D151Y were about 40.7 ?,and the Tm values of E83N was about 39.2 ?,which was lower than that of wild-type(PEL).The Tm values of D92N,E83P and E83V were 44.6?,45.9?and46.3?,which were increased by 3.9?,5.2? and 5.6? respectively.(3)After incubation with 10%ethanol for 24h,the residual activities of E83N,D151Y and WT were 22.9%,36.3%and 33.2%,respectively.However,70%residual enzyme activity of E83P was remained,which was significantly higher than that of wild type(PEL).Also,the residual activities of E83V and D92N were higher than that of wild-Type.(4)The residual activities of WT?E83N?D151Y were almost zero after incubationat 40? for3h,but E83V?E83PandD92Nhadhigher residual activities.(5)E83V?E83P?E83N?D92NandD151 Yshowed differenttolerances in different organic solvents.The residual enzyme activity of WT was 24%under the incubation of methanol,while the residual enzyme activities of E83V,D92N and D151Y were 56%,45%and43%,respectively.After the incubation of ethanol,the residual activity ofWT was only 31%,while the residual activities of E83P?E83V and D92N were 66%,71%and 82%,respectively.After incubation in DMSO,the residual activitiesof WT and E83N were about 43%,while the residual activities of E83V and D151Y were 62%and 55%,which were a little higher than WT,but the residual activities ofE83P and D92N were decreased compared with WT.After incubation in acetone,the residual activities of WT?E83N and E83P were about 33%,while those of E83V?D92N and D151Y were 61%,54%and 62%,which were greatly increased.After incubation in acetonitrile,the residual activity of WT was 32%,the residual activities of E83P?D92N were decreased,while those of E83N?E83V and D151Y were a litter higher than that of WT.