| In order to improve the enzyme properties of the Penicillum expaasum lipase(PEL), the PEL gene was mutated by site-mutagenesis and random mutagenesis. 1. Site-directed mutagenesis of PELIn order to improve the thermostability of Penicillium expansum lipase (PEL), Eleven mutations were created by site-directed mutagenesis. Mutations were generated by overlap extension PCR using the cDNA of wild-type lipase gene(lip07) or a single site mutant lipase ep8 as the template and two special primers that corresponding to mutations. Recombinant vector which contain mutant genes was constructed and electroporated into Pichia pasteris GS115.(1) PEL-P197E single mutant and PEL-ep8-P197E double mutant Double mutant PEL-ep8-P197E was created by mutate Pro to Glu at 197 based on the single mutant PEL-ep8. The double mutant shows a good improvement in thermostability with a Tm of 41.5℃, which is 2.8℃higher than the wild-type PEL and 2.2℃higher than single mutant PEL-ep8.PEL-P197E is a single mutant of PEL at amino acid 197. The Tm of this mutant is 39.5℃which is 0.9℃higher the PEL.After three days methanol induced expression, the enzyme activity of single mutant and double mutant are 556U/mL and 592U/mL respectly.In addition, the substrate specificity of PEL-ep8-P197E is changed.(2) PEL-K152R single mutant and PEL-ep8-K152R double mutantDouble mutant PEL-ep8-K152R was created by mutate Lys to Arg at 152 based on the signal mutant PEL-ep8. The Tm of this mutant is 38.9℃which is similar to that of the wild-type enzyme.PEL-K152R is a single mutant of PEL at amino acid 152. The Tm of this mutant is 38.9℃which is similar to that of the wild-type enzyme.After three days methanol induced expression, the enzyme activity of single mutant and double mutant are 530U/mL and 416U/mL respectly.(3) PEL-P102L single mutant and PEL-ep8-P102L double mutant Double mutant PEL-ep8-P102L was created by mutate Pro to Leu at 102 based on the single mutant PEL-ep8. The double mutant shows a good improvement in thermostability with a Tm of 40.9℃, which is 3.6℃higher than the wild-type PEL and 2.6℃higher than single mutant PEL-ep8. The optimum temperature of PEL-ep8-P102L is 45℃,5℃higher than that of the wild-type enzyme.PEL-P102L is a single mutant of PEL at amino acid 102.After three days methanol induced expression, the enzyme activity of single mutant and double mutant are 300U/mL and 612U/mL respectly.(4) PEL-N166D single mutant and PEL-ep8-N166D double mutantDouble mutant PEL-ep8-N166D was created by mutate Asn to Asp at 166 based on the single mutant PEL-ep8. The double mutant shows reduced in thermostability with a Tm of 36.2℃, which is 1.1℃lower than the wild-type PEL and 2.1℃lower than single mutant PEL-ep8.PEL-N166D is a single mutant of PEL at amino acid 166.After three days methanol induced expression, the enzyme activity of single mutant and double mutant are 236U/mL and 252U/mL respectly.(5) PEL-ep8-P163A double mutantDouble mutant PEL-ep8-P163A was created by mutate Pro to Ala at 163 based on the single mutant PEL-ep8. The double mutant shows a good improvement in thermostability with a Tm of 38.3℃, which is 1.0℃higher than the wild-type PEL and the same to single mutant PEL-ep8.(6) PEL-ep8-K120R,PEL-ep8-M193V double mutantDouble mutant PEL-ep8-K120R was created by mutate Lys to Arg at 120 based on the signal mutant PEL-ep8. The double mutant shows reduced in thermostability with a Tm of 35.8℃, which is 1.5℃lower than the wild-type PEL and 2.5℃lower than single mutant PEL-ep8. The optimum temperature of PEL-ep8-K120R is 35℃,5℃lower than that of the wild-type enzyme.The enzymatic activity of both PEL-ep8-K120R and PEL-ep8-M193V were decreased dramatically.(7) PEL-G205A,PEL-K94D,PEL-K94R,PEL-W169N single mutant All of the four single mutations, PEL-G205A, PEL-K94D, PEL-K94R and PEL-W169N did not show lipase activity when they were tested on an olive oil agar plate and activity measure.2. Random mutagenesis of PEL by error-prone PCRThe PEL gene was mutated by random mutagenesis using error-prone PCR.32 positive clones were identified from among about 8000 transformants by agar plate and two thermostable mutations were obtained from 32 positive clones by a second selection.HR2 shows a good improvement in thermostability with 39.3℃, which is 0.6℃higher than the wild-type PEL. After three days methanol induced expression, the enzyme activity of HR2 is 498U/mL. Only one amino acid substitution of Lys82Arg was identified by DNA sequencing.HR3 shows a good improvement in thermostability with 42.1℃, which is 3.4℃higher than the wild-type PEL. After three days methanol induced expression, the enzyme activity of HR3 is 552U/mL. The DNA sequencing result indicated that there are two substitutions, Leu68Pro and Lys142Arg.In synthesis,15 mutant were constructed by site-directed mutagenesis, and two good mutant were obtained.①PEL-ep8-P197E shows a good improvement in thermostability with a Tm of 41.5℃, which is 2.8℃higher than the wild-type PEL and 2.2℃higher than single mutant PEL-ep8.②PEL-ep8-P102L shows a good improvement in thermostability with a Tm of 40.9℃, which is 3.6℃higher than the wild-type PEL and 2.6℃higher than single mutant PEL-ep8. In addition, one thermostable mutant(HR3) was obtained by error-prone PCR. HR3 shows a good improvement in thermostability with 42.1℃, which is 3.4℃higher than the wild-type PEL, the enzyme activity of HR3 is 552U/mL.
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