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Expression of Penicillium Expansum FS1884 Lipase Gene in Pichia Pastoris GS115 and Its Directed Evolution

Author: LiQiang
Tutor: ShiBiHong
School: Fujian Normal University
Course: Microbiology
Keywords: Penicillium expansum Lipase Site-directed mutagenesis Error-prone PCR Directed evolution
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 100
Quote: 3
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Abstract


The total RNA of Penicillium expansum FS1884 was extracted, and the complete cDNA of the lipase gene with a size of 858 bp was obtained by reverse transcription PCR (RT-PCR). The recombinant expression vector of pPIC3.5K-PEL was constructed and transformed into Pichia pastoris GS115 by electroporation. The recombinant lipase was expressed in P. pastoris and can secret the lipase into the culture medium when induced by methanol. The production of lipase was 0.6mg/ml. The lipase activity was determined by olive emusion plates and NaOH titration method. The recombinant lipase showed the highest specific activity of 2952U/mg, the optimum working temperature of 35℃, and the optimum pH of 9.4.Mutation sites were chosed through gene identity rule. Eight mutation sites which included Leul64Pro, Thr50Cys, Met220Val, Ile102Met, Ile102Val, Ile53His, Arg40Gly, and Ser92Ala were introduced into the wild type lipase gene by one-step site directed mutagenesis method, respectively. And all of the mutants were identified by DNA sequencing. Each mutant lipase was expressed in GS115 when induced by methanol. As the results, the specific activity of the mutant (M220V) recombinant lipase in GS115 (PEL-M220V-GS) was increased by 5% when compared with the wild type recombinant lipase in GS115 (PEL-GS) ; the thermostability of the mutant lipases of PEL-L164P-GS and PEL-I102V-GS were increased when compared with that of PEL-GS; the specific activity of the mutant of PEL-L164P-GS was 47% of that of PEL-GS, but the optimum working temperature of PEL-L164P-GS was reached to 40℃, which was 5℃higher than that of PEL-GS.A random mutant lipase gene libray was construced by error prone PCR technique. 4000 strains of GS115 containing random mutant lipases were primary screened. 300 strains were secondary screened based on lipase activity, thermostability and acid resistance, and 6 mutant strains were finally screened by flask. One mutant strains named ep3 was selected. The optimum working pH of ep3 lipase was pH9.4, which was the same as that of the wild type lipase (PEL-GS); the optimum temperature was 35℃and the specific activity of the lipase was 3440U/mg, which was 17% higher than that of the wild type lipase.

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