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The Expression Of Penicillium Expansum Lipase Gene In Saccharomyces Cerevisiae And Its Molecular Mutant

Posted on:2005-02-09Degree:MasterType:Thesis
Country:ChinaCandidate:S Q LiFull Text:PDF
GTID:2120360122497575Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Expression of two genes which encode mature PEL(Penicillium expansum lipasc) and pro-PEL, optimization of fermentation conditions were studied in this research. Meanwhile, the site-directed mutagenesis technology was used to introduce the mutation into PEL.The genes lip, lip07 encoding mature PEL and pro-PEL were cloned into expression vector respectively, and the rccombinant plasmids pVT102U/a-lip, pVT102U/a -L-lip07 were then transformed into the Saccharomyces cerevisiae S78 competent cell by eleclToporalion, the recombinant strain S78-pVT102U/a -lip, S78-pVT102U/a-L-lip07 was obtained.The active lipase were secreted into the culture after fermentation. The expression product had a molecular mass of 28kDa,which was similar to that of the PEL from Penicullium expansion PF898. The activity of lipasc from S78- pVT102U/a -lip was up to 20U/ml after 72h fermentation; The product of S78-pVT102U/a-L-lip07 was little, only formed the halo on the tributyrin plate.Different strategies (different volume of inoculation, different initial pH, different carbon sources, different medium concentration ) was applied to optimized the S78-pVT102U/a -lip fermentation condition, the optimum conditions as follow: The optimum culture medium composition was 3% yeast extraction, 3% tryptone, 2% sucrose;The volume of inoculation was about 1.3%; the initial pH was 7.0. The activity of lipase was up to 56U/ml under the optimal cultivation conditions.The 227th Serine were replace with proline using overlap extension PCR method. The mutated gene lip-S227P was ligated into the pPPIC3.5K, the recombinants plasmid pPPIC3.5K-lip-S227P was transformed into the Pichia pastoris GS115 competent cell by electroporation. We use plate to check the lipase activity ,the lipase activity is very low, Compared to the wild type , the mutant could not form the clear halo on the olive oil plate but tributyrin plate, it showed that the 227 th Serine was essential for the lipase activity.
Keywords/Search Tags:Penicullium expansion lipasc, Saccharomyces cerevisiae, optimization, expression, mutation
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