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Optimization of Recombinant Bacillus Thuringiensis Pyruvate Dehydrogenase Expression and Structure Prediction

Author: LiYan
Tutor: YuanJunLin
School: Central China Normal University
Course: Biochemistry and Molecular Biology
Keywords: pyruvate dehydrogenase of bacillus thuringiensis expression purification homology structure prediction
CLC: S482.2
Type: Master's thesis
Year: 2008
Downloads: 78
Quote: 0
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Abstract


Pyruvate dehydrogenase multi-enzyme complex（PDHc）,a member of TPP -dependent multi-enzymes,is present in most prokaryote and eukaryote.PDHc catalyses the oxidative decarboxylation of pyruvate,generating acetyl-coenzyme A and providing the primary energy for the organism.Pyruvate dehydrogenase catalyzing the only irreversible step is the first enzyme catalyzing these reactions.If it is inhibited,the organism metabolism will be interferred.In view of its important role in metabolism,the exploitation of inhibitors with low toxicity will create a new area for the research of inhibitor and activator.This study attempts to provide adequate enzymes for the screening of the inhibitors.In our early work,we have successfully expressed the pyruvate dehydrogenase of Bacillus thuringiensis in Escherichia coli.However,the expression product was in the inactive form of inclusion bodies.According to optimize the medium content,medium pH value,cell density,inducer concentration,induction temperature and induction time, the quantity of soluble product can achieve 5.52mg per 100ml culture,the activity 0.272U,when host cultured in TB+M9（1:1）medium（pH6.0-7.5）,induced with 0.04mmol/L IPTG at 21℃for 8h from the cell density 4-4.5(O.D600).Compared to cultured in LB medium（pH 7.2）,induced with lmmol/L IPTG at37℃for 3h from the cell density 0.6(O.D600),the quantity of soluble product achieving 0.945mg per100mL culture,the activity 0.09U,the quantity of soluble product increase about 484%and the activity increase about 200%.By affinity chromatography,every 100mL culture medium will get 0.168 mg recombinant enzyme.In pH6.6 buffer,the enzyme has about 125% relative activity,comparing with in pH7.0 buffer.Vmaxis 16.34μmol/min,Km is 2.14mmol/L.We also analyze the homology of the pyruvate dehydrogenase of Bacillus thuringiensis.The pyruvate dehydrogenase of Bacillus thuringiensis has 99%homology with pyruvate dehydrogenase of Bacillus cereus.Secondary structure prediction shows that,45.45%of helix and 11.23%of stand inαsubunit,43.87%of helix and 16.56% stand of inβsubunit.Hydrophilicity and Hydrophobicity analysis show thatαsubunit is hydrophilic whileβsubunit is hydrophobic.Motif analysis show pyruvate dehydrogenase of Bacillus thuringiensis may be have phosphorylation sites、myristoylation sites、 amidation sites and glycosaminoglycan attachment site.

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