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Cloning, Expression and Application of Acid Phosphatase from Escherichia Coli K12

Author: HuangYing
Tutor: OuLing
School: East China University of Science and Technology
Course: Biochemistry and Molecular Biology
Keywords: Escherichia coli acid phosphatase inosine inosinic acid
CLC: Q78
Type: Master's thesis
Year: 2012
Downloads: 65
Quote: 0
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Abstract


Escherichia coli is able to produce a periplasmic acid phosphatase made of four 26-KDa identical subunits.The enzyme can dephosphorylate various organic phosphomonoesters, it can also catalyze transfer of phosphate from the organic phosphomonoesters to hydroxyl groups of nucleoside in proper condition.The gene AphA encoding acid phosphatase from Escherichia coli K-12 was cloned into expression vector pET-28a after the correct sequencing and alignment with BLAST. The recombinant plasmid (pET-28a-AphA) was transformed into Escherichia coli BL21(DE3). The protein expression of recombinant was little after induction with IPTG identified by SDS-PAGE. The recombinant plasmid (pET-28&-dAphA), which contained partial gene AphA sequence without serial encoding signal petide, was constructed successfully and transformed into E. coli and highly amount of enzyme protein was expressed after induction in 28℃.Through zymogram detection, it was found that the recombinant (pET-28a-AphA) didn’t have activity for the signal peptide could not be cutted correctly, but the recombinant (pET-28a-dAphA) showed high enzyme activity.The resuspended cells of pEY-2%di-AAphA could dephosphorylate pNPP or various 5’nucleotides , The results showed that the optimal temperature was 55℃and optimal pH was 5.0 and pNPP was the best phosphate donor. The hydrolysis activity of acid phosphatase was acitivated by several metal ions.To investgate the impact of temperature, pH and divalent metal ions on the conversion rate of inosinic acid from pNPP and inosine, resupended cells were used as crude enzyme. The results showed that at 37℃and pH 4.0～6.0 the conversion rate of inosinate could reach 30%, but low yield was obtained as the reaction proceeded for IMP was hydrolyzed by enzyme. In order to slow the degration of IMP, addition of 5 mM EDTA into the reaction solution can inhibit hydrolysis activity.Acid phosphatase can also catalyze the transfer of phosphate from pNPP into other nucleosides such as undine and adenosine. The conversion rate can reach 15%.

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