Cloning and characterization of AAD17422 and AAC06164, two novel esterases from Arabidopsis thaliana
Petrova, Yessela Yourieva
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The genes for AAD17422 and AAC06164 from Arabidopsis thaliana have been successfully cloned into pET 16b bacterial vector, and the his-tagged proteins were overexpressed in Escherichia coli. Our study shows that to avoid precipitation of the two proteins, 20% glycerol is required in all purification and storage buffers. In addition, the kinetic constants for the hydrolysis of p-nitrophenyl butyrate by the two proteins were obtained. The Km for AAD17422 was 4800±1800 uM and the Vmax was 56000±20000 nmol/min mg. The Km for AAC06164 was 73±24 uM and the Vmax was 3300±450 nmol/min mg. AAC06164 was more extensively characterized, and its affinity for the butyrate acyl chain moiety over the acetate and decanoate was demonstrated. Finally, the results of the inhibition assays showed that AAC06164 was inhibited by PMSF, partially inhibited by neostigmine and stimulated by DTT. Based on the information about the primary and secondary structure of the protein and kinetic and structural data about other hydrolases, an explanation for the effect of pH and DTT is proposed.
Franklin and Marshall College Archives, Undergraduate Honors Thesis 2005
- F&M Theses Collection