Cloning and characterization cinnamyl alcohol dehydrogenase from Chlorella sorokiniana T89
| Autor: | Jia-Hui Lee, 李加惠 |
|---|---|
| Rok vydání: | 2011 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 99 A cDNA encoding a cinnamyl alcohol dehydrogenase (CAD) gene with 1155 mucleotides was isolated from Chlorella sorokiniana T89. Recombinant CAD protein was expressed in Escherichia coli as a C-terminal histidin-tagged fusion protein and purified by DEAE-Sephacel ion exchange chromatography and TALON affinity chromatography. By these steps, the CAD was purified 1250 fold over the crude protein, and the total recovery was 3%. The optimal pH for the recombinant CAD protein was 6.5, and the optimal temperature was 33 °C. 1 mM Co2+, 1 mM Ni2+ and 1 mM Cu2+ significantly inhibited the activity of the enzyme. 1% 2-Mercaptoethanol, 1 mM dithiothreitol, 1 mM ethylenediaminetetraacetic acid, 0.1% sodium dodecyl sulfateand 2 M urea significantly inhibited the activity of th enzyme. The recombinant CAD protein sequence analysis and prediction three-dimensional structure. There were some features of recombinant CAD protein. First, it may be combination of two zinc ions which related to the structural zinc ion and catalytic zinc ion. Second, the [GX(X)GXXG] motif may be the NADP+-binding domain. Finally, two pairs of disulfide bonds may form between C131 and C134, C75 and C191, respectively. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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