STABILIZATION OF D-AMINO ACID OXIDASES FROM RHODOSPORIDIUM TORULOIDES AND TRIGONOPSIS VARIABILIS BY GENE FUSION AND IMMOBILIZATION
| Autor: | Shih-Juei Wang, 王詩瑞 |
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| Rok vydání: | 2008 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 96 D-Amino acid oxidases from yeast Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) are both homodimeric flavoenzymes. Two of their cDNA genes were connected by a hexanucleotide linker and heterologously expressed in E. coli to produce the corresponding double DAOs (dRtDAO and dTvDAO) made of only a single polypeptide. The specific activities of double DAOs remained similar to those of native dimeric DAOs. The optimal pH for activity and the best pH stability of double DAOs were both observed at pH 8.5. The Tm value for dRtDAO was shifted 5℃ higher while that for dTvDAO was increased only by 2℃, in comparison with the corresponding native counterparts. The improvement in thermal stability was further obtained by immobilization through the biotin-streptavidin binding onto streptavidin-coated beads. Immobilized double DAOs had 6℃ higher Tm values than their soluble counterparts. In the presence of 10 mM H2O2, dRtDAO and dTvDAO exhibited half-lives of about 60 and 45 min, respectively, which were 2 and 1.5 folds more stable than their native DAOs, respectively. The oxidative stability of double DAOs was also further enhanced after immobilization. The immobilized dRtDAO and dTvDAO exhibited half-lives of about 10 and 5 h, respectively, which gave 10- and 7-fold, greater stability than their soluble forms under the same oxidative condition. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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