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Title page for etd-0817107-151040


URN etd-0817107-151040 Statistics This thesis had been viewed 1921 times. Download 12 times.
Author Liao-Ren Jie
Author's Email Address No Public.
Department Bioengineering
Year 2006 Semester 2
Degree Master Type of Document Master's Thesis
Language zh-TW.Big5 Chinese Page Count 84
Title Properties of D-amino acid oxidase
immobilized through His-tag
Keyword
  • DAO
  • His-tag
  • His-tag
  • DAO
  • Abstract D-amino acid oxidase (DAO; EC 1.4.3.3) is one of the dehydrogenase-oxidase enzymes containing FAD as the prosthetic group. DAO catalyzes the specific oxidation of D-amino acids to produce the corresponding α-keto acids with hydrogen peroxide and ammonia as by-products. The most important industrial application of DAO is the production of 7-aminocephalosporanic acid (7-ACA). The immobilization of DAO is often performed using chemical methods. In this thesis, recombinant Rhodosporidium toruloides DAO (RtDAO) and Trigonopsis variabilis DAO (TvDAO) fused C-terminus His-tags were expressed and purified. RtDAO and TvDAO were then immobilized on Ni-NTA Magnetic Agarose Beads through the interaction between His-tag and Ni2+. The effects of temperature, pH, and the presence of hydrogen peroxide were studied for both free and immobilized enzymes. In addition, enzyme kinetics, storage stability, reusability of the immobilized DAO were also discussed. The immobilized RtDAO exhibited significant improvements in stability against thermal and hydrogen peroxide inactivation. After incubation at 50℃ for 1 h, the relative activity of the immobilized RtDAO was 56% while the free RtDAO was completely inactivated. In the presence of 10 mM hydrogen peroxide, the residual activities of free and immobilized RtDAO after 9 h incubation were 21.8 and 72%, respectively. The immobilized RtDAO still retained 35.2% activity even after 15 h of incubation. After storage at room temperature for 10 d, the residual activity of immobilized RtDAO was 73.5% compared to the total loss of activity of the free enzyme. No improvements in stability were observed for immobilized TvDAO.
    Advisor Committee
  • Chi-yang Yu - advisor
  • Files indicate in-campus access at one year and off-campus not accessible
    Date of Defense 2007-07-24 Date of Submission 2007-08-17


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