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Title page for etd-0213115-111259


URN etd-0213115-111259 Statistics This thesis had been viewed 1077 times. Download 0 times.
Author Li-ming Huang
Author's Email Address No Public.
Department Bioengineering
Year 2014 Semester 1
Degree Master Type of Document Master's Thesis
Language zh-TW.Big5 Chinese Page Count 107
Title Biomimetic silicification of yeast D-amino acid oxidases mediated by ubiquitin and ubiquitin fusion protein
Keyword
  • ubiquitin
  • Biomimetic silicification
  • D-amino acid oxidases
  • fusion protein
  • fusion protein
  • D-amino acid oxidases
  • Biomimetic silicification
  • ubiquitin
  • Abstract In this study, ubiquitin and ubiquitin fusion proteins (U-R1, U-R2 and U-R5) heterologously expressed by E. coli were used to mediate biomimetic silicification for immobilization of Rhodosporidum toruloides and Trigonopsis variabilis DAO (RtDAO and TvDAO). The biochemical properties of RtDAO and TvDAO before and after immobilization were aslo investigated. After adjusting the amount of DAO, ubiquitin fusion protein and magnetic nanoparticles (MNP) added, the optimal condition for biomimetic silicification was achieved by sequentially adding 50 mM potassium phosphate buffer (pH 8), 10 ?g DAO, 400??g MNP, 35 ?g ubiquitin fusion protein with a 10:1 molar ratio to DAO added and 0.1 M hydrolyzed trimethylmethoxysilane to make up 100 ?l mixture followed by incubation at 25oC for 5 min. The immobilization efficiency of RtDAO and TvDAO using either ubiquitin or ubiquitin fusion proteins as catalysts were >90% and the specific activities after immobilization were >95% of the original activities. The amount of silica formed with U-R5 as a catalyst was slightly more than that with ubiquitin. The pH optima of RtDAO and TvDAO after immobilization remained the same as those before immobilization , all being 8. The pH stabilities were also similar, but the relative activities of immobilized RtDAO and TvDAO at extreme pH were higher than those of the soluble forms. The temperature optima were all increased from 40 to 45oC. The Tm values of RtDAO and TvDAO were increased by12 and 2~5oC, respectively. With regard to resistance against H2O2 oxidative damage, the half-lives of immobilized RtDAO with ubiquitin and U-R5 were increased by 5.8 and 3.5 folds, respectively, while those of immobilized TvDAO were not improved. The assessment of repeated use indicated that immobilized RtDAO and TvDAO exhibited good reusability.
    Advisor Committee
  • I-Ching Kuan - advisor
  • Chi-Yang Yu - co-chair
  • Min-Kai Chern - co-chair
  • Files indicate in-campus access at 5 years and off-campus access at 5 years
    Date of Defense 2015-01-29 Date of Submission 2015-02-13


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