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The defense date of the thesis is 2013-07-15
The current date is 2019-07-18
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URN etd-0715113-124133 Statistics This thesis had been viewed 1054 times. Download 0 times. Author Sheau-Shyang Chen Author's Email Address No Public. Department Bioengineering Year 2012 Semester 2 Degree Master Type of Document Master's Thesis Language zh-TW.Big5 Chinese Page Count 73 Title Recombinant Arabidopsis ENDO1: its catalytic function and the essential role of N-glycosylation Keyword ENDO1 N-glycosylation N-glycosylation ENDO1 Abstract Nuclease is an enzyme able to cleave the phosphodiester bonds between the nucleotide subunits of nucleic acids. Some nucleases prefer double-stranded nucleotides, while others prefer single-stranded nucleotides. In this study, we investigate the bi-functional nuclease named Endonuclease 1 (ENDO1). The Arabidopsis thaliana At1g11190 gene encoding an endonuclease was isolated and designated ENDO1 has been demonstrated to cleave all types of mismatches with a high efficiency.
We used Agrobacterium-mediated transformation 35SP::ENDO-His tag in A. thaliana to produce stable active enzyme with high yield. The ENDO1 was purified by ammonium sulfate salting out and His tag affinity chromatography. Purified ENDO1 was identified by LC-MS/MS after trypsin digestion. In substrate specificity assay, ENDO1 process activity to digest ssDNA, dsDNA and RNA, however, with a substrate preference for ssDNA and dsDNA. In biochemical characteristic assay, the optimal pH of ENDO1 enzymatic activity was pH 6. The optimal temperature of ENDO1 enzymatic activity was 70 ˚C with dsDNA as substrate, and it was 60 ˚C with ssDNA and RNA as substrate. The thermal stability of ENDO1 enzymatic activity was 60 ˚C. In addition, removing of sugar groups with PNGase F from ENDO1 has affected its enzymatic stability and activity.
Advisor Committee Chin-Wen Ho - advisor
Jei-Fu Shaw - advisor
Files Date of Defense 2013-06-24 Date of Submission 2013-07-15