碩士 / 國立中興大學 / 生物科技學研究所 / 98 / Trehalose has major biological functions in organisms, and has been made widely applicable to fields such as food, cosmetic, and pharmaceutical industries. For industrial applications, the high thermostability of trehalose synthase is the most important property because it can reduce the microbial contamination and lower the production cost. A recombinant trehalose synthase (PTTS) from a hyperacidophilic, thermophilic archaea, Picrophilus torridus maintained high activity at pH 5.0 and 60℃. Also, an N503P mutation on PTTS showed about 39% higher relative activity than that of the wild type at 65℃ for 120 min. To investigate the structural basis for thermostability of PTTS, we expressed recombinant trehalose synthase in E.coli and purified the protein by Ni2+ column. We did the crystallization screening for the purified protein. Besides, purified wild-type and mutated N503P trehalose synthase were used to carry out the experiments of “circular dichroism”, and ”dynamic light scattering” respectively in order to examine the structural transformation of trehalose synthase in high temperature. We found that both wild type and mutation of N503P of α-helix content maintained a major structure under 55℃. In the dynamic light scattering assay, trehalose synthase was probably an oligomer at room temperature. The result of analytical ultracentrifugation showed that wild type protein existed as monomer, dimer and tetramer form under 20℃, so does mutation N503P-PTTS. In the meantime, we designed and expressed several PTTS mutants, and tested the mutants via similar experiments. We found that T251R-PTTS was more therrmostable than wild type PTTS and the ratio of by product glucose of S505R-PTTS was lower than wild type PTTS. Through these studies, we hope the accumulating results could help us to understand the mechanism of thermostability of PTTS.
| Identifer | oai:union.ndltd.org:TW/098NCHU5111018 |
| Date | January 2010 |
| Creators | Chun-Hsiu Hung, 洪春琇 |
| Contributors | Jyung-Hurng Liu, 劉俊宏 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 79 |
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