碩士 / 國立中興大學 / 食品暨應用生物科技學系所 / 99 / Trehalose is a kind of non-reducing disaccharide. It is widespread in organisms capable of survival in stress conditions such as dehydration, heat, cold and radicals. Thus, trehalose is widely applied in cosmetics, pharmaceuticals and food industries. The novel production of trehalose is privileging enzymatic biotransformation. Picrophilus torridus trehalose synthase (PTTS) can convert maltose into trehalose by single step. PTTS with high trehalose conversion rate shows high activity under acid and thermal conditions. It had been over expressed in Escherichia coli Rosetta-gami B(DE3)/pET-23a(+)-PTTS. For the convenience of application in industry, E. coli Rosetta-gami B(DE3)/pET-23a(+)-PTTS was cultivated without addition of antibiotics. The producing crude PTTS were applied for trehalose production. However, the trehalose converted by crude PTTS was degraded. Besides, the productivity of PTTS was also unstable. Therefore, there may be a problem of plasmid instability. To reduce degradation of trehalose, we tried to knock out trehalase gene in E. coli by chromosome engineering. We also tried to enhance the expression of E. coli rare codons and ingrate ptts into chromosome by chromosome engineering to establish recombinant TS06(DE3)/HK-T7PTTS in purpose of stable productivity.
The results showed knocking out of trehalase gene in E. coli reduced trehalose degradation and reached 65% conversion rate by using crude PTTS. Enhancing of expression of E. coli argU and ileX, the expression of E. coli rare codon tRNA of AGG/AGA and AUA was promoted used to replace pRARE. The PTTS productivity was also approved and reached 125 mg/L. By integration ptts into chromosome, the recombinant TS06(DE3)/HK-T7PTTS was created and produced PTTS stably with maximum productivity of 155 mg/L PTTS (4.77 fold productivity of Rosetta-gami B(DE3)/pET-23a(+)-PTTS) with the aim of 1 mM lactose induction. In this study, we established a non-plasmid PTTS-producing strain successively. The recombinant can stable produce PTTS without addition of antibiotics that can applied for trehalose production without purification and show high productivity.
| Identifer | oai:union.ndltd.org:TW/099NCHU5253040 |
| Date | January 2011 |
| Creators | Hsien-Chung Lin, 林憲忠 |
| Contributors | 蕭介夫 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 65 |
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