碩士 / 國立臺灣師範大學 / 生命科學研究所 / 99 / Treholose is a non-reducing disaccharide composed of two glucose residues connected by an α,α-1,1-glucosidic linkage. It is widely present in plants, insects, fungi, and bacteria. Trehalose protects organisms against various environmental stresses, such as freezing, heat, and desiccation. This sugar has many applications such as a sweetener component, preservative, or stabilizer for food, cosmetics, and medicines. It has been produced on an industrial scale by the maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase (MTS-MTH) enzymatic system, and the cost has been reduced by using inexpensive starch to produce the expensive trehalose. Cyanobacteria Nostoc can grow under extreme conditions, and these cells accumulated trehalose upon desiccation. It has been proven that MTS and MTH was upregulated during drought stress in Nostoc punctiforme IAM M-15 and Nostoc flagelliforme. It is theoretically possible that MTS and MTH are involved in dehydration stress response in Nostoc by enhancing the synthesis of trehalose, and the intracellular accumulation of trehalose thus increases the resistances to desiccation. Recently, the complete genome of Nostoc punctiforme PCC 73102 (NP) has been sequenced, and there exist putative MTS and MTH genes whose products have not been characterized yet. We cloned the NPMTS, NPMTH, and NSMTH genes from the genomic DNA of Nostoc punctiforme PCC 73102 and Nostoc sphaeroides which was collected from Wulai of Taiwan. These recombinant enzymes was expressed in Escherichia coli. The recombinant NPMTS showed an optimal temperature of 30℃ and an optimal pH of 8.0. The enzyme was stable after one hour incubation at 10~30℃. The addition of Ca2+, K+, Mg2+, Na+, or Mn2+ had slightly activating effect on NPMTS activity, while the addition of Cu2+ and Zn2+ inhibited the enzyme activity at 1 mM. The transglycosylation activity of NPMTS was higher when using maltohexaose as a substrate than maltopentaose, maltotetraose, maltotriose, and amylase. However, the hydrolysis activity of NPMTS only appeared when using maltotriose as substrate. The Km of NPMTS for maltohexaose was 57.9 mM, and the Vmax was 1.9 U/mg. These results revealed the characterization of NPMTS, and provide important clues for further engineering of NPMTS in biotechnological applications.
| Identifer | oai:union.ndltd.org:TW/099NTNU5112119 |
| Date | January 2010 |
| Creators | Hsin-Yi Tu, 杜欣宜 |
| Contributors | Guan-Chiun Lee, 李冠群 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 113 |
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