碩士 / 國立臺灣師範大學 / 生命科學系 / 98 / Trehalose (α-D-glucopyranosyl-1, 1-α-D-glucopyranoside), a non-reducing disaccharide, existing in various organisms can serve as energy storage, and as protectants of protein and lipid from various stress such as cryoprotectants. It has become as an important compound in foods, cosmetics and pharmaceuticals industrials. Trehalose synthase (TreS) is one of the biosynthesis pathways of trehalose, which reversibly catalyzes the intramolecular transglucosylation (isomerization) of maltose to produce trehalose, as well as the minor side-reaction of irreversible hydrolysis of maltose to make glucose. Because maltose is an inexpensive and readily available sugar, it has great potential to produce trehalose from maltose by utilizing the one-step isomerization reaction of TreS. A number of methods have been currently available to measure TreS activity, including HPLC and trehalase-coupled TreS assay. However, these methods are either time-consuming, expensive or with low substrate specificitied, and this makes them not suitable for characterizing TreS in a high-throughput manner. This proposal established a novel 96-well format assay, maltase-coupled TreS assay to colorimetrically determine the isomerization activity of TreS. We also establish a 96-well format protein purified system for high-throughput TreS purification. With these new colorimetric method and purification method, we can perform the biochemical characterization more efficiently, especially, in the directed enzyme evolution of TreS for screening mutant TreS with improved properties, such as high trehalose yield or high thermostability.
| Identifer | oai:union.ndltd.org:TW/098NTNU5112032 |
| Date | January 2010 |
| Creators | 張紘綸 |
| Contributors | 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 | 95 |
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