碩士 / 國立臺灣海洋大學 / 食品科學系 / 105 / Maltooligosyl trehalose synthase (MTSase) catalyzes an intramolecular transglycosyl reaction to form maltooligosyltrehalose by converting the α-1,4-glucosidic linkage at the reducing end of maltooligosaccharide to an α-1,1-glucosidic linkage. Except the transglycosyl reaction, the hydrolysis activity also exist causing release of glucose by MTSase. The hydrolysis reaction is unfavorable to trehalose production. If the substrate specificity of MTSase could be altered to decrease the hydrolysis activity while maintaining its transglycosyl activity, an increased trehalose yield would be anticipated. In order to decrease hydrolysis activity of MTSase from Sulfolobus acidocaldarius, the mutations Y225L, I253Y, A438T, T439L, Y225L/A438T/T439L, I253Y/A438T/T439L and Y225L/I253Y/A438T/T439L were constructed by site-directed mutagenesis. The wild-type and mutated recombinant DNA was transformed into ClearColi BL21 (DE3)-CodonPlus-RIL to express wild-type and mutant MTSases, respectively. Then the enzymes were purified and studied. The mutant I253Y and A438T were completely inactive. In the modling structure, the two residues were found interacting with T439L.
The mutantions T439L, I253Y/A438T/T439L and Y225L/I253Y/A438T/T439L have no significantly changes of kcat values for transglycosylation, but have lower kM values. And the kcat/kM values for transglycosylation of these mutations were higher than that of wild-type, the hydrolysis activity of these mutations were higher than that of wild-type, but their selective ratios ([kcat/kM (G3)] / [kcat/kM (G5)] × 100%) lower than that of wild-type.
On the other hand, the wild-type MTSase, along with isoamylase and MTHase, were immobilized on NHS-activated Sepharose 4 Fast Flow at the same time, and were studied for trehalose production. We found that the immobilized enzymes have only about half trehalose yield compared to that of free enzymes, and have lower reaction rate. The reaction still not reached equilibrium after 48 h, and we found that the more amounts of enzymes immobilized the higher trehalose yield obtained. Hope that we can find better immobilization condition and use immobilized enzymes to produce trehalose in the future.
| Identifer | oai:union.ndltd.org:TW/105NTOU5253046 |
| Date | January 2017 |
| Creators | Liu, Ya-Wen, 劉雅文 |
| Contributors | Fang, Tsuei-Yun, 方翠筠 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 75 |
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