碩士 / 國立臺灣海洋大學 / 食品科學系 / 92 / Abstract
In order to discuss the effects of aromatic residues of active site on TDFE (trehalosyl dextrin forming enzyme) transglycosyl and hydrolytic activity, this research modified TDFE residues by mutants F405Y, Y290F, Y367F and Y409F. By designing mutation primers, cooperating PCR, digestion with restriction enzyme Dpn I and DNA gel electrophoresis analysis, which obtained the mutant DNA sequences. Carried out transformation mutant DNA by high efficiency competemt cell, colony PCR and sequence dignment that obtained the transform colony with successful mutant DNA. Purifywild-type and mutant TDFE was produced by French-Press, heat precipitation, ion exchange and gel filtration. Wild-type TDFE or F405Y TDFE, Y290F TDFE, Y367F TDFE and Y409F TDFE had increased hydrolysis activity when the chain length of maltooligosaccharides were shorter. The transglycosyl and hydrolytic activity of mutant T290F TDFE and Y367F TDFE reduced by a wild margin than wild-type TDFE. The change of △(△G) (transition-state energy)values explained that mutant Y290F and Y367F TDFE induced the lodd of hydrogen bond between uncharged groups on the substrate and enzyme.Mutant When sued different chain-lengths of maltoologosaccharide (M3-M7), F405Y TDFE had lower 9～25% ratios of hydrolysis to transglycosylation activity than wild-type TDFE. F405Y TDFE remained 73～97% transglycosylation catalysis efficiencies and lost 34% hydrolysis catalysis efficiency of maltotriose than wild-type TDFE.
| Identifer | oai:union.ndltd.org:TW/092NTOU5253063 |
| Date | January 2004 |
| Creators | Yao-Te, Chung, 鍾耀德 |
| Contributors | Tsuei-Yun, Fang, 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 | 114 |
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