碩士 / 國立臺灣海洋大學 / 食品科學系 / 93 / Abstract
The trehalosyl dextrin-forming enzyme (TDFE) mainly catalyzes an intramolecular transglycosyl reaction to form trehalosyl dextrins from dextrins by converting the α-1,4-glucosidic linkage at the reducing end to an α-1,1-glucosidic linkage. The hydrolysis activity exist while the α-1,1-glucosidic linkage couldn’t form by TDFE. In order to study the effects of active site phenylalaninyl residues to the transglycosyl and hydrolytic activities of TDFE, mutants F206W, F206Y, F207Y, F4XXM, F4XXS, and F4XXW were constructed.
The mutant DNA vectors were obtained by PCR amplification. Then the mutant DNA was transformed into E. coli Rosetta (DE3) to express wild-type and mutant TDFEs. The TDFE activity was assayed at 60℃ for 10 min by using 2.8 mM of maltohexaose as substrate in 0.05 M citrate–phosphate buffer (pH 5). The specific activities of purified wild-type, mutant F206W, F206Y, F207Y, F4XXM, F4XXS, and F4XXW TDFEs were 127 U/mg, 6.96 U/mg, 3.79 U/mg, 6.32 U/mg, 127 U/mg, 46.7 U/mg and 100 U/mg, respectively.
The kcat / KM values of F206W, F206Y, and F207Y TDFEs were lower than that of wild-type TDFE for 14 ~ 32 folds. This result explained the residues 206 and 207 were the important residues to the activity of TDFE. All of the kcat / KM values obtained from substrates of maltotetraose, maltopentaose, maltohexaose and maltoheptaose of F4XXM were higher than that of wild-type TDFE. For addition, the selectivity ratio (%) values of F4XXM were lower than that of wild-type TDFE. The selectivity ratio (%) values of F4XXS also lower than that of wild-type TDFE. We presumed F4XXM, and F4XXS TDFE could enhance the yield of trehalose.
Trehalose yields from 10% soluble starch hydrolysis by wild-type, F4XXM, F4XXS, F4XXW, and F4XXY along with isoamylase and TFE at 75℃ were 86.49% (wild-type), 86.56% (F4XXM), 85.07% (F4XXS), 85.84% (F4XXW), and 86.94% (F4XXY) respectively. The F4XXM and F4XXY could enhance the trehalose yield. There was an inverse correlation between the ratio of initial rate of glucose formation to initial rate of trehalose formation and peak trehalose yield.
| Identifer | oai:union.ndltd.org:TW/093NTOU5253048 |
| Date | January 2005 |
| Creators | Ching-Hsing Pan, 潘勁行 |
| 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 | 98 |
Page generated in 0.0017 seconds