碩士 / 國立臺灣海洋大學 / 食品科學系 / 96 / Maltooligosyltrehalose trehalohydrolase (MTHase) mainly hydrolyzes the α-1, 4 linkage adjacent to the α-1, 1 bond of maltooligosyltrehalose to release trehalose, and it can also hydrolyze the α-1, 4 linkage at the reducing end of maltooligosaccharides to release glucose. In order to understand the effects of substrate-binding subsite residues on MTHase hydrolytic activities and substrate selectivity, site-directed mutagenesis was used to construct multiple mutations at the residues related to substrate-bindings in this study. The plasmids contained mutant treZ gene were obtained by PCR using mutation-designed primers. The wild-type and mutant plasmids were then transformed into Escherichia coli BL21(DE3)- CodonPlus to express wild-type and mutant MTHases, respectively. The specific activities of purified wild-type, mutant F332Y/K358E/K362R, F332Y/K358E, F332Y/K362R, K358E/K362R, N384D, N384T and N384V MTHases were 421.7 U/mg, 378.7 U/mg, 414.2 U/mg, 329.9 U/mg, 443.3 U/mg, 68.7 U/mg, 46.6 U/mg and 48.6 U/mg, respectively. All mutant MTHases showed good thermostabilities at 80℃ for 2 hours. These results indicated that the mutant enzymes were well folded and have the same structure as that of wild-type MTHase. Wild-type, F332Y/K358E/K362R, F332Y/K358E, F332Y/K362R, K358E/K362R, and N384D MTHases showed an optimal activity at 85℃, while N384T and N384V MTHases had that at 75℃. All the MTHases showed an optimal activity at pH 5.0. The kinetic studies indicates that wild-type, F332Y/K358E/K362R, F332Y/K358E, F332Y/K362R, and K358E/K362R MTHases had similar catalytic efficiencies (kcat/KM) on G3T and G5, while N384D, N384T and N384V MTHases were smaller than wild-type MTHase. According to kinetic analysis, we chosed F332Y/K358E/K362R, F332Y/K358E, F332Y/K362R and K358E/K362R MTHases to study the trehalose production. The initial rates of glucose formation to trehalose formation had inverse correlation with trehalose yields. Results indicated that the final trehalose yields of all MTHases had no significant difference.
| Identifer | oai:union.ndltd.org:TW/096NTOU5253038 |
| Date | January 2008 |
| Creators | Chia-Hung Kuo, 郭家宏 |
| 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 | 101 |
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