碩士 / 國立臺灣海洋大學 / 食品科學系 / 94 / Abstract
The maltooligosyltrehalose trehalohydrolase (MTHase) was encoded from the treZ gene of Sulfolobus solfataricus, the MTHase mainly hydrolyzes the α-1,4 linkage adjacent to the α-1,1 bond of trehalosyl maltooligosaccharides to release trehalose. Salt bridges are the electrostatic interactions between a positive charge and a negative charge, and have greatly contributed to the protein thermostability. So we investigated the effects of salt bridges on thermostability, optimum temperature, optimum pH and kinetic parameters of MTHase. We compared amino acid sequences of homological MTHases and used site-directed mutagenesis technology to create six mutant MTHases with a loss of salt bridge and two mutant MTHases with an addition of salt bridge. All of the mutant enzymes with a loss of salt bridge had a decreased optimum temperature compard to that of wild-type. The three mutant enzymes, RxxxG, DxxxN, and ExxxQ MTHases had an optimum activity at temperature of 75℃, and the other enzymes with a loss of salt bridge are 80℃. LxxxK MTHase is the same as that of wild-type MTHase in optimum temperature, but it had an increased relative activity compard to that of wild-type. Optimum pH of the wild-type and mutant MTHases are pH 5.0. In respect of thermostability, the relative activities of RxxxG and ExxxQ MTHases are 28.7% and 23.7% at 95℃, they are lower than that of wild-type MTHase. Only LxxxK MTHase had an increased thermostability in mutant enzymes with an addition of salt bridge, its thermostability is better than wild-type MTHase. According to the values of Kd, this result explained RxxxG and ExxxQ MTHases were sensitive and LxxxK MTHase was tolerance to temperature. Except mutant ExxxQ MTHases, the kinetic parameters indicated that the catalytic efficiencies and kcat of mutant MTHases were lower than that of wild-type MTHases. The kinetic parameters of RxxxG and DxxxN MTHases were similar and were lowest in all of the mutant MTHases. This result explained the salt bridge Rxxx-Dxxx play an important role to maintain the catalysis ability of MTHases. Only catalytic efficiency of ExxxQ MTHase was higher than that of wild-type. The less changes in Δ(ΔG) values for maltotriosyltrehalose hydrolysis by all of the mutants suggest that there were no loss of hydrogen bond between the substrate and MTHases.
| Identifer | oai:union.ndltd.org:TW/094NTOU5253082 |
| Date | January 2006 |
| Creators | Bo-Yi Li, 李柏逸 |
| 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 | 97 |
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