碩士 / 國立臺灣海洋大學 / 食品科學系 / 98 / The maltooligosyltrehalose synthase (MTSase) catalyzes an intramolecular transglycosyl reaction of maltooligosaccharide and produced maltooligosyltrehalose. MTSase expression from Sulfolobus solfataricus ATCC 35092 treY gene is much lower than that of S. acidocaldarius ATCC 33909. In order to improve the expression of MTSase from S. solfataricus ATCC 35092 treY gene, Splicing Overlap Extension (SOE) was used to produce five different recombinant treY genes. The expressions of recombinant MTSases of SA2, SA3, and SA4 are increased obviously, and their appropriate IPTG concentrations were 0.2, 0.1 and 0.1 mM, respectively. The amount of mRNA transcribed from SA2 treY gene is higher than that of wild-type. But the thermostabilities of recombinant SA2, SA3, and SA4 MTSases are decreased about 15℃ than that of wild-type MTSase. SOE was carried out again to reduce the sequence differences between SA2 and wild-type MTSases. The amino acid sequence of resulting SA21 MTSase only has 19 amino acids different from that of wild-type. The appropriate IPTG concentration for SA21 induction is 0.5 mM and thermostability of recombinant SA21 MTSase is also decreased about 15℃ than that of wild-type. After heat treatment, the recovered enzyme activities of wild-type, SA2, SA3, SA4, and SA21 are 4.38, 1.77, 1.64, 1.73, and 2.37 U/g cells, respectively. Then, SA21 MTSase was subjected to site-directed mutagenesis to produce SA21-G107W/G131E. The appropriate IPTG concentration is 0.2 mM, and thermostability of recombinant SA21-G107W/G131E MTSase is better than other recombinants. The recovered enzyme activity of SA21-G107W/G131E is 3.28 U/g cells.
| Identifer | oai:union.ndltd.org:TW/098NTOU5253046 |
| Date | January 2010 |
| Creators | Yu-Jen Jeng, 鄭宇軫 |
| 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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