碩士 / 國立臺灣海洋大學 / 食品科學系 / 92 / We have successfully cloned treZ gene from Sulfolobus solfataricus ATCC35092 to an expression vector (pET-15b) which possesses a T7 promoter in the upstream of its multiple cloning sites. The recombinant treZ gene was expressed well under the T7 expression system in the E. coli BL21(DE3) codonplus-RIL. IPTG induction did not affect the expression of recombinant trehalose formining enzyme (TFE).
In order to purify TFE by metal chelating chromatography, treZ gene was fused in frame with the His-Tag coding sequence on the expression vector; therefore, the expressed target protein possessed a His-Tag on its N-terminal region. To express a wild-type TFE, the pET-15b-treZ was digested with NcoI and XhoI, made blunt by mung bean nuclease, ligated again to remove His-tag coding sequence, resulting a recombinant clone designated as pET-15b-△H-treZ.
In order to detect the activity of TFE, we use trehalosyl maltotetraose as substrate which was prepared from maltohexaose by trehalosyl dextrins forming enzyme (TDFE). The TDFE reaction was followed by a 0.2 N NaOH treatment at boiling water bath for 2 h, and then the trehalosyl maltotetraose was purified by Amberlite IRA-900 and IR-120A ion exchanger, finally HPLC equipped with Nucleosil 5 NH2 column was used to determine the concentration of trehalosyl maltotetraose.
Wild-type TFE have been purified by heat treatment and ion exchange chromatography. The purified wild-type TFE exhibited optimum activity at 85℃ and pH 5.5. The enzyme was quite stable at the temperature up to 80℃ for 2h. The enzyme remained stable in a pH range of 3.5-11. The activity of TFE would be inhibited by Hg2+ ion. The chain lengths of trehalosyl maltooligosaccharides also had an effect on the TFE activity. In addition, the TFE also could hydrolyze the α-1,4 linkages of maltooligosaccharide to release glucose, maltose, and maltooligosaccharides with shorter chain lengths. The hydrolysis rates on maltooligosaccharides, however, were much slower than the trehalose formation rates on trehalosyl maltooligosaccharides.
| Identifer | oai:union.ndltd.org:TW/092NTOU5253064 |
| Date | January 2004 |
| Creators | Meng-Shin Guo, 郭孟欣 |
| 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 | 69 |
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