碩士 / 國立臺灣海洋大學 / 食品科學系 / 95 / Maltooligosyltrehalose trehalohydrolase (MTHase) mainly hydrolyzes the α-1,4 linkage adjacent to the α-1,1 bond of maltooligosyltrehalose to release trehalose, and it also hydrolyzes the α-1, 4 linkage at the reducing end of maltooligosaccharides to release glucose leading to the decrease of the trehalose yield. BvtreZ gene from Brevibacterium helvolum ATCC 11822 was cloned and inserted into an expression vector pET21b. The stop codon of BvtreZ gene was changed to glycine codon by site-directed mutagenesis, therefore, the expressed BvMTHase possessed a His-Tag on its C-terminal region. The recombinant BvMTHase was expressed in the host E. coli BL21 (DE3) and was purified by Ni2+-affinity chromatography. The purified recombinant BvMTHase exhibited optimum activity at 40℃ and pH 6.0. The enzyme was quite stable at 30~40℃ and pH 5.5~6.5. The activity of recombinant BvMTHase would be completely inhibited by 4 mM Cu2+ and Zn2+ ions.
The SStreZ and SAtreZ gene from pET-15b-△H-TreZ recombinant plasmid and Sulfolobus acidocaldarius ATCC 33909, respectively, were amplified and inserted into an expression vector pET21b. The BvtreZ, SStreZ and SAtreZ genes were used as template DNA in genetic recombination. These three amino acid sequences were 40~60% identical. DNA family shuffling was first carried out according to Stemmer’s method. The DNA fragments of 50-100 bp were prepared by DNase I cleavage. Since the sequence homology is not high, cannot be amplified by this method. In addition, DNA family shuffling was also carried out according to Kikuchi’s method using DNA fragments of 100-300 bp prepared by different restriction enzyme digestion. The fragments are reassembled into full-length genes of 1.5 kb, and inserted into an expression vector pET21b. The sequencing result shows that the sequences of RMTHase only contain partial original template, and RMTHase even lost the conserved regions of α-amylase family regions.
| Identifer | oai:union.ndltd.org:TW/095NTOU5253072 |
| Date | January 2007 |
| Creators | Hui-Chun Su, 蘇惠君 |
| 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 | 112 |
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