碩士 / 國立中正大學 / 化學暨生物化學研究所 / 107 / This thesis focus on the synthetic application of α1,3/4-fucosyltransferase from Helicobacter pylori DSM6709 (FucTIII). We have successfully constructed three types of recombinant FucTIII with different expression vectors (pET28a、pTXB1、pMAL-c2X) for protein overexpression comparsion. Among these protein expression systems, pMAL-c2X system with C-terminal (His)6-tag was found to increase the water-solubility of FucTIII with the optimal overexpression yield of 6 mg/L cell culture. Linear human milk oligosaccharides and tri-saccharides (GlcNAc-LacNAc with type I and type II linkages) were prepared as FucTIII acceptors. FucTIII was able to construct various fucosylated glycans such as human milk oligosaccharides (LNFP V, LNFP VI, LNDFH II, LNnDFH II), Lewis antigens (Lex, Ley, Lea, Leb, sLex, sLea) and their derivatives. According to enzyme kinetics parameters, the catalytic efficiency (kcat/Km) of FucTIII on type II LacNAc (22) was 50 times higher than type I LacNAc (21). Furthermore, the catalytic efficiency of FucTIII on trisaccharides was 30-40 times higher than disaccharides ((21) v.s. (23); (22) v.s. (24)). The results revealed that the additional GlcNAc on the non-reducing end of the acceptors would enhance the catalytic efficiency of FucTIII.
| Identifer | oai:union.ndltd.org:TW/107CCU00065006 |
| Date | January 2018 |
| Creators | 蔡騰緯 |
| Contributors | Yu, Ching-Ching, 游景晴 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 351 |
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