碩士 / 國立臺灣師範大學 / 生命科學研究所 / 101 / In mammals, trehalase, sucrase-isomaltase and maltase-glucoamylase are the major α-glycosidases of the intestinal brush border membranes. These enzymes are responsible for the degradation of di- and oligosaccharides into monosaccharides, and are crucial for the energy-intake. Trehalase (EC 3.2.1.28) hydrolyses α,α-trehalose (1-α-D-glucopyranosyl α-D-glucopyranoside) to two glucose molecules. The intestinal trehalase is involved in the hydrolysis of ingested trehalose which is found mainly in many nutrient foods. The dual protective properties of trehalose (as a chemical chaperone and an inducer of autophagy) have encouraged pharmaceutical application of the disaccharide in neurodegenerative diseases caused by protein aggregation process. Therefore, it is theoretically possible to increase intestinal absorption of trehalose through inhibiting intestinal trehalase activity, and thus increase in trehalose content in blood or brain. This may in turn alleviate neurological protein deposition diseases. The protein structure, catalytic mechanism and specific inhibitors of human intestinal trehalase (hTreH) have not been elucidated. In the present study, a cDNA fragment encoding the mature form of hTreH was cloned and recombinant hTreH was expressed in Escherichia coli. However,the recombinant hTreH was expressed as an inclusion body. Protein refolding through dialysis and on-column refolding process were performed. The refolded enzyme showed very low specific activity. To prevent protein misfolding through the formation of incorrect intra- or inter-molecular disulfide bonds and thus increase its solubility, based on tertiary structure modeling, several predicted non-disulfide-bonding cysteine residues in hTreH were replaced with serine by site-directed mutagenesis. Four cysteine residues in hTreH were changed into serine, which are predicted to be distant from each other and may not form disulfide bonds with each other. However, the mutant proteins were also expressed as inclusion bodies, and the refolded enzymes still showed no activity. Several trehalose analogs were biochemically characterized as mammalian trehalase inhibitors, and they can be as potential therapeutics for the protein deposition-mediated diseases.
Keyword: trehalose, trehalase, trehalase inhibitor, recombinant protein expression
| Identifer | oai:union.ndltd.org:TW/101NTNU5112002 |
| Date | January 2013 |
| Creators | Yin-Jung Huang, 黃胤榮 |
| Contributors | Guan-Chiun Lee, 李冠群 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 143 |
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