碩士 / 國立臺灣師範大學 / 生命科學研究所 / 100 / Trehalose, a non-reducing disaccharide, existing in various organisms can serve as energy storage and as a protectant of protein and lipid against various stresses. It has become as an important compound in foods, cosmetics and pharmaceuticals industries. Trehalose synthase (TS) is one of the biosynthesis pathways of trehalose, which reversibly catalyzes the intramolecular transglucosylation (isomerization) of maltose to produce trehalose, also known as maltose isomerase, as well as the side reaction of irreversible hydrolysis of maltose to produce two glucose molecules. However, the structure as well as the enzymatic mechanism of TS has not been determined. Sucrose isomerase (SI) and TS have isomerase activity and belong to the same subfamily of the α-amylase family. Three-dimensional structures of three SIs have been determined. The tertiary structural model of Deinococcus radiodurans trehalose synthase (DrTS) which has been characterized as a cold-active enzyme was built using a SI structure as template. The overall DrTS structure is highly conserved with α-amylase family. It possesses a central catalytic domain formed by a (β/α)8-barrel structure, a loop rich subdomain and two antiparallel β-sheet domains. The DrTS-substrate complex model was also built by docking with its substrate maltose. 21 amino acid residues located in close contact with maltose within the 5 Å cut-off distance have been identified and may play important roles in substrate binding. By amino sequence and three-dimensional structure alignments with the SIs, nine of the 21 residues were identified as different from SIs and may play important roles in the TS function. Among these nine distinct residues, Thr154, Phe174, and Gln254 are not conserved in the TS family from various species. To deduce the three residues are relative with biochemical properties and conversion rate of TS, site-specific saturation mutations of these residues were performed to screen for mutant enzymes which exhibit high isomerization activity. A high-throughput purification and assay system was developed to screen Thr154 random libraries, resulting that 2 coloies with higher activies than the wild type, showing 3-fold increased activity, DNA sequencing showed that Thr154 were altered to Phe. Furthermore, the Asn317 was predicted to interact with the water molecule which may participate in the hydrolysis side-reaction. The Arg316 may influence the catalytic water binding whereas it is non-identical in the SIs. The Asn253 was predicted to be able to prevent hydrolysis by blocking the entrance of the active site pocket. To reduce hydrolysis activity of the side reaction, the hydrophilic Asn317 were altered to hydrophobic Phe, Leu or Ala by site-directed mutagenesis. The non-identical and positively charged of Arg316 to nonpolar Gly may result in reducing hydrolysis side-reaction, and the Asn254 were altered to aromatic Phe.The results showed that the activities of N317A, N317F, N317L and R316G mutants were signicantly decreased, and N253F mutant led to a complete loss in activity. In addition, two approaches were also performed to improve the conversion rate of DrTS. One is coupling TS with glucose isomerase (GI) which converts and thus removes the side-reaction product glucose into fructose to reduce the product inhibition. The results indicated that the trehalose conversion rate of GI-coupled DrTS was enhanced. The other one is inhibiting the reversed reaction of TS by adding α-glucosidase inhibitors (validoxylamine A, validamycin A, kanamycin A, acarbose or azactidine), or trehalose analogs (lactose, lactulose or isomaltulose). The results indicated that both the forward and reversed reactions of DrTS were inhibited by validoxylamine A, validamycin A, kanamycin A and acarbose. However, azactidine, lactose, lactulose and isomaltulose almost had no inhibition effect on the forward and reversed reaction of DrTS. In general, the results provide feasible methods to improve the conversion rate of DrTS for industrial application of trehalose production.
| Identifer | oai:union.ndltd.org:TW/100NTNU5112115 |
| Date | January 2011 |
| Creators | Hsin -Hao Tseng, 曾信豪 |
| 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 | 118 |
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