Immobilization of L-Arabinose Isomerase from Thermoanaerobacterium saccharolyticum NTOU1 and Using Site-Directed Mutagenesis to Enhance Its Reaction Rate
| Autor: | Tsai, Chia-Hua, 蔡佳樺 |
|---|---|
| Rok vydání: | 2017 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 105 D-Tagatose is an isomer of D-galactose and it has some properties such as low calories (1.5 kcal/g), high sweetness (92% as sweet as sucrose) and low glycemic index. With less by-products, eco-friendly and less costly advantages, production of D-tagatose from D-galactose using L-arabinose isomerase (L-AI) has been studied intensively in recent years. L-AI from Thermoanaerobacterium saccharolyticum NTOU1 (TsAI) has been characterized in a previous study and it has good thermostability. However, the reaction rate of TsAI was inferior to some L-AIs from other microorganisms. This study attempted to improve the reaction rate of TsAI by protein engineering and produce D-tagatose using immobilized cells for increasing its applicability to industry. The structure of TsAI was modeled by SWISS-MODEL and the modeling structure was further docked with D-galactose. Mutant residues were selected by analyzing the relative residues of binding site which might interact with substrate and mutations were made by site-directed mutagenesis. According to enzyme kinetics, the kcat of mutant F270N was eight times larger than that of wild-type TsAI at 50℃. It revealed that F270N was more suitable for producing D-tagatose with decreasing browning of D-tagatose. To improve reusability and stability, the recombinant Escherichia coli ClearColi BL21 (DE3) cells harboring TsAI were immobilized with alginate. The optimal conditions for immobilization were conducted with 5% alginate, 200 mM CaCl2, 0.05% Triton X-100 and 50 g/L cell mass. On the optimal conditions, the activity of immobilized cells harboring wild-type TsAI was 16.1 U/g cell and immobilized efficiency was 20.5% at 55℃; the activity of immobilized cells harboring F270N TsAI was 29.8 U/g cell and immobilized efficiency was 33.2% at the same temperature. It concluded that the immobilized cells harboring F270N was presented with higher activity and efficiency at 55℃. To further exploring the industrial application, production of D-tagatose from 10% D-galactose was performed at 55℃ by immobilized cells in packed-bed reactor. The immobilized cells harboring wild-type TsAI could reach reaction equilibrium in 36 h at about 30% conversion yield and the immobilized cells harboring F270N TsAI could reached reaction equilibrium in 6 h at about 40% conversion yield in the same condition. In summary, it had advantages to produce D-tagatoe by immobilized cells harboring F270N with higher reaction rate and higher conversion yield than those of wild-type TsAI. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|