| Glycyrrhizin (GL) is a major component of licorice which is the most important traditional Chinese medicine. Enzymatic modification of glycyrrhizin to glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) with higher bioavailability has important research value and good prospect in application. Ionic liquids (ILs) are a new class of solvents composed of ions, which are liquid at room temperature and can be designed by modification of either the cation or anion. They have good stability, strong solubility and negligible evaporation, etc. It is very important to take the green and ecofriendly ionic liquids as the reaction medium in replace of volatile and toxic traditional solvents. In this thesis we attempt to make a high-effective utilization of recombined enzyme to catalyze GL to GAMG in the presence of ionic liquids. Firstly, the ionic liquids were synthesized and screened for late use. Then, the catalytic activities, the chemical bond selectivity, thermal stability and the kinetics of enzymatic reaction were investigated in the ionic liquids-containing systems. The main results are as follows:(1) Synthesis of ionic liquids:Firstly, Used N-methylimidazole, pyridine and bromoalkanes as raw materials, the intermediate of four types of ionic liquids, namely, 1-ethyl-3-methylimidazolium bromide salt ([EMIM]Br), 1-butyl-3-methylimidazolium bromide salt ([BMIM]Br), N-ethylpyridine bromide salt ([EPy]Br) and N-butylpyridine bromide salt ([BPy]Br) were synthesized under the conditions of ultrasound wave and no solvent. They have been characterized by their melting points, elemental analysis, IR,1H NMR and 13C NMR. Secondly, using [EMIM]Br4,[BMIM]Br,[EPy]Br and [BPy]Br as intermediate, the hydrophilic ionic liquids [BMIM]BF4, [EMIM]BF4, [EPy]BF4, [BPy]BF4 and hydrophobic ionic liquid [BMIM]PF6 were prepared by the way of anion exchange. Finally, the five types of ionic liquids were characterized by elemental analysis, IR,1H NMR and 13C NMR. By the measure of their viscosities, we found that the temperature had great influence on the viscosity of ionic liquids. Their viscosities decreased quickly in the case of gradual rise for temperature. When the temperature is 30 to 40℃, the viscosities decreased nearly linearly along with the rise of temperature. The decreasement rate of the viscosities was getting smaller and smaller when the temperature was raised continuously.(2) Screening and optimization of ionic liquids-containing reaction system:When the ionic liquid pure solvents were used as the reaction media for hydrolysis of GL to GAMG catalyzed byβ-D-glucuronidase, all the conversions of substrate GL were very low (all less than 20%) and much less than that of GL in buffer, the conversion of GL in buffer was 46.01%. When 10%(v/v) ionic liquid/buffer mixed solvents were used as the reaction media, all the conversions of GL were increased to 2-3 fold of that in pure ionic liquids. Of the six media tested,10%(v/v) [BMIM]PF6/buffer biphasic system and 10%(v/v) [EMIM]BF4/buffer homogeneous system showed higher conversions,57.13%and 54.23%, respectively, much more than that in buffer system. Therefore, they had been chosen as the suitable ionic liquid-containing media. After preliminary optimization, the initial reaction rate, the conversion of GL, the GAMG yield and the chemical bond selectivity (Scb) reached 2.9 mMh-1,71.71%,72.62% and 98.76% in the 40%(v/v) [BMIM]PF6/buffer biphasic system, respectively.(3) Catalytical characteristic ofβ-D-glucuronidase in [BMIM]PF6/buffer biphasic system:β-D-glucuronidase showed high catalytical efficiency for hydrolysis of GL to GAMG in [BMIM]PF6/buffer biphasic system. Many factors showed influence on the initial reaction rate and chemical bond selectivity. The optimized reaction conditions in [BMIM]PF6/buffer biphasic system were [BMIM]PF6 content 40%, pH 6.0, temperature 50℃, substrate concentration 6 mM and shaking speed 200 rpm. Under these optimal conditions the initial reaction rate, the GAMG yield and the Scb reached 3.15 mMh-1,74.36% and 98.12%, respectively. The desired product was distributed in the upper buffer phase in the [BMIM]PF6/buffer biphasic system, while the by-product glycyrrhetinic acid (GA) distributed in the lower layer of ionic liquid, so GAMG and GA were separated automaticly. The recyclability of [BMIM]PF6 was also studied and found to be reusable for 5 batches still with high recovery percentage (≥92%). Additionally, higher Vmax, lower apparent Km and Ea were achieved in [BMIM]PF6/buffer biphasic system, they are 3.14 mMh-1,1.21 mM and 25.97 kJmol-1, respectively.(4) Catalytical characteristic ofβ-D-glucuronidase in [EMIM]BF4/buffer homogeneous system:The hydrolysis of GL to GAMG catalyzed byβ-D-glucuronidase performed efficiently in the [EMIM]BF4/buffer homogeneous system. Their catalytic efficiency was influenced by the concentration of ionic liquid, the buffer pH, the reaction temperature and the substrate concentration. By optimizing the reaction, we found out the optimized conditions were [EMIM]BF4 content 11%, pH 5.4, temperature 45℃and substrate concentration 4 mM. After 24 h for the reaction under the above conditions, the initial reaction rate, the GAMG yield and the Scb reached 2.76 mMh-1,95.31%and 61.81%, respectively. In 11%(v/v) [EMIM]BF4/buffer medium, the thermal stability ofβ-D-glucuronidase with the presence of substrate is higher than those without the presence of substrate. Compared with the reaction conducted in the buffer system, the Km (0.85 mM), Vmax (2.22 mMh-1) and Ea (9.96 kJmol-1) were lower, but the value for Vmax/Km was 2.04 fold of that in the buffer system.This research showed that faster reaction rate, higher product yield and stronger chemical bond selectivity were obtained for the hydrolysis of GL to GAMG catalyzed byβ-D-glucuronidase in the green and environmental-friendly IL-containing systems. These related results are of great significance for the development and utilization of important renewable resource licorice. |
PDF Full Text Request