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Use of biological method modified natural products to get more value compounds has become a research hotspot of bio-chemical and pharmaceutical industry. The glycyrrhizic acid modified single-glucuronide the glycyrrhetinic acid (GAMG) in the body has a good solubility and transmembrane transport capacity and high sweetness, low in calories, more secure, which makes the GAMG have even broader application prospects. Task Force pre-separation filter out a Penicillium purpurogenum Penicillium purpurogenumLi-3, can be transformed into the glycyrrhizic acid directional generated GAMG, and cloned the gene expression of p-glucuronidase enzyme Pgus constructed beta-glucuronic acid glucosidase The expression systems prokaryotic E.coli and eukaryotic P.pastoris, three different conversion types of bacteria. For biotransformation substrate licorice acid readily soluble in water, licorice acid in the aqueous phase and low conversion efficiency, this paper attempts to use of microbial whole-cell catalyst systems containing ionic liquids transformed licorice acid generation GAMG and pure water phase, the non-aqueous organic solvent phase system compares whole cell transformation model to create a system of ion-containing liquid reaction medium ScS ionic liquid transforming glycyrrhizic acid to the reaction conditions and the selectivity of the whole cell, and how it affects beta - glucuronidase enzyme catalytic properties and structure and function to do a preliminary inquiry. The results are as follows: (1) the whole-cell catalyst and the reaction medium selection: OK Penicillium purpurogenum P.purpurogenum Li-3 (w-PGUS), recombinant Pichia pastoris P. pastoris (the r-PGUS-P) and recombinant E. coli Escherichia coli BL21 (the r-PGUS-E) three whole-cell harvest time, were induced enzyme production 72,48 and 6 h, corresponding to the whole-cell enzyme activity were 1360,1102 and 428 U · kg-1. Of the three cells in the 12 kinds of non-aqueous media into the glycyrrhizic acid generated GAMG the reaction, the results show that the selected hydrophobic ionic liquid 1 - butyl - 3 - methylimidazolium hexafluorophosphate ([Bmim] PF6 ) biocompatibility best the whole cell the w-PGUS and r-PGUS,-P showed a good catalytic effect. (2) whole-cell w-PGUS water / [bmim] PF6 two-phase system into the glycyrrhizic acid generated GAMG reaction: To determine the whole-cell enzyme the w - PGUS catalytic generation GAMG of the optimal reaction conditions, ionic liquids and water with ratio to 1:1, pH 5.2, reaction temperature 30 ° C, the cell density of 60 g L-1. Under these conditions, the reaction 60 h product GAMG the rate of 87.63%. Preliminary screening by the immobilization carrier in order to improve the operation stability of the whole cell, and select the alginate-immobilized carrier carried the w-PGUS of whole cell immobilized to ScS immobilized of w-PGUS cells in water / [Bmim] PF6 two-phase medium system transformation the glycyrrhizic acid generating reaction GAMG, and to buffer single-phase system as a control. The ScS several factors, such as the concentration of ionic liquid, the pH of the buffer solution, the reaction temperature, substrate concentration on the reaction. Determined in water / [BMIM] PF6 two-phase system, the optimum ionic liquid added in a proportion, the pH of the buffer solution, the reaction temperature, substrate concentration were 10%, 5.8,35 ° C and 6.0mmol · L-1. Single phase system in the buffer, the buffer, pH, reaction temperature, substrate concentration 5.0,35 ° C and 3.6mmol · L-1, respectively. The addition of ionic liquids to some extent, improve the the glycyrrhizic acid conversion rate and the effect of pH and temperature stability. Immobilized cells reused 3 times in a two phase system in the water / [BMIM] PF6, glycyrrhizic acid conversion was still maintained at 77.36%, and helps improve the stability of the cell manipulation after cell immobilization. (3) Restructuring the Pichia pastoris r-PGUS-P cells in the water / [Bmim] PF6 two-phase system into the glycyrrhizic acid generated GAMG the reaction: OK [Bmim] PF6 and water the optimum ratio of 2:8 (v / v), the optimum buffer, pH, reaction temperature, substrate concentration, and cells added in an amount of 5.4,45 ° C, 6.0mmol · L-1 and 8.0 g · L-1. Under this condition, the reaction of 58 h, the product yield and chemical bond selectivity (GAMG SCB) were 69.6% and 67.2%, respectively, compared with pure water phase reaction system increased by 12.4% and 12.61%. Ionic liquid recycled 7 times, the recycling rate of 93.47%. Products and by-products effectively separated in the two-phase system, as a follow-up product separation and purification of convenience. (4) ionic liquid enzymatic characteristics and the structure and function of the p-glucuronic acid glycosides. Examine the impact of the ionic liquid on the characteristics of p-glucuronidase enzyme-catalyzed reaction. The results showed that the water / [Bmim] PF6 two-phase system in optimal substrate material 4 - nitrophenyl-beta-D-pyran the galactosidase (pNPG) 10.0 mmol · L-1, compared to buffer single-phase system 1 to determine the kinetics and thermodynamics of the system parameters, Vmax 0.153 mmol · L-1 · min-1, Km 0.620 mmol · L-1, the apparent activation energy Ea 35.386 kJ · mol-1. Buffer single-phase reaction system Vmax 0.094 mmol · L-1 · min-1, Km to 0.773 mmol · L-1 the Ea to 48.199 kJ mol-1, the ionic liquid [Bmim] PF6 reduce the reaction activation energy and enhance the reaction rate. Hydrophobic ionic liquid [BMIM] PF6 catalytic behavior has a greater impact. By fluorescence spectroscopy and circular dichroism analysis of ionic liquid enzyme protein beta-glucuronide glycosides, studies show that ionic liquid treatment induced stretching and secondary changes in the structure of the enzyme protein molecules, a spiral structure to reduce beta-fold increased. Speculated that the reasons for the ionic liquid can promote enzyme structure shift, the hydrophobic ionic liquid allows to reduce the moisture of the protein microenvironment around to lead enzyme conformational toward the rigid structure evolution, which is the key to change enzyme catalytic properties.
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