| Glycyrrhizin (GL), the biologically active compound of liquorices, can be hydrolyzed into glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) by whole cells containing (3-glucuronidase. GAMG is widely used in the pharmaceutical and food industries due to its stronger physiological functions, higher biological activity, more favorable sweetness, and lower caloric value as compared with GL. Due to these reasons, GAMG is considered to be the potential substitute of GL. Different methods have been developed to synthesize the multifunctional product of GAMG by chemical and biological transformations. Biotransformation has the advantages of good options for mild reaction conditions, structural modification, and simple operational procedure. Biological methods with many advantages are therefore an appropriate alternative in the catalysis of various reactions, such as the hydrolysis of GL. However, the low water solubilities of both GL and GAMG great limit the large-scale industrial production of GAMG. Medium engineering for biocatalytic reactions is an imperative approach for the synthesis of biologically active compounds, particularly when the biological conversion efficiency in an aqueous system is low.(1) The hydrolysis of glycyrrhizin (GL) to glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) catalyzed by whole-cell biocatalysts in systems containing non-conventional solvents were performed. Three types of whole-cell biocatalysts including wild-type Penicillium purpurogenum Li-3 (w-PGUS), and two recombinant strains, Escherichia coli BL21 and Pichia pastoris GS115 were used. The biotransformation of GL to GAMG by w-PGUS in 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF6)/water biphasic system was mainly studied because w-PGUS showed higher GAMG yield and relative activity in this system than the other two whole-cell biocatalysts. Under the optimized conditions of a pH 5.2 buffer,6.0 mM substrate concentration, 30℃temperature, and 60g·L-1 cell concentration, a GAMG yield of 87.63% was achieved after 60 h. [Bmim]PF6 remained at higher recovery percentage even after eight reaction cycles of repeated use; the biotransformation activity of w-PGUS remained almost unaffected even after two batch reaction cycles.(2) The hydrolysis of glycyrrhizin (GL) to glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) catalyzed by immobilized cell in system containing 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF6) was performed. The reactions in [Bmim]PF6/buffer biphasic system were mainly studied, and with the buffer single-phase system as control. The optimal conditions of reaction in [Bmim]PF6/buffer biphasic system were determined, the optimum ionic liquid concentration, buffer pH, substrate concentration, temperature were 10%,5.8,6.0 mM,35℃, respectively. The optimal conditions of reaction in buffer single-phase systems were determined, the optimum pH, substrate concentration, temperature were 5.0,3.6 mmol·L-1,35℃respectively. The conversion of GL is higher in the biphasic system (82.6%) compared with the single-phase buffer system (76.3%).(3) The recombinant Pichia pastoris (r-PGUS-P) designated in this work expressingβ-glucuronidase, was used as whole-cell biocatalyst catalyst glycyrrhizin (GL) to glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) in non-aqueous solvents systems. The results showed that PGUS-P gave the highest yield of GAMG in ionic liquid [Bmim]PF6/water (2:8, v/v)system. Then, we mainly studied the biotransformation of GL to GAMG by the whole cell of PGUS-P in [Bmim]PF6/water biphasic system. The optimal conditions of this reaction were determined, the optimum pH, substrate concentration, temperature and cell content were 5.4,6.0 mmol·L-1,45℃and 8.0 g·L-1, respectively. Under these optimized conditions,69.6% yield of GAMG and 67.2% of the chemical bond selectivity (Scb) was achieved after 58 h reaction time that compare to aqueous phasic increase 12.4% and 12.61%, respectively. Simultaneously, the ionic liquid [Bmim]PF6 also remained at higher recovery percentage of about 93.47% during repeated use for 7 reaction cycles. [Bmim]PF6 has good reusability that could remarkably improve the efficiency of biotransformation. Especially, product GAMG and byproduct GA was got separated in biphasic system automatically that bring convenience for separation and purification. The combination of whole cells and ionic liquid is a promising approach for the economical and industrial production of GAMG.(4) These studies prompted us to understand why water immiscible ILs stabilize bond selectivity ofβ-glucuronidase by looking at structure/function relationships. According to the distinct physicochemical properties of these solvents, the objectives were to study the ionization state of the substrates in media, then its influence on the efficiency and the selectivity of the enzymatic acylation. The observed evolution of a-helix toβ-sheet in enzyme in the assayed water-immiscible ILs to reach another active and stable enzyme conformation might be explained as a function of both the amino acid sequence and the solvent accessibility of each amino acid residue. In this study, it is assumed that the function of enzyme during biotransfonnation process is directly correlated with simultaneous structural changes in the protein. An aqueous solution of free-enzyme molecules added to the IL phase could be considered as being included but not dissolved in the medium, providing a suitable microenvironment for the catalytic action and enhanced enzyme stability... |
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