Biosynthesis of glycyrrhetic acid 3-O-mono-β-d-glucuronide catalyzed by β-d-glucuronidase with enhanced bond selectivity in an ionic liquid/buffer biphasic system
The bond selective hydrolysis of glycyrrhizin (GL) to glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG) catalyzed by recombinant β-d-glucuronidase from Escherichia coli BL21 (PGUS-E) was successfully performed in an ionic liquid (IL)/buffer biphasic system. Five ILs were analyzed, however, a hydrophobic IL 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF6) showed the best biocompatibility with PGUS-E. An obvious enhancement in the initial reaction rate, substrate conversion, GAMG yield and chemical bond selectivity (Scb) was observed using 40% (v/v) [BMIM]PF6/buffer as the reaction medium when compared to the acetate buffer medium. Under the optimized conditions (pH 6.0, temperature 50 °C, substrate concentration 6 mM and shaking speed 200 rpm), the initial reaction rate, the GAMG yield and the Scb reached 3.15 mM h−1, 74.36% and 98.12%, respectively. The recyclability of [BMIM]PF6 was also studied and found to be reusable for five batches with high recovery percentage (≥92%). Furthermore, the desired product and byproduct were easily separated since they were distributed in different phases. Additionally, higher Vmax (3.14 versus 2.24 mM h−1), lower apparent Km (1.21 versus 1.80 mM) and Ea (25.97 versus 32.60 kJ mol−1) were achieved in [BMIM]PF6/buffer biphasic system than that in monophasic buffer system.
Journal: Process Biochemistry - Volume 45, Issue 12, December 2010, Pages 1916–1922