Bioconversion of glycyrrhizin in a natural intensive sweetener by immobilized β-glucuronidase

The demand for natural sweeteners as alternatives to sucrose is growing rapidly, driving research into enzymatic bioconversion methods for more efficient production. Glycyrrhizin (GL) is approximately 190 times sweeter than sucrose, but its excessive consumption has been linked to adverse health effects. Its hydrolysis yields glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG), a compound nearly 1000 times sweeter than sucrose and with improved sensory and solubility properties. However, the selective conversion of GL into GAMG remains a challenge due to the simultaneous formation of glycyrrhetinic acid (GA) as a byproduct. This study aimed to characterize β-D-glucuronidase (β-GUS) from three different sources (bovine liver, recombinant Escherichia coli, and limpets, Patella vulgata) to identify the most efficient biocatalyst for the selective hydrolysis of GL. β-D-Glucuronidases were characterized for optimal pH, temperature, and catalytic properties using the synthetic substrate 4-nitrophenyl-β-D-glucuronide (pNPG). The enzymes were tested for bioconversion of GL. Among them, β-GUS from Patella vulgata showed the highest efficiency, reducing GL content by 82 % in 72 h, producing 57 mg/L of GAMG and 46 mg/L of GA in 6 h at 40 °C. β-GUS was immobilized on chitosan beads, enhancing specific activity and storage stability (retaining 70 % of initial activity after 30 days). Maximum GAMG production was 61 mg/L in 48 h at 40 °C, with a 58 % GL reduction, and 40 mg/L in 6 h at 80 °C, with a 45 % GL reduction. Irrespective of the treatment temperature, the immobilized biocatalyst allowed the production of the intensive sweetener in a nearly pure form.