Biosynthesis of glycyrrhetinic acid 3-O-mono-β-d-glucuronide by free and immobilized Aspergillus terreus β-d-glucuronidase

Enzymatic conversion of glycyrrhizinic acid (GL) into glycyrrhetinic acid 3-O-mono-β-d-glucuronide (GAMG) was achieved using β-d-glucuronidase from Aspergillus terreus I. The partially purified β-d-glucuronidase was immobilized by entrapment in Ca-alginate beads and the immobilization yield was about 83% at 2% alginate concentration. The pH-activity profile was widened upon immobilization. The optimum temperature was shifted from 40 to 45 °C and the apparent activation energy (Ea) was increased from 7.3 to 17.3 kcal/mol by immobilization. The immobilized enzyme exhibited higher thermal stability compared to the free form. The half-life values of the immobilized and free enzyme at 60 °C were 124.9 and 33.8 min, respectively. Also, immobilization eliminates the inhibitory effect of Cu2+ on GAMG production. The value of Michaelis-constant Km of the immobilized enzyme (1.4 mg/ml) was greater than that of the free form (0.88 mg/ml), whereas, Vmax was smaller for the immobilized enzyme. The durability of the immobilized β-d-glucuronidases in repeated use was studied. The immobilized enzyme retained about 40% of its original activity after 4 cycles.