Biotransformation of baicalin and glycyrrhizic acid using immobilized Fe(3)O(4)@Chitosan@β-glucuronidase.

β-Glucuronidase can hydrolyze β-glucuronic acid-containing glycosides, such as baicalin and glycyrrhizic acid. In this study, the β-glucuronidase gene from Lactobacillus rhamnosus was cloned and expressed in Escherichia coli. The resulting recombinant protein, designated LrhGUS, exhibited a molecular weight of approximately 72 kDa. The hydrolysis pathway of glycyrrhizic acid by recombinant LrhGUS proceeded as follows: glycyrrhizic acid → glycyrrhetinic acid monoglucuronide (GMAG) → glycyrrhetinic acid (GA), achieving a conversion rate of 90.38% with 2 mg/ml glycyrrhizic acid. Additionally, LrhGUS hydrolyzed baicalin into baicalein with a conversion rate of 94.64% using 20 mg/ml baicalin. Magnetic chitosan microspheres were utilized as carriers for immobilizing recombinant LrhGUS. Response surface methodology was employed to optimize immobilization conditions, which were determined to be a glutaraldehyde concentration of 1%, an enzyme loading of 0.8 mg/g bead, and a crosslinking temperature of 25 °C. The optimal temperature and pH for the immobilized enzyme were identical to those of the free enzyme; however, the immobilized enzyme demonstrated superior stability compared to the free enzyme. Notably, under acidic conditions, the pH stability of immobilized LrhGUS was significantly higher than that of the free enzyme. After incubation at 80 °C for 12 h, the thermal stability of the immobilized enzyme improved by approximately 50% relative to the free enzyme. Moreover, the immobilized LrhGUS exhibited excellent reusability, maintaining approximately 30% enzyme activity after seven cycles. Using the immobilized enzyme, baicalein was successfully prepared on a 1 g scale, while GAMG and GA were prepared on a 100 mg scale. These findings provide a robust foundation for the potential industrial application of β-glucuronidase. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-025-04220-w.