Construction of a CaHPO₄-PGUS1 hybrid nanoflower through protein-inorganic self-assembly, and its application in glycyrrhetinic acid 3-O-mono-β-d-glucuronide preparation

Glycyrrhetinic acid 3-O-mono-β-d-glucuronide (GAMG), an important pharmaceutical intermediate and functional sweetener, has broad applications in the food and medical industries. A green and cost-effective method for its preparation is highly desired. Using site-directed mutagenesis, we previously obtained a variant of β-glucuronidase from Aspergillus oryzae Li-3 (PGUS1), which can specifically transform glycyrrhizin (GL) into GAMG. In this study, a facile method was established to prepare a CaHPO₄-PGUS1 hybrid nanoflower for enzyme immobilization, based on protein-inorganic hybrid self-assembly. Under optimal conditions, 1.2 mg of a CaHPO₄-PGUS1 hybrid nanoflower precipitate with 71.2% immobilization efficiency, 35.60 mg·g⁻¹ loading capacity, and 118% relative activity was obtained. Confocal laser scanning microscope and scanning electron microscope results showed that the enzyme was encapsulated in the CaHPO₄-PGUS1 hybrid nanoflower. Moreover, the thermostability of the CaHPO₄-PGUS1 hybrid nanoflower at 55°C was improved, and its half-life increased by 1.3 folds. Additionally, the CaHPO₄-PGUS1 hybrid nanoflower was used for the preparation of GAMG through GL hydrolysis, with the conversion rate of 92% in 8 h, and after eight consecutive runs, it had 60% of its original activity. [Figure not available: see fulltext.].