Engineering robust β-glucuronidase via protein assembly and immobilization for enhanced glycyrrhizin hydrolysis

β-Glucuronidase (GUS) plays a crucial role in modifying glycyrrhizin (GL) to enhance its physicochemical properties and biological activities. However, most enzymes suffer from poor thermostability and high production costs, limiting their industrial applications. In this study, we utilized the SpyTag-SpyCatcher pair to assemble tetrameric Aspergillus oryzae GUS (PGUS) into higher-order structures. The thermostability of PGUS was significantly improved by optimizing the assembly sites. Furthermore, we coated an organosilica network (OSN) onto Escherichia coli cells expressing PGUS assemblies using tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES), constructing an immobilized PGUS system. This integrated approach not only enhanced PGUS thermostability without compromising its catalytic activity but also improved the reusability and long-term operational stability, offering a sustainable solution for industrial biocatalysis.