Site-specific crosslinking and assembly of tetrameric β-glucuronidase improve glycyrrhizin hydrolysis

In this study, eight nonconserved residues with exposed surfaces and flexible conformations of the homotetrameric PGUS (β-glucuronidase from Aspergillus oryzae Li-3) were identified. Single-point mutation into cysteine enabled the thiol-maleimide reaction and site-specific protein assembly using a two-arm polyethylene glycol (PEG)-maleimide crosslinker (Mal₂ ). The Mal₂ (1k) (with 1 kDa PEG spacer)-crosslinked PGUS assemblies showed low crosslinking efficiency and unimproved thermostability except for G194C-Mal₂ (1k). To improve the crosslinking efficiency, a lengthened crosslinker Mal₂ (2k) (with 2 kDa PEG spacer) was used to produce PGUS assembly and a highly improved thermostability was achieved with a half-life of 47.2-169.2 min at 70°C, which is 1.04-3.74 times that of wild type PGUS. It is found that the thermostability of PGUS assembly was closely associated with the formation of inter-tetramer assembly and intratetramer crosslinking, rather than the PEGylation of the enzyme. Therefore, the four-arm PEG-maleimide crosslinker Mal₄ (2k) (with 2 kDa PEG spacer) was employed to simultaneously increase the inter-tetramer assembly and intratetramer crosslinking, and the resulting PGUS assemblies showed further improved thermostabilities compared with Mal₂ (2k)-crosslinked assemblies. Finally, the application of PGUS assemblies with significantly improved thermostability to the bioconversion of GL proved that the PGUS assembly is a strong catalyst for glycyrrhizin (GL) hydrolysis in industrial applications.