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

Abstract 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 (Mal 2 ). The Mal 2 (1k) (with 1 kDa PEG spacer)‐crosslinked PGUS assemblies showed low crosslinking efficiency and unimproved thermostability except for G194C‐Mal 2 (1k). To improve the crosslinking efficiency, a lengthened crosslinker Mal 2 (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 Mal 4 (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 Mal 2 (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.