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Single\u2010point mutation into cysteine enabled the thiol\u2010maleimide reaction and site\u2010specific protein assembly using a two\u2010arm polyethylene glycol (PEG)\u2010maleimide crosslinker (Mal<jats:sub>2<\/jats:sub>). The Mal<jats:sub>2<\/jats:sub>(1k) (with 1\u2009kDa PEG spacer)\u2010crosslinked PGUS assemblies showed low crosslinking efficiency and unimproved thermostability except for G194C\u2010Mal<jats:sub>2<\/jats:sub>(1k). To improve the crosslinking efficiency, a lengthened crosslinker Mal<jats:sub>2<\/jats:sub>(2k) (with 2\u2009kDa PEG spacer) was used to produce PGUS assembly and a highly improved thermostability was achieved with a half\u2010life of 47.2\u2013169.2\u2009min at 70\u00b0C, which is 1.04\u20133.74 times that of wild type PGUS. It is found that the thermostability of PGUS assembly was closely associated with the formation of inter\u2010tetramer assembly and intratetramer crosslinking, rather than the PEGylation of the enzyme. Therefore, the four\u2010arm PEG\u2010maleimide crosslinker Mal<jats:sub>4<\/jats:sub>(2k) (with 2\u2009kDa PEG spacer) was employed to simultaneously increase the inter\u2010tetramer assembly and intratetramer crosslinking, and the resulting PGUS assemblies showed further improved thermostabilities compared with Mal<jats:sub>2<\/jats:sub>(2k)\u2010crosslinked 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.<\/jats:p>","DOI":"10.1002\/bit.28556","type":"journal-article","created":{"date-parts":[[2023,9,14]],"date-time":"2023-09-14T13:52:49Z","timestamp":1694699569000},"page":"3570-3584","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Site\u2010specific crosslinking and assembly of tetrameric \u03b2\u2010glucuronidase improve glycyrrhizin hydrolysis"],"prefix":"10.1002","volume":"120","author":[{"given":"Qibin","family":"Wang","sequence":"first","affiliation":[{"name":"Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering Ministry of Industry and Information Technology, Institute of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology Beijing P.R. 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