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However, the application of BVMOs is restrained by cofactor dependency and enzyme instability combined with water\u2010related downsides such as low substrate loading, low oxygen capacity, and water\u2010induced side reactions. Herein, we described a redox\u2010neutral linear cascade with in\u2010situ cofactor regeneration catalyzed by fused alcohol dehydrogenase and cyclohexanone monooxygenase in aqueous and microaqueous organic media. The cascade conditions have been optimized regarding substrate concentrations as well as the amounts of enzymes and cofactors with the Design of Experiments (DoE). The carrier\u2010free immobilization technique, crosslinked enzyme aggregates (CLEAs), was applied to fusion enzymes. The resultant fusion CLEAs were proven to function in microaqueous organic systems, in which the enzyme ratios, water contents (0.5\u20135\u2005vol.\u2009%), and stability have been systematically studied. The fusion CLEAs showed promising operational (up to 5 cycles) and storage stability.<\/jats:p>","DOI":"10.1002\/cbic.202200794","type":"journal-article","created":{"date-parts":[[2023,2,7]],"date-time":"2023-02-07T07:55:10Z","timestamp":1675756510000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Crosslinked Aggregates of Fusion Enzymes in Microaqueous Organic Media"],"prefix":"10.1002","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1628-037X","authenticated-orcid":false,"given":"Guillem","family":"Vernet","sequence":"first","affiliation":[{"name":"Institute of Technical Chemistry Leibniz University Hannover  Callinstr. 5 30167 Hannover Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2435-1748","authenticated-orcid":false,"given":"Yu","family":"Ma","sequence":"additional","affiliation":[{"name":"Institute of Technical Chemistry Leibniz 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