Cross-Linked Enzyme-Adhered Nanoparticles (CLEANs) for Continuous-Flow Bioproduction

Nanostructured but micro-sized biocatalysts were created by bottom-up technology using multi-functionalized silica nanoparticles (NPs) as nano-sized building blocks to form cross-linked enzyme-adhered nanoparticles (CLEANs) as robust micro-sized particles with beneficial internal structure and good mechanical properties. Systematic surface modification of NPs with a grafting mixture consisting of organosilanes with reactive (aminopropyl) and inert (e. g., vinyl, propyl, phenyl, or octyl) functions resulted in functional NPs enabling cross-linking agents, such as glutardialdehyde or bisepoxides (glycerol diglycidyl ether, neopentylglycol diglycidyl ether, and poly(propylene glycol) diglycidyl ether), to bind and cross-link enzymes covalently and to form macroporous microparticles. These CLEANs were able to diminish several weaknesses of traditional cross-linked enzyme aggregates as biocatalysts, such as poor mechanical resistance, difficult recovery, and storage, strengthening their use for packed-bed enzyme reactors. Lipase B from Candida antarctica (CaLB) was selected as model enzyme for development of robust CLEANs, which were successfully tested for various industrially relevant applications including a kinetic resolution of a racemic alcohol and the production of various natural fragrance compounds under continuous-flow conditions.