Programming a Biofilm-Mediated Multienzyme-Assembly-Cascade System for the Biocatalytic Production of Glucosamine from Chitin.

Chitin is used as an essential raw material for the production of glucosamine (GlcN). In this study, we adopted three key enzymes, isolated from Thermococcus kodakaraensis KOD1, that catalyze the sequential conversion of α-chitin into GlcN and developed a multienzyme-assembly-cascade (MAC) system immobilized in a bacterial biofilm, which enabled a multistep one-pot reaction. Specifically, the SpyTag-SpyCatcher and SnoopTag-SnoopCatcher pairs provided covalent and specific binding force to fix enzymes to the biofilm one by one and assemble close enzyme cascades. The MAC system showed great catalytic activity, converting 79.02 ± 3.61% of α-chitin into GlcN with little byproducts, which was 2.09 times that of GlcN catalyzed by a mixture of pure enzymes. The system also exhibited good temperature and pH stability. Notably, 90% of enzyme activity was retained after 6 rounds of reuse, and appreciable activity remained after 17 rounds.