Synergistic construction of structurally uniform, highly stable, and recyclable β-glucosidase aggregates via surface...

The development of carrier-free immobilized enzymes with outstanding thermal stability is crucial for efficient biocatalysis under extreme industrial conditions. In this study, we propose a synergistic strategy combining protein surface carboxyl engineering with catalytically active inclusion bodies (CatIBs) crosslinking to construct highly stable β-glucosidase cross-linked enzyme aggregates (CLEAs). Using β-glucosidase TsBgl1 from Thermotoga sp. KOL6 as a model, site-directed mutagenesis was employed to introduce glutamate and aspartate residues on the enzyme surface, significantly increasing the number of surface carboxyl groups available for crosslinking. The engineered genes were efficiently expressed in E. coli, and the resulting CatIBs were readily purified by low-speed centrifugation at 4000 rpm. Subsequent EDC-NHS-mediated crosslinking with polyethylenimine yielded catalytically active inclusion body-based cross-linked enzyme aggregates (CLCatIBs) with uniform morphology and high activity recovery. The multi-site mutant E7D3 CLCatIBs exhibited remarkable thermal stability, retaining 80 % of activity after incubation at 80 °C for 60 min and 50 % after 40 min at 90 °C, while maintaining over 75 % of initial activity after six reuse cycles. In high-temperature cellobiose hydrolysis, E7D3 CLCatIBs achieved a 1.7-fold increase in glucose conversion compared to the wild-type. This work provides an efficient and scalable approach for constructing carrier-free immobilized enzymes with enhanced thermal stability, demonstrating significant potential for industrial applications.