Carrier-free immobilization of α-galactosidase using novel cross-linkers: Robust inclusion body-derived CLEAs for efficient hydrolysis of raffinose family oligosaccharides in soymilk

α-Galactosidases with high activity and stability are crucial for industrial applications, such as the hydrolysis of raffinose family oligosaccharides (RFOs) in soymilk. In this study, four novel cross-linkers, including dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), bis(sulfosuccinimidyl)suberate (BS 3 ), and bis- N -succinimidyl-(pentaethylene glycol) ester [BS(PEG) 5 ], were first applied to directly cross-link active α-galactosidase inclusion bodies. This simple approach generated stable and recyclable cross-linked enzyme aggregates (CLEAs) without precipitation or the need for carrier matrices. The resulting CLEAs showed 95–100% activity recovery, far exceeding that of glutaraldehyde (45%). All CLEAs exhibited broad pH and thermal stability, holding over 94% of their initial activity after 10 batches for RFOs hydrolysis in soymilk. Specifically, DMS-CLEAs maintained nearly 100% hydrolysis efficiency throughout 10 cycles. CLEAs prepared with the short-arm cross-linkers demonstrated superior enzyme activity recovery compared to those formed with the long-arm ones. These findings highlight the potential of α-galactosidase CLEAs for legume-based food processing.