Immobilization of laccase via cross-linked enzyme aggregates prepared using genipin as a natural cross-linker

Genipin is a nontoxic natural cross-linker that was successfully used to prepare cross-linked enzyme aggregates (CLEAs) of Trametes versicolor laccase. The recovered activity of CLEAs was influenced by the co-solvent type, genipin concentration, cross-linking time, preparation pH, and bovine serum albumin (BSA; amino group feeder) concentration. The characteristics of CLEAs prepared using genipin under optimal conditions (genipin-BSA-CLEAs) were compared with those of typical CLEAs prepared using glutaraldehyde or dextran polyaldehyde. Genipin-BSA-CLEAs were nano-sized (average diameter, approximately 700 nm), had a ball-like shape, showed a narrow size distribution, and exhibited the highest substrate affinity among the prepared CLEAs. The thermal stability of genipin-BSA-CLEAs was 6.8-fold higher than that of free laccase, and their pH stability was also much higher than that of free laccase in the tested range. Additionally, genipin-BSA-CLEAs retained 85% of their initial activity after 10 cycles of reuse. Particularly, genipin-BSA-CLEAs showed higher thermal and pH stability than CLEAs that were cross-linked using glutaraldehyde. Therefore, genipin represents an alternative to toxic compounds such as glutaraldehyde during cross-linking to prepare CLEAs.