Repeated-batch operation for the synthesis of lactulose with β-galactosidase immobilized by aggregation and crosslinking

•Operational stability of CLAGs was 8.7 times higher than in non-reactive conditions.•At fructose/lactose molar ratio of 4, 98 batches were performed (2145 h).•Enzyme stability was strongly affected by the sugars distribution in reaction medium.•Repeated-batch operation allowed a significant increase in lactulose production.

Synthesis of lactulose under repeated-batch operation was done with cross-linked aggregates of Aspergillus oryzae β-galactosidase (CLAGs). The effect of the crosslinking agent to enzyme mass ratio and cross-linking time were first evaluated. Best results were obtained at 5.5 g de glutaraldehyde/g enzyme at 5 h of cross-linking, obtaining a specific activity of 15,000 IU g−1, with 30% immobilization yield. CLAG was more stable than the free enzyme under non-reactive conditions with a half-life of 123 h at 50 °C and when operated in repeated-batch mode, yield and productivity was 3.8 and 4.3 times higher. Maximum number of batches was determined considering biocatalyst replacement at 50% residual activity. 98 and 27 batches could be performed under such criterion at fructose/lactose molar ratio of 4 and 20 respectively, reflecting that enzyme stability is strongly affected by the sugars distribution in the reaction medium.

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