Lactose-assisted immobilization of β-galactosidase with enhanced transglycosylation performance

The construction of immobilized β-galactosidase with structural robustness and enhanced transglycosylation activity represents a promising approach to elevate the functional properties of dairy products. In this study, the β-galactosidase from Kluyveromyces lactis was immobilized onto Duolite A568, a macroporous anion-exchange resin, through electrostatic adsorption followed by glutaraldehyde crosslinking. The immobilization conditions were optimized, and the retention of the maximum enzymatic activity was obtained after incubation at pH 4.5, with an enzyme concentration of 2.0 mg/mL for 60 min, resulting in a high enzyme loading capacity of ∼27 mg/g resin. Post-crosslinking with 0.1 % glutaraldehyde remarkably enhanced the relative activity of the immobilized enzyme to 144 % compared to the non-crosslinked adsorbed enzyme. Notably, a novel sugar-assisted immobilization strategy was introduced by incorporating sugars at specific immobilization stages. Comparatively, the lactose-assisted crosslinking approach exhibited exceptional performance, yielding immobilized β-galactosidase (labelled as ACP-β-galactosidase) with a relative activity of 226 %. ACP-β-galactosidase maintained high activity over a broad pH range (4.0-10.0) and retained over 80 % of its initial activity after ten catalytic cycles, indicating high operational stability. Further investigation on the transglycosylation activity of the ACP-β-galactosidase in terms of the yield of lactulose was conducted in the presence of fructose. The ACP-β-galactosidase achieved a lactulose yield of 60.45 g/L (conversion ratio: 24.92 %), markedly higher than that of the free enzyme (39.55 g/L, 16.23 %). This study not only provides a robust lactose-assisted immobilization strategy for constructing high-performance β-galactosidase biocatalysts but also presents a new trial for altering the lactulose biosynthesis through immobilization-induced modulation of β-galactosidase functionality.