Improvement of chitosan derivatization for the immobilization of bacillus circulans β-galactosidase and its further application in galacto-oligosaccharide synthesis.

Chitosan was derivatized by two methodologies to design a robust biocatalyst of immobilized Bacillus circulans β-galactosidase from a low-cost support for its further application in the synthesis of galacto-oligosaccharides (GOS). In the first one, chitosan was derivatized by cross-linking with glutaraldehyde and activated with epichlorohydrin; in the second one, cross-linking and activation were done with epichlorohydrin in a two-step process, favoring first support cross-linking and then support functionalization (C-EPI-EPI). Epoxy groups were hydrolyzed and oxidized, obtaining two supports activated with different aldehyde concentrations (100-250 μmol/g). The expressed activity and stability of the immobilized biocatalysts varied according to the derivatization methodology, showing that both the cross-linking agent and the activation degree are key parameters in the final biocatalyst performance. The best compromise between expressed activity and thermal stability was obtained using C-EPI-EPI with 200 μmol of aldehyde groups per gram of support. The immobilization conditions were optimized, obtaining a biocatalyst with 280 IU/g, immobilization yields in terms of activity and protein of 17.3 ± 0.4 and 61.5 ± 3.9%, respectively, and a high thermal stability, with a half-life of 449 times the value of the soluble enzyme. The biocatalyst was applied to the synthesis of GOS in repeated batch operation without affecting the product composition. Four successive batches were required for obtaining a cumulative specific productivity higher than the one obtained with the soluble enzyme.