Synthesis of galactooligosaccharides by CBD fusion β-galactosidase immobilized on cellulose

► The fusion of Lactobacillus bulgaricus β-galactosidase with cellulose binding domain. ► The immobilization of the fusion enzyme on cellulose. ► The maximal galactooligosaccharides yield by the cellulose immobilized enzyme reached 49%. ► The immobilized enzyme could be recycled for 20 batches with oligosaccharide yields all above 40%. The β-galactosidase gene (bgaL3) was cloned from Lactobacillus bulgaricus L3 and fused with cellulose binding domain (CBD) using pET-35b (+) vector in Escherichia coli. The resulting fusion protein (CBD-BgaL3) was directly adsorbed onto microcrystalline cellulose with a high immobilization efficiency of 61%. A gram of cellulose was found to absorb 97.6U of enzyme in the solution containing 100mM NaCl (pH 5.8) at room temperature for 20min. The enzymatic and transglycosylation characteristics of the immobilized CBD-BgaL3 were similar to the free form. Using the immobilized enzyme as the catalyst, the yield of galactooligosaccharides (GOS) reached a maximum of 49% (w/w) from 400g/L lactose (pH 7.6) at 45°C for 75min, with a high productivity of 156.8g/L/h. Reusability assay was subsequently performed under the same reaction conditions. The immobilized enzyme could retain over 85% activity after twenty batches with the GOS yields all above 40%.