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

• DOI: 10.1016/j.biortech.2012.03.108
• Publication Date: 2012-04-06T10:16:16Z
• Authors: Lili Lu; Shuze Xu; Renfei Zhao; Dayu Zhang; Zhengyi Li; Yumei Li; Min Xiao
• Keywords: 0106 biological sciences; Time Factors; Recombinant Fusion Proteins; Molecular Sequence Data; Temperature; Galactose; Oligosaccharides; Hydrogen-Ion Concentration; Sodium Chloride; Enzymes, Immobilized; beta-Galactosidase; 01 natural sciences; Protein Structure, Tertiary; Kinetics; Lactobacillus; Batch Cell Culture Techniques; Enzyme Stability; Electrophoresis, Polyacrylamide Gel; Recycling; Adsorption; Cloning, Molecular; Cellulose

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 97.6 U of enzyme in the solution containing 100mM NaCl (pH 5.8) at room temperature for 20 min. 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 400 g/L lactose (pH 7.6) at 45 °C for 75 min, with a high productivity of 156.8 g/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%.