Abstract: This study introduced a new source of bifidobacterial enzyme for the synthesis of galactooligosaccharide (GOS). We investigated the bioinformatics analysis, heterologous expression, enzymatic properties and application in the synthesis of GOS of a newly identified β-galactosidase (β-galATCC29521) derived from Bifidobacterium bifidum ATCC 29521. Bioinformatic analysis showed that β-galATCC29521 belonged to the glycoside hydrolase family 2, without a signal peptide or a transmembrane region. The optimal medium for β-galATCC29521 expression was composed of 10 g/L sucrose, 12 g/L tryptone, and 24 g/L yeast powder, and after 16 h of culture induced by 0.05 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) at 27 ℃, the activity of β-galATCC29521 was 318.03 U/mL. The crude enzyme was purified to electrophoretic homogeneity through Ni-IDA affinity chromatography. The optimal pH and temperature for β-galATCC29521 were 7.5 and 40 ℃, respectively. The purified enzyme was stable within the pH range of 6.5–9.0 and the temperature range of 10–30 ℃. Its activity was significantly enhanced in the presence of Mn2+ and Mg2+ but inhibited in the presence of Cu2+ and Fe3+. When using whey as the substrate, the yield of GOS synthesized by β-galATCC29521 was 29.83%. The results indicated that β-galATCC29521 holds potential in the synthesis of GOS.

Key words: galactooligosaccharide; β-galactosidase; Bifidobacterium bifidum ATCC 29521; fermentation optimization; enzymatic properties