A novel β-galactosidase from anaerobic thermophilic Caldicellulosiruptor saccharolyticus DSM 8903 with high potential for lactulose production.

Abstract

Background: Lactulose is a rare disaccharide with applications in the pharmaceutical, food, and animal feed industries. It can be synthesized through a transgalactosylation reaction between fructose and lactose, mediated by β-galactosidases. However, the need for high excess fructose to drive lactulose synthesis, along with relatively low yields and selectivity, has posed challenges for commercial production. This study extensively characterizes a novel lactulose-producing glycoside hydrolase family 2 (GH2) β-galactosidase from Caldicellulosiruptor saccharolyticus expressed in Escherichia coli (CsBgl2A).

Results: The enzyme exhibited high activity across a broad pH and temperature range, maintaining more than 95% of its maximal activity between pH 6.0-9.0 and 40-55 °C. Under optimal conditions - 750 g L^-1 initial sugar concentration, a fructose-to-lactose molar ratio of 4, 6 μmol L^-1 enzyme, pH 7.0, and 55 °C - the reaction produced a maximum lactulose concentration of 81.9 g L^-1 at 3 h, with 32.5% lactulose yield, 10.9% lactulose-to-total sugars yield, and 61.6% lactose conversion. By 6 h, nearly all lactose had been hydrolyzed.

Conclusion: CsBgl2A is among the few β-galactosidases that achieve high lactulose yields with low fructose-to-lactose ratios. This enzyme is thus promising for producing low lactose dairy products and for cost-effective lactulose synthesis. © 2025 Society of Chemical Industry.