Researchers in China have developed a coenzyme-free in vitro enzymatic pathway that produces galacto- N -biose (GNB), a sugar building block found in the gut’s protective mucus layer, from lactose, an abundant low-value byproduct of the dairy industry. The research was led by Prof. Yi-Heng P. J. Zhang at the Tianjin Institute of Industrial Biotechnology (TIB), Chinese Academy of Sciences (CAS). The study was published in the September 25 issue of the Chinese Journal of Biotechnology .
GNB is a functional disaccharide in which galactose and N -acetylgalactosamine (GalNAc) are joined by a β-1,3 glycosidic bond. It forms part of the sugar chains attached to gastrointestinal mucins, the proteins that help build the intestinal mucus barrier. Previous studies suggest that GNB can support beneficial gut bacteria, including bifidobacteria, and have reported neuroprotective effects. These properties make GNB a promising ingredient for functional foods, infant nutrition fortification and biomedical research.
The production of GNB remains costly. Some enzymatic biosynthesis routes consume ATP or require complex systems to regenerate UDP-sugar donors. Others rely on specialized sugar donors and face competing hydrolysis reactions that reduce conversion. These increase production costs and require complicate process development.
“We wanted to simplify GNB synthesis without the need for added ATP and UDP-sugar donors,” Prof. Zhang said. “By coupling glucose removal, we developed a minimal (shortest) three-enzyme machine that runs entirely on cheap starting materials toward the target product.”
The pathway works in a one-pot. First, a lactose phosphorylase, in fact a cellobiose phosphorylase from the thermophilic bacterium Thermoclostridium caenicola , used here for its activity toward lactose, converts lactose into galactose 1-phosphate and glucose. Next, a thermostable lacto- N -biose phosphorylase transfers the galactose unit onto GalNAc to form GNB, releasing inorganic phosphate that is recycled back into the first step. Because the phosphorylation reaction is reversible and tends to stall at equilibrium, the researchers added a third, auxiliary thermophilic enzyme, polyphosphate glucokinase (PPGK), which uses low-cost polyphosphate to remove glucose, pulling the overall reaction toward GNB production.
After intensively optimizing temperature, pH and the dosage of the rate-limiting enzyme, the researchers obtained 7.6 mM GNB within four hours at 55 o C and pH 6.5, achieved a 76% molar yield relative to the initial GalNAc. GNB concentrations remained approximately stable through eight hours. Notably, the cascade precisely rearranges the β-1,4 bond of lactose into the β-1,3 bond of GNB without the chemical protection and deprotection steps.
“Lactose is widely available from dairy processing, making it an attractive starting material for functional-sugar synthesis,” said Prof. Zhang. " This study provides a new foundation for further developing less costly GNB production and exploring similar strategies for other functional oligosaccharides."
The approach remains at the laboratory stage. Further work would improve the activity and stability of the key enzymes, and explore enzyme immobilization to enable catalyst reuse. These developments would help establish in vitro biomanufacturing at a large scale.
The study extends the application of in vitro BioTransformation (ivBT) platform, a new biomanufacturing platform pioneered by Zhang's team, wherein in vitro multi-enzyme molecular machines can surpass microbial cell factories.
Other contributors include Hao Fang from Tianjin University of Science and Technology in Tianjin, China, Yuan Li from the Tianjin Institute of Industrial Biotechnology in Tianjin, China, and so on.
This work was supported by the National Key Research and Development Program of China (2022YFC3401700).
Chinese Journal of Biotechnology
Coenzyme-free in vitro biotransformation for the synthesis of galacto-N-biose
25-Sep-2026