Tianjin Institute: three-enzyme in vitro pathway makes galacto-N-biose from lactose at 76% yield
Researchers at the Tianjin Institute of Industrial Biotechnology built a laboratory-stage cascade that makes galacto-N-biose from lactose and GalNAc without ATP or UDP-sugars.
Researchers led by Prof. Yi-Heng P. J. Zhang at the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, described an in vitro enzymatic pathway that synthesizes galacto-N-biose (GNB) from lactose and N-acetylgalactosamine (GalNAc) without ATP or UDP-sugars. According to the PubMed record of the study, published in the Chinese Journal of Biotechnology on 25 September, the pathway reached a molar yield of 76% with 10 mmol/L GalNAc doi.org
PubMeddoi.orgOpen source ↗. The work remains at the laboratory stage.
GNB is a disaccharide in which galactose and GalNAc are joined by a β-1,3 glycosidic bond. It is a core structural unit of O-glycan and a component of gastrointestinal mucins, and it plays a role in regulating intestinal microecology and maintaining the mucosal barrier doi.org
PubMeddoi.orgOpen source ↗. Earlier studies, as cited in the institute's release, suggest that GNB can support beneficial gut bacteria, including bifidobacteria, and have reported neuroprotective effects. Traditional enzymatic routes to GNB depend on a supply of ATP and UDP-sugars; other routes use specialized sugar donors and face competing hydrolysis reactions
doi.org
PubMeddoi.orgOpen source ↗.
Three enzymes in one pot
The pathway runs in a single vessel and uses two core enzymes and one auxiliary enzyme. Lactose phosphorylase (LacP), in this case a cellobiose phosphorylase from the thermophilic bacterium Thermoclostridium caenicola chosen for its activity toward lactose, converts lactose into galactose 1-phosphate and glucose. Lacto-N-biose phosphorylase (LnbP), a thermostable enzyme, then transfers the galactose unit onto GalNAc to form GNB. This step releases inorganic phosphate, which returns to the first reaction doi.org
PubMeddoi.orgOpen source ↗.
The cascade rearranges the β-1,4 bond of lactose into the β-1,3 bond of GNB, with no chemical protection and deprotection steps. Because the phosphorylation reaction is reversible and tends to stall at equilibrium, the team added the third enzyme, polyphosphate glucokinase (PPGK). It uses polyphosphate to remove the byproduct glucose, which shifts the equilibrium toward GNB and also regenerates inorganic phosphate doi.org
PubMeddoi.orgOpen source ↗.
Yield and conditions
The authors optimized the reaction temperature, pH and the dose of the rate-limiting enzyme. They obtained 7.6 mM GNB within four hours at 55 °C and pH 6.5, a 76% molar yield relative to the initial GalNAc. GNB concentration stayed approximately stable through eight hours.
The authors state that the in vitro route provides a technical basis for low-cost, large-scale production of GNB and other functional oligosaccharides doi.org
PubMeddoi.orgOpen source ↗. According to the release, further work would aim to improve the activity and stability of the key enzymes and to test enzyme immobilization so the catalysts can be reused. The study extends the in vitro BioTransformation (ivBT) platform developed by Zhang's team.
