Efficient One-Pot Synthesis of Uridine Diphosphate Galactose Employing a Trienzyme System

The limited availability of high-cost nucleotide sugars is a significant constraint on the application of their downstream products (glycosides and prebiotics) in the food or pharmaceutical industry. To better solve the problem, this study presented a one-pot approach for the biosynthesis of UDP-Gal using a thermophilic multienzyme system consisting of GalK, UGPase, and PPase. Under optimal conditions, a 2 h reaction resulted in a UTP conversion rate of 87.4%. In a fed-batch reaction with Gal/ATP = 20 mM:10 mM, UDP-Gal accumulated to 33.76 mM with a space-time yield (STY) of 6.36 g/L·h–1 after the second feeding. In repetitive batch synthesis, the average yield of UDP-Gal over 8 cycles reached 10.80 g/L with a very low biocatalyst loading of 0.002 genzymes/gproduct. Interestingly, Galk (Tth0595) could synthesize Gal-1P using ADP as a donor of phosphate groups, which had never been reported before. This approach possessed the benefits of high synthesis efficiency, low cost, and superior reaction system stability, and it provided new insights into the rapid one-pot synthesis of UDP-Gal and high-value glycosidic compounds

Enhanced Synthesis of Alkyl Galactopyranoside by <i>Thermotoga naphthophila</i> β‑Galactosidase Catalyzed Transglycosylation: Kinetic Insight of a Functionalized Ionic Liquid-Mediated System

Green synthesis is of pivotal importance for environmental sustainability. This work reports a novel approach to synthesize an array of alkyl galactopyranosides using thermophilic β-galactosidase from <i>Thermotoga naphthophila</i> RKU-10 (TN1577) as biocatalyst and milk processing waste lactose as galactosyl donor. Ammoeng 102 (only 2.5% addition of total reaction volume), a functionalized ionic liquid (IL) containing tetraaminum cation with C₁₈ acyl and oligoethylene glycol, is identified as the most promising one from a variety of structurally diverse ILs, affording a 2.37-fold increase in octyl galactopyranoside yield compared to the buffer system. Up to 18.2 g L^–1 octyl galactopyranoside could be produced in 7 h, which is significantly higher than any previous report in terms of time-space efficiency. Kinetic study and COSMO-RS <i>in silico</i> predictions elucidate that the thermophilic nature of TN1577 β-galactosidase, increased solubility of substrate, suppression of hydrolysis, and excellent biocompatibility of Ammoeng 102 with enzyme (allowing TN1577 β-galactosidase to perform optimal catalysis up to 95 °C) are the main driving forces. The general applicability of the Ammoeng 102 system is verified, by which a series of alkyl galactopyranosides are successfully synthesized with <i>n</i>-butanol to <i>n</i>-tetradecanol as alkyl acceptors and lactose as galactosyl donor