Cell-Free Multi-Enzyme Synthesis and Purification of Uridine Diphosphate Galactose.

High costs and low availability of UDP-galactose hampers the enzymatic synthesis of valuable oligosaccharides such as human milk oligosaccharides. Here, we report the development of a platform for the scalable, biocatalytic synthesis and purification of UDP-galactose. UDP-galactose was produced with a titer of 48 mM (27.2 g/L) in a small-scale batch process (200 µL) within 24 h using 0.02 genzyme /gproduct . Through in-situ ATP regeneration, the amount of ATP (0.6 mM) supplemented was around 240-fold lower than the stoichiometric equivalent required to achieve the final product yield. Chromatographic purification using porous graphic carbon adsorbent yielded UDP-galactose with a purity of 92 %. The synthesis was transferred to 1 L preparative scale production in a stirred tank bioreactor. To further reduce the synthesis costs here, the supernatant of cell lysates was used bypassing expensive purification of enzymes. Here, 23.4 g/L UDP-galactose were produced within 23 h with a synthesis yield of 71 % and a biocatalyst load of 0.05 gtotal_protein /gproduct . The costs for substrates per gram of UDP-galactose synthesized were around 0.26 €/g.

Combined biosynthetic pathway for de novo production of UDP-galactose: catalysis with multiple enzymes immobilized on agarose beads.

Regeneration of sugar nucleotides is a critical step in the biosynthetic pathway for the formation of oligosaccharides. To alleviate the difficulties in the production of sugar nucleotides, we have developed a method to produce uridine diphosphate galactose (UDP-galactose). The combined biosynthetic pathway, which involves seven enzymes, is composed of three parts: i) the main pathway to form UDP-galactose from galactose, with the enzymes galactokinase, galactose-1-phosphate uridyltransferase, UDP-glucose pyrophosphorylase, and inorganic pyrophosphatase, ii) the uridine triphosphate supply pathway catalyzed by uridine monophosphate (UMP) kinase and nucleotide diphosphate kinase, and iii) the adenosine triphosphate (ATP) regeneration pathway catalyzed by polyphosphate kinase with polyphosphate added as an energy resource. All of the enzymes were expressed individually and immobilized through their hexahistidine tags onto nickel agarose beads ("super beads"). The reaction requires a stoichiometric amount of UMP and galactose, and catalytic amounts of ATP and glucose 1-phosphate, all inexpensive starting materials. After continuous circulation of the reaction mixture through the super-bead column for 48 h, 50 % of the UMP was converted into UDP-galactose. The results show that de novo production of UDP-galactose on the super-bead column is more efficient than in solution because of the stability of the immobilized enzymes.

Concentration of white blood cell UDPgalactose and UDPglucose determined by high performance liquid chromatography.

We have applied a HPLC method to separate and quantitate UDPgalactose (UDPGal) and UDPglucose (UDPGlu) in human white blood cells (WBCs). Trichloroacetic acid-treated, protein-free filtrates were chromatographed on an anion-exchange column (Dionex CarboPac PA-1) using a gradient of 20-40% potassium phosphate buffer (pH 4.5). Recoveries of UDPGal and UDPGlu ranged from 93 to 106%, and the method was linear over a wide range of WBC protein concentrations. Volumes of blood as low as 2.5 ml (2.2 mg WBC protein) could be used to achieve quantitative recovery of the sugar nucleotides. The mean values and standard deviations of UDPGal and UDPGlu in 33 normal individuals ranging in age from 1 day to 65 years were 12.4 +/- 4.2 and 31.5 +/- 9.3 mumol/100 g protein, respectively. No statistical differences in UDPGal and UDPGlu values were observed between children and adults. No correlation was established between the concentrations of UDPGal and UDPGlu and either total WBC count or the number of lymphocytes obtained from Coulter counter analysis. There was no relationship between the concentrations of UDPGal and UDPGlu in WBCs and RBCs which were prepared from the same blood specimen.

Reconstitution into proteoliposomes and partial purification of the Golgi apparatus membrane UDP-galactose, UDP-xylose, and UDP-glucuronic acid transport activities.

Previous studies in vitro on proteoglycan biosynthesis from our laboratory have shown that nucleotide sugar precursors of all the sugars of the linkage oligosaccharides (xylose, galactose, and glucuronic acid) and of the glycosaminoglycans (N-acetylglucosamine, N-galactosamine, and glucuronic acid) are transported by specific carriers into the lumen of Golgi vesicles. More recently, we also reported the reconstitution in phosphatidylcholine liposomes of detergent-solubilized Golgi membrane proteins containing transport activities of CMP-sialic acid and adenosine-3'-phosphate-5'-phosphosulfate. We have now completed the successful reconstitution into liposomes of the Golgi membrane transport activities of UDP-galactose, UDP-xylose, and UDP-glucuronic acid. Transport of these nucleotide sugars into Golgi protein proteoliposomes occurred with the same affinity, temperature dependence, and sensitivity to inhibitors as observed with intact Golgi vesicles. Preloading of proteoliposomes with UMP, the putative antiporter for Golgi vesicle transport of these nucleotide sugars, stimulated transport of the nucleotide sugars by 2-3-fold. Transport of UDP-xylose into Golgi protein proteoliposomes was dependent on the presence of endogenous Golgi membrane lipids while that of UDP-galactose and UDP-glucuronic acid was not. This suggests a possible stabilizing or regulatory role for Golgi lipids on the UDP-xylose translocator. Finally, we have also shown that detergent-solubilized Golgi membrane translocator proteins can be partially purified by an ion-exchange chromatographic step before successful reconstitution into liposomes, demonstrating that this reconstitution approach can be used for the biochemical purification of these transporters.

Synthesis, purification, and characterization of 2,4,6-trinitrophenyl-UDP-galactose: a fluorescent substrate for galactosyltransferase.

Glycosyltransferases enzymatically transfer monosaccharides from sugar-nucleotides to complex oligosaccharide chains and, as cell surface molecules, exhibit receptor-like activity. We have modified the substate UDP-galactose to produce a compound that has useful absorbance and fluorescence properties upon binding to galactosyltransferase (GalTase). Using strategies similar to those for preparing fluorescent ATP analogs, we were able to synthesize 2,4,6-trinitrophenyl-5'-UDP-galactose (TUG). In solution, the absorbance properties of TUG are pH dependent, with absorbance maxima at 260, 408, and 453 nm and an isobestic point at 353 nm. In the presence of soluble GalTase extracted from bovine milk, TUG exhibited an excitation maximum at 368 nm with emission maxima at 436 and 533 nm; in the absence of GalTase only the 533-nm peak was present. Under enzymatic conditions, TUG acted as a competitive substrate of UDP-galactose with GalTase. Under noncatalytic conditions, the fluorescence emission of TUG at 436 nm increased monotonically with Gal-Tase concentration, with a half-maximal response at approximately 4 microM. This compound may be useful for quantifying GalTase function as both an enzyme and a cell adhesion molecule.

Synthesis and rapid purification of UDP-[6-3H]galactose.

UDP[6-3H]galactose of high specificity can be obtained by oxidation of the C-6 hydroxymethyl group of UDP-galactose by galactose oxidase and subsequent reduction by sodium borotritide. One-step purification of the nucleotide sugar involves anion-exchange chromatography on a Pharmacia Mono Q column. Radiolabeled UDP-N-acetylgalactosamine can also be synthesized and purified by this procedure. Both nucleotide sugars can be used for sugar incorporation studies using the appropriate glycosyltransferase.