Dextransucrase Reaction Research Articles

The mechanism of acceptor reactions of leuconostoc mesenteroides B-512F dextransucrase

Reactions of dextransucrase and sucrose in the presence of sugars (acceptors) of low molecular weight have been observed to give a dextran of low molecular weight and a series of oligosaccharides. The acceptor reaction of dextransucrase was examined in the absence and presence of sucrose by using d-[ 14C]glucose, d-[ 14C]fructose, and 14C-reducing-end labeled maltose as acceptors. A purified dextransucrase was pre-incubated with sucrose, and the resulting d-fructose and unreacted sucrose were removed from the enzyme by chromatography on columns of Bio-Gel P-6. The enzyme, which migrated at the void volume, was collected and referred to as “charged enzyme”. The charged enzyme was incubated with 14C-acceptor in the absence of sucrose. Each of the three acceptors gave two fractions of labeled products, a high molecular weight product, identified as dextran, and a product of low molecular weight that was an oligosaccharide. It was found that all three of the acceptors were incorporated into the products at the reducing end. Similar results were obtained when the reactions were performed in the presence of sucrose, but higher yields of labeled products were obtained and a series of homologous oligosaccharides was produced when d-glucose or maltose was the acceptor. We propose that the acceptor reaction proceeds by nucleophilic displacement of glucosyl and dextranosyl groups from a covalent enzyme-complex by a specific, acceptor hydroxyl group, and that this reaction effects a glycosidic linkage between the d-glucosyl and dextranosyl groups and the acceptor. We conclude that the acceptor reactions serve to terminate polymerization of dextran by displacing the growing dextran chain from the active site of the enzyme; the acceptors, thus, do not initiate dextran polymerization by acting as primers.

Preparation of isomaltose oligosaccharides labelled with 14C in the non-reducing terminal unit, and their use in studies of dextranase activity

A series of end-labelled isomaltose oligosaccharides was prepared by the reaction of dextran-sucrase with sucrose- 14 C in the presence of excess of unlabelled isomaltose saccharides as alternative acceptor. The main product of each reaction contained one more D-glucose residue than the acceptor substrate, and the label was located at the non-reducing end. The end-labelled saccharides were used to determine the specificity of a bacterial dextranase that required five or more consecutive α-(1→6)- D-glucosidic linkages in the substrate. The third linkage from the reducing end of isomaltohexaose ( IM 6) and of other substrates with longer chains ( IM 7 and IM 8) was the most susceptible to attack, and the products from higher oligosaccharides were IM 3, IM 4, and IM 5. Isomaltopentaose ( IM 5) was further hydrolysed to IM 3 and IM 2 when a 35-fold excess of enzyme was added, but there was no action on IM 4, IM 3, or IM 2 under these conditions. It was concluded that the dextranase hydrolysed linkages penultimate to either end of the chain only with difficulty, and that end linkages were completely resistant to attack.

Über die Spezifität von Dextransaccharase aus Leuconostoc mesenteroides B512F

The specifity of the reaction of dextransucrase from leuconostoc mesenteroides B 512 F has been studied, and several methylated sucroses have been tested for substrate properties. It has been reported lately that dextran itself could act as a substrate and that dextransucrase not only produces dextran but according to multiple enzyme function also builds up levan. Chromatographic investigations of the reaction products as well as their hydrolysis substances lead to the conclusion that sucrose is the only substrate for dextransucrase, and no levan ist produced in this reaction. These results are discussed in connexion with our reaction mechanism of the enzymatic dextran syntheses, which was outlined in a recent paper.