Abstract

The acceptor reaction of dextransucrase consists of the transfer of d-glucosyl groups from
sucrose to other carbohydrates, and occurs at the expense of dextran synthesis. In the present
study, solutions of [14C]sucrose and of each of seventeen acceptor sugars were digested with
highly purified Leuconostoc mesenteroides B-512F dextransucrase. The products were
separated by paper chromatography, and quantitated by liquid scintillation counting. Maltose
was the most effective acceptor; its products, members of an isomaltodextrinyl-maltose series
(d.p. 3 to 6), accounted for >75% of the d-glucosyl groups of sucrose. Other acceptors giving
rise to a similar series of oligosaccharide products were (in order of decreasing
effectiveness): isomaltose, nigerose, methyl α-d-glucoside, 1,5-anhydro-d-glucitol, d-glucose,
turanose, methyl β-d-glucoside, cellobiose, and l-sorbose. Lactose, raffinose, melibiose, d-
galactose, and d-xylose each gave a single, mono-d-glucosylated product; d-fructose and d-
mannose each gave a pair of mono-d-glucosylated (disaccharide) products. Another series of
digests contained sucrose and various proportions of maltose. As the level of maltose
increased, the size of the largest oligosaccharide acceptor-product decreased, and less dextran
was produced. The virtual absence of high-d.p. (8 to 13) oligosaccharide products in all
acceptor digests is interpreted as evidence against a role for acceptors as primers of dextran
synthesis.

Abstract
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.

Abstract

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. Abstract

The specific growth rate, the maximum specific dextransucrase production rate and the
maximum dextransucrase yield during the dextransucrase fermentation byLeuconostoc
mesenteroides were all unaffected by both the pH of the culture medium, maintained at 5.5 or
6.7, and the mode of pH control, by adding NaOH or K2HPO4.