Streptococcus mutans Dextransucrase: Functioning of Primer Dextran and Endogenous Dextranase in Water-Soluble and Water-Insoluble Glucan Synthesis

Abstract

The extracellular enzyme activities ofStreptococcus mutans 6715 that synthesize glucans from sucrose were concentrated and partially purified by ammonium sulfate precipitation and gel permeation column chromatography. Polyacrylamide gel analysis demonstrated that all of the major proteins precipitated by ammonium sulfate were quantitatively recovered in the high-molecularweight, enzyme-containing aggregates found in the void volume of the gel column. Anion-exchange column chromatography was used to fractionate the aggregates into preparations, a and /3, which produced water-insoluble and water-soluble glucans, respectively. Polyacrylamide gel analysis showed that a and ,3 contained unique proteins and dextransucrase (EC 2.4.1.5) activities. Studies on the time course of glucan synthesis by a demonstrated that this enzyme preparation contained dextranase activity, which partially degraded nascent alcohol-insoluble glucan into alcohol-soluble products that were subsequently reincorporated into insoluble product. The ,3 enzyme preparation contained no detectable dextranase activity. Mixing experiments in the absence of primer dextran demonstrated that the dextranase activity present in a could modify glucan production by /. CsCl density gradient analysis of product glucans demonstrated that exogenous primer dextrans were used as acceptor molecules by both the a and /8 enzyme preparations, and that water-soluble glucans synthesized by /8 could be converted into water-insoluble glucans by a.It is proposed that the structural heterogeneity of the native glucans produced from sucrose by S. mutans is a result of the concerted action of glucan-forming dextransucrases and endohydrolytic dextranase activity. 31). A significant effect of the presence of exogenous dextranase (Spicaria violaceae) during glucan synthesis by S. mutans dextransucrase on the quantity of a-(1--+3) linkages in the product glucans has been reported (7). Thus, the endogenous S. mutans dextranase may con-637 on July 16, 2020 by guest http://iai.asm.org/ Downloaded from cates the position of the bovine serum albumin standard run in an adjacent lane of the gel.

Effect of sucrose in culture media on the location of glucosyltransferase of Streptococcus mutans and cell adherence to glass surfaces

“…It has also been found that addition of glucan to extracellular GTase resulted in the formation of salt-stable enzyme-glucan complexes, indicating a high affinity of GTase for glucans (4-6). Germaine et al (5) have emphasized the roles of glucan and enzyme aggregation in enzyme binding to the cell surface. Furthermore, it is of interest to note that many "purified" GTase preparations contain a tightly bound carbohydrate (probably glucan) moiety (2,4,20).…”

Glucansucrases: mechanism of action and structure–function relationships

“…The necessity of a primer was first accepted in relation to glycogen synthesis mechanism studies carried out at the same period [43]. This hypothesis has been supported by the fact that addition of exogenous glucan has an activating effect on glucan synthesis [50, 52, 53]. However, glucansucrases are active enzymes in the absence of any exogenous primer.…”

Exocellular polysaccharides produced by lactic acid bacteria

“…It was not established if the a-l-3 linkages were part of the main chains, or if two types of glucan synthesizing enzymes were present in S. mutans and $. sanguis, one synthesizing a-l-6-linked (soluble) and the other a-l-3-1inked (insoluble) polymer [46][47][48][49][50]. In the last 10 years knowledge of enzymes involved in mutan synthesis has increased and it is clear now that several glucosyltransferases are produced by mutans streptococci.…”