Gwen J. Walker

Metabolism of the Polysaccharides of Human Dental Plaque

An investigation into occurrence of dextranase in human oral streptococci revealed that cell extracts of 8 out of 25 strains contained an enzyme, α-1,6-glucan glucohydrolase, that hydrolyzed the α-(1 → 6)-glucosidic linkages in dextran and isomaltose. Glucose was the only product of low molecular weight released by this enzyme. Six strains contained a different dextranase that released oligosaccharides of the isomaltose series from dextran. In the case of Streptococcus mutans IB16, it was established that the main oligosaccharide products were isomaltotetraose and isomaltopentaose. Cell-free filtrates from 14 of the 25 strains synthesized extracellular polysaccharides from sucrose, and eleven of the filtrates produced water-insoluble glucans. Dextranase occurred in five of the strains that synthesized glucan and in five strains that were unable to synthesize extracellular polysaccharides. All the insoluble glucans were partially hydrolyzed by a fungal α-(1 → 3)-glucanase. None of the cell-extracts or cell-free filtrates was able to hydrolyze α-1,3-glucan.

Degradation of dextrans by an α-1,6-glucan glucohydrolase from Streptococcus mitis

Abstract A dextran-glucosidase (α-1,6-glucan glucohydrolase) found in cell extracts of several strains of Streptococcus mitis has been purified, and some of its properties have been investigated. Dextran-glucosidase hydrolysed isomaltose and isomaltose saccharides at comparable rates, and a comparison of K m values for isomaltose, isomalto-pentaose and dextran showed that the enzyme had equal affinity for these substrates despite their different chain-length. The action pattern of the enzyme was not completely characteristic of an exo-glucanase or of a glucosidase. Its ability to act on polymers, and its complete specificity for α-(1→6)- D -glucosidic linkages were properties associated with exo-glucanases, whereas its transferring ability, the retention of configuration, and inhibition by nojirimycin supported its classification as a glucosidase. Dextrans were incompletely degraded by the enzyme, the extent of hydrolysis being related to the proportion of α-(1→6)- D -glucosidic linkages. It was concluded that the α-(1→4)- α-(1→3)-, and α-(1→2)- D -glucosidic linkages that occur in bacterial dextrans were resistant to the action of dextran-glucosidase, and arrested the further enzymic degradation of dextran. The use of the enzyme in investigations of the fine structure of dextrans is discussed.

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

Abstract 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.

Metabolism of the Polysaccharides of Human Dental Plaque: Release of Dextranase in Batch Cultures of Streptococcus mutans

Dextranase activity was determined in cell extracts and cell-free filtrates of Streptococcus mutans strains which had been grown in batch culture. Exo-dextranase activity was located chiefly in cell extracts, whereas endo-dextranase was mainly extracellular. Release of endo-dextranase began early in the exponential phase of growth, and ended when the concentration of residual sugar was low. Thus, dextranase expression was associated with rapidly growing cells, the yield of dextranase was increased several fold when the initial concentration of D-glucose in the medium was changed from 0.5% to 2%. The endo-dextranase was not stable at pH 5, and control of the pH of the culture was essential to preserve active dextranase during overnight growth. Strain Ingbritt (serotype c) and serotype d strains were the best dextranase producers; other strains (serotypes a, b, c, e and f) displayed much lower activity. The ability to produce endo-dextranase, and to synthesize alpha-D-glucans with a high proportion of (1 leads to 3)-linked sequences, appeared to be related properties. The possibility is discussed that the release of two enzymes, namely endo-dextranase and the D-glucosyltransferase (GTF-I) that synthesizes (1 leads to 3)-alpha-D-glucan, are factors that contribute to the cariogenicity of S. mutans serotype d.

Application of high-performance liquid chromatography to a study of branching in dextrans

Abstract The length of the side chains in dextrans has been examined by enzymic hydrolysis and l.c. The linear (IM n ) and branched (B n ) oligosaccharide-products of endodextranase activity were separated by l.c. in water. The branched fractions B 5 B 8 , obtained from Leuconostoc mesenteroides NRRL B-512(F) dextran were resolved into two components by the same system. Treatment of the isolated components of B 5 with (1→6)-α- d -glucan glucohydrolase, showed that B 5 -1, the oligosaccharide eluted in the first peak, was completely hydrolysed to d -glucose and B 4 , whereas B 5 -2 was not a substrate. The two components of B 6 B 8 were all hydrolysed to d -glucose and B 5 -2. From a knowledge of the specificity of the two dextranases, together with the results of methylation analysis, it was concluded that the B n -1 series were 3 3 -α-isomaltosylisomaltosaccharides, and that the B n -2 series were 3 3 -α- d -glucosylisomaltosaccharides. The products are consistent with the structures previously proposed for B 5 B 8 , and confirm directly that side chains containing two or more glucose residues occur in B-512(F) dextran. The B n -1 series was obtained neither from Streptococcus viridans NRRL B-1351 dextran nor from a chemically synthesized, branched dextran in which the (1→3) branch linkages attached d -glucosyl side-chains exclusively. A determination of B n -2 oligosaccharides and B 4 (3 3 -α- d -glucosylisomaltotriose) in the final products indicates the proportion of glucosyl side-chains in dextrans.

Metabolism of the polysaccharides of human dental plaque. : Part II. Purification and properties of cladosporium resinae (1→3)-α-D-glucanase, and the enzymic hydrolysis of glucans synthesised by extracellular D-glucosyltransferases of oral streptococci

Cladosporium resinae (1 leads to 3)-alpha-D-glucanase has been characterized as an endoglucanase capable of completely hydrolysing insoluble (1 leads to 3)-alpha-D-glucans isolated from fungal cell-walls. D-Glucose was the major product, but a small amount of nigerose was also produced. The enzyme was specific for the hydrolysis of (1 leads to 3) bonds that occur in sequence, and nigerotetraose was the smallest substrate that was rapidly attacked. Isolated (1 leads to 3)-alpha-D-glucosidic linkages that occur in mycodextran, isolichein, dextrans, and oligosaccharides derived from dextran were not hydrolysed. Insoluble glucan synthesised from sucrose by culture filtrates of Streptococcus spp. were all hydrolysed to various limits; the range was 11-61%. A soluble glucan, synthesised by an extracellular D-glucosyltransferase of S. mutans OMZ176, was not a substrate, whereas insoluble glucans synthesised by a different D-glucosyltransferase, isolated from S. mutans strains OMZ176 and K1-R, were extensively hydrolysed (84 and 92%, respectively). It is suggested that dextranase-CB, a bacterial endo(1 leads to 6)-alpha-D-glucanase that does not release D-glucose from any substrate, could be used together with C. resinae (1 leads to 3)-alpha-D-glucanase to determine the relative proportions of (1 leads to 6)-linked to (1 leads to 3)-linked sequences of D-glucose residues in the insoluble glucans produce by oral streptococci. The simultaneous action of the two D-glucanoses was highly effective in solubilizing the glucans.

Purification and substrate specificity of an endo-dextranase of Streptococcus mutans K1-R

Abstract An extracellular endo-dextranase has been isolated from Streptococcus mutans K1-R. Incubation of cell-free culture fluid with sucrose permitted the removal of a large proportion of the extracellular d -glucosyltransferases by irreversible adsorption onto the insoluble glucans that these enzymes synthesize from sucrose. The remaining d -glucosyltransferases were separated from dextranase by precipitation with ammonium sulphate, chromatography on hydroxylapatite and DEAE-cellulose, followed by filtration on Ultrogel. The major products of action of the purified dextranase on (1→6)-α- d -glucans were isomaltotriose (IM 3 ), isomaltotetraose (IM 4 ), and isomaltopentaose (IM 5 ). Further hydrolysis of IM 4 and IM 5 occurred after prolonged incubation with excess of enzyme, to give d -glucose, IM 2 , and IM 3 . The relative rate of hydrolysis of isomaltose saccharides fell sharply with decreasing chainlength from IM 12 to IM 5 . The hydrolysis of dextrans containing 96% or more of (1→6)-α- d -glucosidic linkages, expressed as apparent conversion into IM 3 , was virtually complete, and substrates such as Streptococcus sanguis glucan, containing sequences of (1→6)-α- d -glucosidic linkages, were also effectively hydrolyzed. Dextranase activity towards the soluble glucan of Streptococcus mutans was limited, and there was no action on the insoluble glucan synthesized by S. mutans sucrose 3- d -glucosyltransferase.

Action of α-1,6-glucan glucohydrolase on oligosaccharides derived from dextran

Abstract The action of α-1,6-glucan glucohydrolase on α-(1→6)- D -glucosidic linkages in oligosaccharides that also contain an α-(1→2)-, α-(1→3)-, or α-(1→4)- D -glucosidic linkage has been investigated. The enzyme could hydrolyse α-(1→6)- D -glucosidic linkages from the non-reducing end, including those adjacent to an anomalous linkage. α-(1→6)- D -Glucosidic linkages at branch points were not hydrolysed, and the enzyme could neither hydrolyse nor by-pass the anomalous linkages. These properties of α-1,6-glucan glucohydrolase explain the limited hydrolysis of dextrans by the exo-enzyme. Hydrolysis of the main chain of α-(1→6)- D -glucans will always stop one D -glucose residue away from a branch point. The extent of hydrolysis by α-1,6-glucan glucohydrolase of some oligosaccharide products of the action on dextran of Penicillium funiculosum and P. lilacinum dextranase, respectively, has been compared. Differences in the specificity of the two endo-dextranases were revealed. The Penicillium enzymes may hydrolyse dextran B-512 to produce branched oligosaccharides that retain the same 1-unit and 2-unit side-chains that occur in dextran.

Inducible and constitutive formation of fructanase in batch and continuous cultures of Streptococcus mutans

The production of extracellular beta-D-fructanase by several strains of Streptococcus mutans was studied in continuous culture. When glucose was the limiting nutrient, S. mutans K1-R and OMZ176 accumulated fructanase to maximum levels at low growth rates (dilution rate 0.05-0.10 h-1), due to the longer residence times of the bacteria in the culture vessel under these conditions. Extracellular fructanase activity was greater than has been previously reported for batch cultures. The rate of fructanase production for both S. mutans strains K1-R and OMZ176 increased with increasing growth rate when glucose was limiting. Under conditions of glucose sufficiency, the rate of fructanase production was always lower than in cultures where glucose was limiting, irrespective of the growth rate. Cultures of S. mutans Ingbritt (serotype c) grown with sorbitol- or glucose-limitation synthesized fructanase at a very low basal rate. When fructose was the limiting carbohydrate the enzyme was induced with a maximum rate of production occurring at a dilution rate of 0.40 h-1. Strains of S. mutans from other serotypes (a, d, d/g) were either not affected by changing the limiting sugar from glucose to fructose or else fructanase activity was slightly decreased in the fructose-limited medium. Fructanases from various strains of S. mutans readily hydrolysed (2----6)-beta-D-fructans, but all possessed the ability to hydrolyse (2----1)-beta-D-fructans to varying degrees.

The action pattern of Penicillium lilacinum dextranase

The product distributions resulting from the action of Penicillium lilacinum dextranase on end-labelled oligosaccharides of the isomaltose series have been determined. The initial rates of formation of labelled products were measured for isomaltotriose up to isomalto-octaose, and the molar proportions and radioactivity of the final products from isomaltotriose up to isomaltohexaose were determined. D-Glucose was released only from isomaltotriose and isomaltotetraose, by hydrolysis of the first linkage from the reducing end (linkage 1); the terminal bonds of higher members of the series were not attacked. All oligosaccharides except isomaltotriose were hydrolyzed at more than one linkage. The main points of attack on isomaltotetraose up to isomalto-octaose were at linkage 2, and at the third linkage from the non-reducing end; these two positions coincide for isomaltopentaose. The degradation of isomaltotriose up to isomalto-octaose was entirely hydrolytic. The enzyme also catalyzed an extremely slow, concentration-dependent degradation of isomaltose, and this may have occurred via a condensation to isomaltotetraose, followed by hydrolysis of linkage 1 to give D-glucose and isomaltotriose.