Optimization of conditions for the efficient production of mutan in streptococcal cultures and post-culture liquids

The strain Streptococcus sobrinus CCUG 21020 was found to produce water-insoluble and adhesive mutan. The factors influencing both stages of the mutan production, i.e. streptococcal cultures and glucan synthesis in post-culture supernatants were standardized. The application of optimized process parameters for mutan production on a larger scale made it possible to obtain approximately 2.2 g of water-insoluble glucan per 11 of culture supernate--this productivity was higher than the best reported in the literature. It was shown that some of the tested beet sugars might be successfully utilized as substitutes for pure sucrose in the process of mutan synthesis. Nuclear magnetic resonance analyses confirmed that the insoluble biopolymer synthesized by a mixture of crude glucosyltransferases was a mixed-linkage (1-->3), (1-->6)-alpha-D-glucan (the so-called mutan) with a greater proportion of 1,3 to 1,6 linkages.     

A comparative study of the extracellular glucosyl- and fructosyltransferases from cariogenic and non-cariogenic Streptococcus mutans strains of two different serotypes

Extracellular proteins from continuous cultures of serotype c and g Streptococcus mutans strains were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Gels stained with raffinose after electrophoresis revealed that although serotype c strains secrete two fructosyltransferases of molecular mass 68 kDa and 79 kDa, no fructosyltransferase was secreted by the serotype g strain K1. A sucrose activity stain was used to detect two glucosyltransferases (GTF) of molecular mass 162 kDa (bifunctional 1,6-alpha-D-glucan 3-alpha- and 6-alpha GTF or 'dextransucrase') and 153 kDa (a 1,3-alpha-D-glucan 3-alpha-GTF) in samples from cariogenic serotype c strains. Neither the 153 kDa protein nor the corresponding GTF activity was secreted by the non-cariogenic mutant C 67-25. The molecular masses of the corresponding 1,3-alpha and 1,6-alpha-GTF proteins from the serotype g strain K1 were 164 kDa and 158 kDa, respectively. All of the GTF proteins were degraded to discrete bands of lower molecular mass on storage at 4 degrees C even after extensive purification. The results provide an explanation for several outstanding controversies in the GTF literature.     

Purification and characterization of a third glucosyltransferase from Streptococcus mutans serotype g

Streptococcus mutans strain AHT (serotype g) secretes at least two glucosyltransferases with different pI values. A novel glucosyltransferase with a pI of 5.8 was purified 244-fold from the ammonium sulphate fraction by DEAE-cellulose chromatography, FPLC (Mono Q column, Pharmacia) and hydrophobic chromatography. The enzyme preparation gave a single protein band on analysis by both PAGE and SDS-PAGE, and did not form multiple protein bands detectable by IEF. The Mr was estimated to be about 130,000 by SDS-PAGE and about 135,000 by ultracentrifugal analysis. The apparent Km value and pH optimum of the enzyme were 3.9 +/- 0.2 mM (mean +/- SD) and about 4.7, respectively. The enzyme synthesized water-soluble glucan from sucrose, and the glucan consisted of over 90 mol% 1,6-alpha-D-glucosidic linkages. The enzyme activity was not stimulated by primer dextran. Anti-enzyme serum produced a single precipitin band with the purified enzyme preparation, whereas it did not react with either of the other two known glucosyltransferases.     

Three kinds of extracellular glucosyltransferases from Streptococcus mutans 6715 (serotype g)

In addition to the 1,3-alpha-D-glucan synthetase (pI 4.9) and the highly-branched 1,6-alpha-D-glucan synthetase (pI 3.9-4.1), Streptococcus mutans 6715 (serotype g) was found to secrete the third glucosyltransferase in multiple forms (pI 5.5-7.0), which exhibited 87% 1,6-alpha-bond-, 6% 1,3-alpha-bond- and 7% 1,3,6-branch-forming activities. The production of this enzyme was extremely enhanced when the organism was grown in Tween 80-supplemented medium. The 3 glucosyltransferases from the same organism were enzymatically and immunologically distinct from each other, and they were commonly found among the serotype g strains.     

Purification and properties of extracellular glucosyltransferases from Streptococcus mutans serotype a

Extracellular glucosyltransferases (sucrose: 1,6-alpha-D-glucan 3-alpha- and 6-alpha-glucosyltransferase) of Streptococcus mutans HS6 (serotype a) were purified from the culture supernatant by DEAE-Sepharose chromatography, ConA-Sepharose chromatography and chromatofocusing. The enzymes I and II with specific activities of 6.20 and 5.86 i.u. mg-1, respectively, exhibited slightly different isoelectric points (pI 4.5 and 4.2) and the molecular weights were estimated to be 161000 and 174000, respectively, by SDS-PAGE. The enzymes had the same optimum pH of 5.5 and the same Km values of 1.3 mM for sucrose and of 83 microM-glucose equivalent for dextran T10. By double immunodiffusion test on agar, these enzymes were immunologically identical to each other. Analysis by GLC of the glucans synthesized de novo from sucrose by the enzymes (I and II) established that they were 1,6-alpha-D-glucans with 20 and 24.5 mol% 1,3,6-branch points, respectively. Both are therefore bifunctional enzymes.     

Mechanism of Streptococcus mutans glucosyltransferases: hybrid-enzyme analysis.

Streptococcus mutans GS5 expresses three glucosyltransferases (GTFs): GTF-I and GTF-SI, which synthesize water-insoluble glucans in a primer-independent manner, and GTF-S, which is responsible for the formation of primer-dependent soluble glucan. The amino acid sequences of the GTF-I and GTF-S enzymes exhibit approximately 50% sequence identity. Various hybrid genes were constructed from the structural genes for the enzymes, and their products were analyzed. Three different approaches were used to construct the hybrid enzymes: (i) ligation of DNA fragments containing compatible endonuclease restriction sites of the two genes at homologous positions; (ii) in vivo recombination between the homologous regions of each gene; and (iii) random fusion of DNA fragments from each gene generated following exonuclease III digestion of tandemly arranged fragments corresponding to the two functional domains of each enzyme. Hybrid GTFs composed of the sucrose-binding domain of one enzyme (GTF-I or GTF-S) with the glucan-binding domain of the other synthesized insoluble glucan exclusively in the absence of primer dextran. Insoluble glucan synthesis by some, but not all, of the GTF-S:GTF-I chimeric enzymes was stimulated by primer dextran T10 addition. In addition, glucan binding by the former but not latter group of hybrid GTFs was demonstrated. These results suggest that the glucan-binding domain alone does not solely determine primer dependence or independence or the structure of the resulting glucan product, although this carboxyl-terminal domain containing direct repeating units does appear to play a significant role in primer dependence.     

Isolation and sequence of an active-site peptide containing a catalytic aspartic acid from two Streptococcus sobrinus alpha-glucosyltransferases

An active-site peptide containing an aspartic acid implicated in catalysis has been isolated and sequenced from two Streptococcus sobrinus extracellular glucosyltransferases: sucrose:1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase (GTase-I) and sucrose:1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase (GTase-S). The sequenced peptides, tagged with radiolabeled glucose, were isolated from a pepsin digest of a stabilized glucosylenzyme complex prepared by rapidly denaturing a reaction of enzyme and radiolabeled sucrose. The glucosyl linkage had previously been characterized as a beta-anomer bound to an active-site carboxyl group. Purified GTase-I and GTase-S glucosyl-peptides had the following similar but not identical sequences: GTase-I, Asp-Ser-Ile-Arg-Val-Asp-Ala-Val-Asp; and GTase-S, Asp-Gly-Val-Arg-Val-Asp-Ala-Val-Asp. Each has 3 aspartic acids as potential sites of glucose conjugation, but the relevant residue was not identified in sequence analysis because the highly base-labile glucosyl bond was cleaved in the first sequence cycle. As an alternative, the GTase-I glucosyl-peptide was partially digested at the N terminus with cathepsin C and at the C terminus with carboxypeptidase P. Analysis of the truncated products by fast atom bombardment mass spectrometry localized the glucosyl group to Asp-6 i the GTase-I peptide. In the native enzyme, this sequence is found near the N terminus, well-removed from the glucan-binding site located on a 60-kDa domain at the C terminus. The catalysis-dependent method of incorporating a glucosyl label implicates the aspartic acid as the residue involved in stabilizing an oxocarbonium ion transition state. The peptide segment is highly conserved and homologous to a peptide from sucrase-isomaltase labeled by site-directed irreversible inhibition and peptide segments common to a broad array of alpha-glucosidases and related transferases.     

Purification and characterization of basic glucosyltransferase from Streptococcus mutans serotype c

Streptococcus mutans Ingbritt (serotype c) was found to secrete basic glucosyltransferase (sucrose: 1,6-alpha-D-glucan 3-alpha and 6-alpha-glucosyltransferase). The enzyme preparation obtained by ethanol fractionation, DEAE Bio-Gel A chromatography, chromatofocusing and preparative isoelectric focusing was composed of three isozymes with slightly different isoelectric points (pI 8.1-8.4). The molecular weight was estimated to be 151000 by SDS-polyacrylamide gel electrophoresis. The specific activity of the enzyme was 9.8 IU per mg of protein and the optimum pH was 6.5. The enzyme was activated 2.4-fold by commercial dextran T10, and had Km values of 7.1 micro M for the dextran and 4.3 mM for sucrose. Glucan was de novo synthesized from sucrose by the enzyme and found to be 1,6-alpha-D-glucan with 17.7% of 1,3,6-branching structure by a gas-liquid chromatography-mass spectroscopy.     

Structure and enzymatic properties of genetically truncated forms of the water-insoluble glucan-synthesizing glucosyltransferase from Streptococcus sobrinus.

Glucosyltransferase-I (GTF-I: 175 kDa) of a cariogenic bacterium, Streptococcus sobrinus 6715, mediates the conversion of water-soluble dextran (alpha-1,6-glucan) into a water-insoluble form by making numerous alpha-1,3-glucan branches along the dextran chains with sucrose as the glucosyl donor. The structures and catalytic properties were compared for two GTF-I fragments, GTF-I' (138 kDa) and GS (110 kDa). Both lack the N-terminal 84 residues of GTF-I. While GTF-I' still contains four of the six C-terminal repeats characteristic of streptococcal glucosyltransferases, GS lacks all of them. Electron microscopy of negatively stained samples indicated a double-domain structure for GTF-I', consisting of a spherical head with a smaller spherical tail, which was occasionally seen as a long extension. GS was seen just as the head portion of GTF-I'. In the absence of dextran, both fragments simply hydrolyzed sucrose with similar K(m) and k(cat) values at low concentrations (<5 mM). At higher sucrose concentrations (>10 mM), however, GTF-I' exhibited glucosyl transfer activity to form insoluble alpha-1, 3-glucans. So did GS, but less efficiently. Dextran increased the rate and efficiency of the glucosyl transfer by GTF-I'. On removal of the C-terminal repeats of GTF-I' by mild trypsin treatment, this dextran-stimulated transfer was completely lost and the dextran-independent transfer became less efficient. These results indicate that the N-terminal two-thirds of the GTF-I sequence are organized as a structurally and functionally independent domain to catalyze not only sucrose hydrolysis but also glucosyl transfer to form alpha-1,3-glucan chains, although not efficiently; the C-terminal repeat increases the efficiency of the intrinsic glucosyl transfer by the N-terminal domain as well as rendering the whole molecule primer-dependent for far more efficient insoluble glucan synthesis.     

Insolubilization of exogenous primer dextran with 1,3-α-d-glucan synthase from Streptococcus mutans

Abstract The water-insolubilization mechanism of exogenous primer dextran with 1,3-α- d -glucan synthase (EC 2.4.1.-) from Streptococcus mutans was studied. The 1,3-α- d -glucan synthase solution, containing sucrose and exogenous primer dextran, was incubated briefly. Water-insoluble glucan was synthesized. At the same time, water-soluble glucan, mainly derived from exogenous primer dextran, decreased. Linkage analysis data of glucan produced revealed that 1,3-α- d -glucoside bonds increased. Exogenous primer dextran was changed by the action of 1,3-α- d -glucan synthase to water-insoluble glucan. The results suggest that in a short-term reaction system of outside primer-insertion type, the 1,6-α- d -glucoside bond forms the main chain of water-insoluble glucan.     

Stimulation of various functions in murine peritoneal macrophages by glucans produced by glucosyltransferases from Streptococcus mutans

The glucan that was produced by glucosyltransferases (GTFs) from Streptococcus mutans was examined for its stimulating functions toward murine peritoneal macrophages. Soluble glucan was obtained by the reaction with cell-free crude GTFs and sucrose, followed by ethanol precipitation, dispersion in water and re-precipitation by ethanol. Soluble glucan, those average molecular weight was about 3 x 10(5), was composed of mixture of alpha-1,6 and alpha-1,3 linkages in a 3:1 ratio. When 30 and 60 microg/ml of the glucan was incubated with peritoneal macrophages, the lysosomal phosphatase activity was increased in a dose-dependent manner, indicating that soluble glucan may activate macrophages. To examine its effects on the various functions of macrophages, soluble glucan was orally administered daily at a level of 100 mg/kg of body weight to C57BL/6 mice. Significant stimulation of the production of H2O2 by the macrophages was observed without any increase in NO production. The production of tumor necrosis factor-alpha (TNF-alpha) by the macrophages was also stimulated from 538.73-555.06 pg/ml to 585.73-596.40 pg/ml during 15 days of oral administration of soluble glucan. The cytotoxicity of peritoneal macrophages against B16 tumor cells was significantly enhanced by 25-38% during 15 days of oral administration. These results may indicate that soluble glucan stimulates the immune functions of macrophages.     

Purification and characterization of cell-associated glucosyltransferase synthesizing insoluble glucan from Streptococcus mutans serotype c

Streptococcus mutans Ingbritt (serotype c) was shown to have a significant amount of cell-associated glucosyltransferase activity which synthesizes water-insoluble glucan from sucrose. The enzyme was extracted from the washed cells with SDS, renatured with Triton X-100, adsorbed to 1,3-alpha-D-glucan gel, and then eluted with SDS. The enzyme preparation was electrophoretically homogeneous, and the specific activity was 7.3 i.u. (mg protein)-1. The enzyme had an Mr of 158,000 as determined by SDS-PAGE, and was a strongly hydrophilic protein, as judged by its amino acid composition. The enzyme gradually aggregated in the absence of SDS. The enzyme had an optimum pH of 6.5 and a Km value of 16.3 mm for sucrose. Activity was stimulated 1.7-fold by dextran T10, but was not stimulated by high concentrations of ammonium sulphate. Below a sodium phosphate buffer concentration of 50 mm, activity was reduced by 75%. This enzyme synthesized an insoluble D-glucan consisting of 76 mol% 1,3-alpha-linked glucose and 24 mol% 1,6-alpha-linked glucose.     

Purification and properties of extracellular glucosyltransferase synthesizing 1,6-, 1,3-alpha-D-glucan from Streptococcus mutans serotype a

An extracellular glucosyltransferase (sucrose: 1,6-, 1,3-alpha-D-glucan 3-alpha- and 6-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by DEAE-Sepharose chromatography and preparative isoelectric focusing. The molecular weight measured by SDS-PAGE was 159 000 and the isoelectric point was pH 4.9. The specific activity was 89.7 i.u. (mg protein)-1 and the optimum pH was 6.0. The Km value for sucrose was 4.9 mM and the enzyme activity was not stimulated by exogenous dextran T10. Glucan was synthesized de novo from sucrose by the purified enzyme and consisted of 49.1 mol% 1,6-alpha-linked glucose and 33.9 mol% 1,3-alpha-linked glucose, with 13.6 mol% terminal glucose and 3.3 mol% 1,3,6-alpha-branched glucose.     

Purification of a fourth glucosyltransferase from Streptococcus sobrinus.

Recently, we found a novel primer-independent, water-soluble glucan synthase as a fourth glucosyltransferase (GTF) in a culture supernatant of strain AHT-k of Streptococcus sobrinus (Y. Yamashita, N. Hanada, and T. Takehara, Biochem. Biophys. Res. Commun. 150:687-693, 1988). In the present study, four kinds of purified GTFs, including the novel GTF, were prepared. They were composed of two primer-dependent GTFs and two primer-independent GTFs. Of the primer-dependent GTFs, one was a water-insoluble glucan synthase and the other was a water-soluble glucan synthase; both of the primer-independent GTFs were water-soluble glucan synthases (GTF-Sis). Using antisera against four purified GTFs, we concluded that the immunological properties of each were completely different from those of the others. Additionally, it was shown that the novel GTF-Si, which was previously shown to have a molecular weight of 137,000, was proteolytically degraded and could be isolated at a molecular weight of 152,000 and that Streptococcus cricetus secreted an enzyme that immunologically cross-reacted with GTF-Si. While the product of the novel GTF-Si was not an effective primer for both of the primer-dependent enzymes (water-soluble and -insoluble glucan synthases), the product of the enzyme affected the molecular size of the products of the other GTF-Sis.     

Origin and function of the multiple extracellular glucosyltransferase species from cultures of a serotype c strain of Streptococcus mutans

Two methods were used to purify the bifunctional extracellular enzyme sucrose: (1-6)- and (1-3)-alpha-D-glucan-6-alpha-D-glucosyltransferase (EC 2.4.1.5; dextransucrase) from continuous cultures of a serotype c strain of Streptococcus mutans. The first method, based on a previously published report, involved Sepharose 6B gel filtration and DEAE cellulose anion exchange chromatography. This resulted in a dextransucrase preparation with an apparent molecular mass of 162 kDa and a specific activity of 125 mg of glucan formed from sucrose h-1 (mg of protein)-1, at 37 degrees C. It was almost homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The ratio of carbohydrate to protein was 0.14 and the recovery was 14% relative to the total glucosyltransferase activity in the original culture fluid. In the subsequently preferred method, hydroxyapatite-Ultrogel was used to purify dextransucrase with a 24% yield. The specific activity, 197 mg of glucan formed h-1 (mg of protein)-1, was the highest yet reported and this preparation contained less than 0.5 glucose-equivalent per subunit of molecular mass 162 kDa. Dextransucrase is therefore not a glycoprotein. Exogenous dextran stimulated activity, but was not essential for activity. The purified protein slowly degraded to multiple lower molecular mass forms during storage at 4 degrees C and 87% of the activity was lost after 20 days. The molecular mass of the most prominent, active degradation product was 140 kDa, similar to that of one of the multiple forms of dextransucrase detected in other laboratories. Preparations in which either the 140-kDa or the 162-kDa species predominated catalyzed the synthesis of a water-soluble glucan with sucrose alone, but catalyzed that of an insoluble glucan with sucrose and a high concentration of either (NH4)2SO4 or polyethylene glycol. The water-insoluble glucan was shown to lack sequences of 1,3-alpha-linked glycosyl residues typical of the insoluble glucan, mutan, which has been implicated in dental caries. We conclude that mutan is synthesized by the concerted action of two independent glucosyltransferases rather than by interconvertible forms of a single enzyme, as was proposed previously.     

The caries inhibitory effects of GOS-sugar in vitro and in rat experiments

The caries inhibitory activity of GOS-sugar (panose- and maltose-rich sugar mixture) was examined and compared with that of sucrose, maltose, or glucose in in vitro and in vivo experiments. Streptococcus mutans MT8148R (serotype c) and Streptococcus sobrinus 6715 (g) did ferment GOS-sugar and produce acid in a similar way as with maltose and glucose. However, GOS-sugar could not be a substrate for the glucosyltransferases (GTases) of these mutans streptococci to synthesize the water-insoluble glucan. Also, it significantly inhibited not only the synthesis of water-insoluble glucan from sucrose by the crude GTases but also the sucrose-dependent adherence of these cells to a glass surface. In particular, adherence of growing cells of 6715 was markedly inhibited by the presence of GOS-sugar. GOS-sugar was found to induce significant but minimal dental caries in SPF rats infected with either MT8148R or 6715. Furthermore, the replacement of half of the dietary sucrose content with GOS-sugar resulted in a significant reduction of caries development in rats infected with strain 6715.     

Effect of partially hydrolyzed soluble glucan produced by glucosyltrasferases of Streptococcus mutans on stimulating human T cell

Soluble glucan, which was obtained from action of glucosyltransferases (GTFs) of Streptococcus mutans on sucrose, was partially hydrolyzed by acetic acid and examined for human T lymphoblast (MOLT-4) stimulating activity. Addition of the partially hydrolyzed glucan (15-60 microg/ml) stimulated human T cell (39-65%) in a dose dependant manner according to MTT assay. Production of interleukine-2 (IL-2) and interleukine-2 receptor (IL-2R) from T cell was increased by 44.5 and 25%, respectively, by addition of partially hydrolyzed glucan (15 microg/ml). These results indicate that stimulation of human T cells by hydrolyzed glucan is probably caused by its effects on stimulating gene expression of IL-2 and IL-2R of human T cell.     

Activity of branched dextrans in the acceptor reaction of a glucosyltransferase (GTF-I) from Streptococcus mutans OMZ176

The ability of several native and chemically synthesized, branched dextrans to stimulate the activity of an alpha-D-glucosyltransferase (GTF-I) of Streptococcus mutans has been compared. The enzyme catalysed the transfer of glucosyl residues from sucrose with the formation of water-insoluble (1----3)-alpha-D-glucan. The rate of this reaction was greatly increased in the presence of dextran, and the extent of stimulation was negatively correlated with the degree of branching of the added dextran. The results refute the concept that growth of water-insoluble glucan occurs from the multiple, non-reducing termini of dextran acceptors.     

Further studies of primer-independent phosphorylase isozymes in the algae

Both Oscillatoria princeps and Cyanidium caldarium contain phosphorylase isozymes that can cause the synthesis of polyglucan from glucose-1-phosphate in the absence of added maltodextrin ‘primer’. In addition, O. princeps contains a primer-dependent phosphorylase isozyme. When the phosphorylase fractions isolated from extracts of the algae were treated with α-amylase, the primer-independent isozyme became primer-dependent and shifted from the position it was normally found at after polyacrylamide gel electrophoresis. This primer-independent isozyme became less mobile towards the anode, and was found at the locus usually occupied by the primer-dependent isozyme. It was not possible to restore its mobility towards the anode and its primer-independent properties by preincubation with maltoheptaose. The indication is that this isozyme is a glucoprotein and that the glucan component is chemically bonded to the protein.     

TreX from Sulfolobus solfataricus ATCC 35092 displays isoamylase and 4-alpha-glucanotransferase activities

A treX in the trehalose biosynthesis gene cluster of Sulfolobus solfataricus ATCC 35092 has been reported to produce TreX, which hydrolyzes the alpha-1,6-branch portion of amylopectin and glycogen. TreX exhibited 4-alpha-D-glucan transferase activity, catalyzing the transfer of alpha-1,4-glucan oligosaccharides from one molecule to another in the case of linear maltooligosaccharides (G3-G7), and it produced cyclic glucans from amylopectin and amylose like 4-alpha-glucanotransferase. These results suggest that TreX is a novel isoamylase possessing the properties of 4-alpha-glucanotransferase.