Characterisation of extracellular polysaccharides produced by Crypthecodinium cohnii

The valuable polyunsaturated fatty acid, docosahexaenoic acid, can be produced by cultivation of the heterotrophic microalga, Crypthecodinium cohnii. During batch growth of C. cohnii on glucose, sea salt and yeast extract for 5 days, so far unreported extracellular polysaccharides were produced. These caused an increased viscosity and a strong drop in the maximum oxygen transfer. The viscosity increased most markedly as cells entered the stationary phase. The polysaccharides varied in size (from 6 kDa to >1,660 kDa) and monomer distribution. A high molecular mass fraction (from 100 kDa to >1,660 kDa) and a medium molecular mass fraction (6-48 kDa) were prepared. The high molecular mass fraction contained (on a molar basis) 71.7% glucose, 13.1% galactose and 3.8% mannose, whereas the medium molecular mass fraction contained 37.7% glucose, 19.8% galactose and 28.1% mannose. Other monomers present in both fractions were fucose, uronic acid and xylose. Monomers were coupled mainly via alpha-(1-3) links. Increased viscosity due to polysaccharide production complicates the development of commercial, high cell-density processes for the production of docosahexaenoic acid.     

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.     

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.     

Partial characterization of extracellular polysaccharides produced by cyanobacterium Arthrospira platensis

In this study, the physico-chemical characteristics of extracellular polysaccharides (EPS) produced by Arthrospira platensis were evaluated. Elemental analysis and a bicinchoninic acid (BCA) reaction indicated that the EPS were heteropolysaccharides that contain carbohydrate (13%) and protein (55%) moieties. Analysis of the infrared spectrum and elemental analysis revealed the presence of a sulfate group (0.5%). The UV-visible spectrum showed high UV absorption at 190∼230 nm and a shoulder at 260∼280 nm. In addition, this spectrum indicated that EPS can form aggregates with mycosporine-like amino acids and/or scytonemin. Gas chromatography analysis of the carbohydrate portion of the EPS indicated that it was composed of seven neutral sugars: galactose (14.9%), xylose (14.3%), glucose (13.2%), frucose (13.2%), rhamnose (3.7%), arabinose (1%), and mannose (0.3%) and two uronic acids, galacturonic acid (13.5%) and glucuronic acid (0.9%).     

Evaluation of three individual glucosyltransferases produced by Streptococcus mutans using monoclonal antibodies

Abstract We previously established murine hybridomas producing a monoclonal antibody monospecific against three glucosyl-transferases (I, SI and S) of Streptococcus mutans which contribute to dental caries formation. Here, we developed a new immunochemical technique (cross-dot system) with which individual levels of glucosyltransferases expressed by S. mutans can be evaluated. We also examined glucosyltransferase production and in vitro artificial plaque formation by a reference strain and several clinical isolates of S. mutans . The findings indicate that the levels of glucosyltransferases produced greatly vary with the cells and the culture medium, and that the cells producing high levels of both glucosyltransferase-SI and glucosyltransferase-I enzymes may possess high in vitro artificial plaque forming ability. We suggest that the cross-dot system will be useful for estimating the cariogenic potential of S. mutans isolates.     

Electrophoretic studies of extracellular glucosyltransferases and fructosyltransferases from seventeen strains of Streptococcus mutans

Streptococcus mutans was classified by the electrophoretic properties of glucosyltransferases (GTases) and fructosyltransferases (FTases). The cells of serotypes a, d and g did not release extracellular FTases, although those from other serotypes did. The enzymes from cells of serotypes d and g synthesized a good deal of insoluble polysaccharide compared with other serotypes. The enzymes were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and polyacrylamide gel-isoelectric focussing (PAG-IEF). Gels were stained for their activity and protein content. Enzymes belonging to the same serotype gave the same specific pattern on both gels. The seven serotypes could be classified into the following four groups: serotypes d and g, serotype a, serotypes c, e and f, and serotype b. The results agree well with some previous reports based on other methods. The molecular weights of three GTase bands were 156K, 146K and 135K, and of four kinds of FTase bands were 108K, 95K, 80K and 76K. The isoelectric points of main enzymes were 4.25, 4.60, 5.00, 5.55 and 5.70. Those of FTases were 4.25 and 4.60.Abbreviations GTase glucosyltransferase - FTase fructosyltransferase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PAG-IEF polyacrylamide gel-isoelectric focussing - PAS periodic acid-Schiff     

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.     

Identification and characterisation of two extracellular proteases of Streptococcus mutans

Abstract Streptococcus mutans was shown to produce two extracellular proteases capable of degrading both gelatin and collagen-like substrates. These enzymes have molecular masses of 52 and 50 kDa when analysed by SDS-PAGE. Both enzymes were inhibited by EDTA, but not by a range of other inhibitors with different specificities, indicating that they are metalloproteases. The activity of EDTA-inactivated enzymes could be restored by the addition of manganese and zinc. The identical inhibition and restoration profiles of the two enzymes suggest that one of the proteases may be a degradation product of the other.     

Binding of Streptococcus mutans to extracellular matrix molecules and fibrinogen

We have determined the ability of Streptococcus mutans cells to bind to extracellular matrix (ECM) molecules and fibrinogen. S. mutans cells were found to bind fibronectin, laminin, collagen type I, and fibrinogen. An isogenic S. mutans strain with a defect in the expression of the major surface protein of S. mutans, antigen I/II, possessed a reduced ability to bind fibronectin, collagen, and fibrinogen but not laminin, suggesting that antigen I/II contributes during pathological processes to the interaction of S. mutans cells with fibronectin, collagen type I, and fibrinogen.     

Characterisation of the extracellular polysaccharides produced by isolates of the fungus Acremonium

Solid state (13)C NMR studies of the extracellular glucans from the fungi Acremonium persicinum C38 (QM107a) and Acremonium sp. strain C106 indicated a backbone of (1-->3)-beta-linked glucosyl residues with single (1-->6)-beta-linked glucosyl side branches for both glucans. Analyses of enzymatic digestion products suggested that the average branching frequency for the A. persicinum glucan (66.7% branched) was much higher than that of the Acremonium sp. strain C106 glucan (28.6% branched). The solid state (13)C NMR spectra also indicated that both glucans are amorphous polymers with no crystalline regions, and the individual chains are probably arranged as triple helices.     

Neutral sugar composition of extracellular polysaccharides produced by strains of Butyrivibrio fibrisolvens

The extracellular polysaccharides (EPSs) produced by 37 isolates presently classified as Butyrivibrio species (or more specifically as Butyrivibrio fibrisolvens) were purified from glucose-grown cultures. The neutral sugar compositions of these EPSs were determined by both thin-layer and gas-liquid chromatographic techniques. Results showed that while the neutral sugar composition of the EPS was constant for a given strain, it varied considerably between strains. In addition, several acidic components in the EPS, of both known and unknown structure, were detected artifactually as acetylated lactones, the acetylated alditols derived from these lactone(s), or both. Two novel components, L-altrose and the acidic sugar 4-O-[1-carboxyethyl]-D-galactose, were common constituents of the EPS from some strains of B. fibrisolvens. These and other EPS compositional features were used to sort isolates of B. fibrisolvens into groups which may have taxonomic significance. A scheme for sorting isolates into these groups, and the relative relationships between groups, is proposed.     

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

Extracellular 1,3-alpha-D-glucan synthase (sucrose: 1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by ultrafiltration, DEAE-Sepharose chromatography and preparative isoelectric focusing. The enzyme had a molecular weight of 158 000 by SDS-PAGE and an isoelectric point of pH 5.2. The specific activity of the enzyme was 48.3 i.u. (mg protein)-1. The Km for sucrose was 1.2 mM and the activity was optimal at pH 6.0. The enzyme activity was stimulated about 20-fold in the presence of dextran T10. Glucan was synthesized de novo from sucrose by the enzyme and characterized as a linear 1,3-alpha-D-glucan by GC-MS.     

Purification and properties of bacteriolytic enzymes from Bacillus licheniformis YS-1005 against Streptococcus mutans

To find a novel lytic enzyme against cariogenic Streptococci, strains showing strong lytic activity have been screened from soil using Streptococcus mutans. A strain identified as Bacillus licheniformis secreted two kinds of lytic enzymes, which were purified by methanol precipitation, CM-cellulose chromatography, gel filtration, and hydroxyapatite chromatography. The molecular weights of these two enzymes, L27 and L45, were 27,000 and 45,000, respectively. Optimum pH and temperature of both enzymes for lytic activity were pH 8 and 37 degrees C. L27 and L45 digest the peptide linkage between L-Ala and D-Glu in peptidoglycan of Streptococcus mutans. The lytic activity was highly specific for Streptococcus mutans, suggesting their potential use as a dental care product.     

Characteristics of a high molecular weight extracellular protein of Streptococcus mutans

A high molecular weight protein antigen, designated P1, has been isolated from the culture fluid of chemostat-grown Streptococcus mutans strain Ingbritt and shown to be free of other antigens including glucosyltransferase. Antiserum against the protein was used in rocket immunoelectrophoresis to confirm and extend the previous observation that there were major differences in the amount of the protein produced under different growth conditions. Physico-chemical and serological studies indicated that protein P1 was indistinguishable from antigens B, I/II and IF isolated in other laboratories. Mammalian tissue cross-reactivity of protein P1 was demonstrated by binding of antiserum to P1 to sections of normal rabbit tissues, particularly heart. There was also a statistically significant increase in the number of mononuclear leucocytes in heart tissue of rabbits which had been injected with protein P1, when compared with the levels in control uninjected rabbits; injection with whole cells of S. mutans Ingbritt did not produce this effect.     

Degradation of lignocellulose by extracellular enzymes produced by Thermomonospora fusca BD25

Degradation products from the addition of extracellular enzymes from Thermomonospora fusca BD25 to ball-milled wheat straw, oat spelt xylan and solubilised kraft pulps were characterised by HPLC and TLC. Overall, a high percentage hydrolysis of oat spelt xylan (28.9%) occurred after 26 h incubation. However, the rates of hydrolysis of ball-milled wheat straw and kraft pulp were approximately 4-6-fold less than xylan hydrolysis, although the total percentage hydrolysis of available substrate was similar (22.2% and 25.9% respectively). Incubation of kraft pulp and ball-milled wheat straw by crude extracellular enzymes of T. fusca BD25 resulted in the detection of aromatic compounds at concentrations of 0.6 microg ml(-1) and 8.7 microg ml(-1), respectively. Hydrolysis of oat spelt xylan by T. fusca BD25 extracellular enzymes yielded a mixture of xylose, xylotriose and putative substituted-xylotriose, while the products of ball-milled wheat straw hydrolysis were xylose, glucose and a small oligomer present in the digest. The results highlight the ability of culture supernatant from T. fusca to release both simple sugars and aromatic compounds from lignocellulosic substrates and suggest a role for this organism in the biobleaching of pulp.     

Galactose Metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-β-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

Galactose metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-beta-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

Malolactic fermentation by Streptococcus mutans

Streptococcus mutans and certain other oral lactic-acid bacteria were found to have the ability to carry out malolactic fermentation involving decarboxylation of L-malate to yield L-lactic acid and concomitant reduction in acidity. The activity was inducible by L-malate in S. mutans UA159 growing in suspensions or biofilms. The optimal pH for the fermentation was c. 4.0 for both suspensions and biofilms, although the pH optimum for malolactic enzyme in permeabilized cells of S. mutans UA159 was close to 5.5. Although malate did not serve as a catabolite for growth of S. mutans, it did serve to protect the organism against acid killing and to maintain ATP pool levels during starvation. Alkalinization associated with malolactic fermentation resulted in pH rise or increased need to add standardized HCl solution to maintain a set pH value in pH-stat experiments. The net conclusion is that malate has the potential to be effective for alkalinization of dental plaque, although the fermentation is sensitive to fluoride and triclosan, which are commonly added to oral care products.