Effects of dextranase on experimental caries in the rat.
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Animals↗
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In-vivo effects of dextranase on plaque and caries.
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Animal Feed↗
Cloning and sequence analysis of the gene for glucodextranase from Arthrobacter globiformis T-3044 and expression in Escherichia coli cells.
The gld gene for glucodextranase from Arthrobacter globiformis T-3044 was cloned by using a combination of gene walking and probe methods and expressed on the recombinant plasmid pGD8, which was constructed with pUC118, in Escherichia coli cells. The enzyme gene consisted of a unique open reading frame of 3,153 bp. The comparison of the DNA sequence data with the N-terminal and 6 internal amino acid sequences of the purified enzyme secreted from A. globiformis T-3044 suggested the enzyme was translated from mRNA as a secretory precursor with a signal peptide of 28 amino acids residues. The deduced amino acids sequence of the mature enzyme contained 1,023 residues, resulting in a polypeptide with a molecular mass of 107,475 daltons. The deduced sequence showed about 38% identity to that of the glucoamylase from Clostridium sp. G0005. The glucodextranase activity of transformant harboring pGD8 was about 40 mU/ml at 30 degrees C for a 16-h culture. Although the GDase that was produced from the transformant was shorter than authentic GDase by 2 amino acid residues at the N-terminal end side, its enzymatic properties were almost same as the authentic one. Two kinds of genes, dex1 and dex2, for endo-dextranases from A. globiformis T-3044 were also cloned into Escherichia coli cells. The N-terminal of the purified endo-dextranase from A. globiformis T-3044 agreed with the deduced amino acid sequence, after the 33rd alanine residue, of only the dex1 gene for edo-dextranase. This result suggests that the endo-dextranase is translated from mRNA as a secretory precursor with a signal peptide of 32 amino acids residues. The deduced sequence of endo-dextranase 1 and endo-dextranase 2 showed about 93% and 65% identity with that of known endo-dextranase from Arthrobacter sp. CB-8, respectively.
Amino Acid Sequence↗
Lectin analysis of Trypanosoma congolense bloodstream trypomastigote and culture procyclic surface saccharides by agglutination and electron microscopic technics.
Living, intact bloodstream trypomastigotes and culture procyclic forms of Trypanosoma congolense were tested for aggulination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and fucose binding protein (FBP). Similar experiments were conducted with living bloodstream and culture forms treated with trypsin or dextranase. Parasites were incubated for 30 min at 25 C in various concentrations of each lectin, then examined for agglutination by dark-field microscopy. Control preparations consisted of parasites incubated alone or with 0.5 M of the specific competing sugar, with or without the corresponding lectin. Electron-microscopic localization of lectin binding sites on the surface of intact and dextranase-treated bloodstream and intact culture forms was accomplished with Con A, reacted with horseradish peroxidase (HRP) and then diaminobenzidine (DAB). In addition, FBP and SBA were coupled to HRP, then utilized for the localization of binding saccharides on the surface of bloodstream forms by the DAB technic. Similar studies were conducted with culture procyclics incubated with WGA-, SBA-, PP- or FBP-HRP conjugates and then reacted with DAB. Controls were utilized to confirm the sugar specificity of all positive reactions. Intact living bloodstream forms were agglutinated in a concentration-dependent manner with all the lectins tested. Agglutination levels were scored as Con A greater than FBP greater than WGA = PP = SBA. Sugars resembling alpha-D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and alpha-L-fucose are evidently present on the surface of the parasites. No agglutination was noted in any control preparations. Identical lectin-induced agglutinations were obtained with trypsin- or dextranase-treated bloodstream forms. Trypsin disrupted but did not entirely remove the surface coat of bloodstream forms, while dextranase did not alter the ultrastructure of the parasites. Con A-, SBA- and FBP-binding saccharides were distributed uniformly on the surface coat of intact bloodstream forms; a similar distribution of Con A receptors was noted also on the surface of dextranase-treated cells. No lectin-binding saccharides were visualized by electron microscopy on any control preparations. Intact, trypsin- or dextranase-treated, procyclics were agglutinated in a concentration-dependent fashion by Con A and WGA, but not by the other lectins tested. Control preparations did not agglutinate and the enzymes did not affect the ultrastructure of the parasites. Con A- and WGA-specifically binding saccharides were uniformly distributed on intact procyclics and control preparations were lectin-negative. Thus, T. congolense procyclics retained surface saccharides resembling alpha-D-mannose and N-acetyl-D-glucosamine but lost sugars resembling N-acetyl-D-galactosamine (or D-galactose) and alpha-L-fucose...
Agglutination Tests↗
Microbial dextran-hydrolyzing enzymes: fundamentals and applications.
Dextran is a chemically and physically complex polymer, breakdown of which is carried out by a variety of endo- and exodextranases. Enzymes in many groups can be classified as dextranases according to function: such enzymes include dextranhydrolases, glucodextranases, exoisomaltohydrolases, exoisomaltotriohydrases, and branched-dextran exo-1,2-alpha-glucosidases. Cycloisomalto-oligosaccharide glucanotransferase does not formally belong to the dextranases even though its side reaction produces hydrolyzed dextrans. A new classification system for glycosylhydrolases and glycosyltransferases, which is based on amino acid sequence similarities, divides the dextranases into five families. However, this classification is still incomplete since sequence information is missing for many of the enzymes that have been biochemically characterized as dextranases. Dextran-degrading enzymes have been isolated from a wide range of microorganisms. The major characteristics of these enzymes, the methods for analyzing their activities and biological roles, analysis of primary sequence data, and three-dimensional structures of dextranases have been dealt with in this review. Dextranases are promising for future use in various scientific and biotechnological applications.
Bacteria↗
Enzymatic modification of glycocalyx in the treatment of experimental endocarditis due to viridans streptococci.
The presence of abundant surface polysaccharide, or glycocalyx, on viridans streptococci has been associated with failure to eradicate the organism from experimental cardiac vegetations during penicillin treatment. The role of glycocalyx in retarding sterilization was tested by in vivo administration of dextranase, an endohydrolase that attacks internally situated alpha (1-6) linkages. Dextranase and penicillin, either singly or in combination, were used to treat experimental endocarditis. After two days of therapy, 100% of animals treated with penicillin or dextranase alone had infected vegetations, whereas only 25% treated with penicillin and dextranase had infected vegetations (P less than .01). After five days of therapy, 100% of the animals treated with penicillin had infected vegetations, versus none that were treated with penicillin and dextranase (P less than .01). We conclude that glycocalyx acts to retard antibiotic activity in vegetations and that partial enzymatic digestion of the glycocalyx facilitates penicillin sterilization of the infected valve.
Animals↗
Enzymolysis of glomerular immune deposits in vivo with dextranase/protease ameliorates proteinuria, hematuria, and mesangial proliferation in murine experimental IgA nephropathy.
The therapeutic effects of saccharolytic and proteolytic enzymes were investigated in models of IgA nephropathy. Mesangial glomerulonephritis was induced in mice by intravenous injection of preformed soluble immune complexes of dextran sulfate and either IgA (J 558) or IgM (MOPC 104 E) anti-dextran MAb (passive model) or by immunization with DEAE dextran (active model). In the passive model, only 30-40% of dextranase-treated mice given IgA or IgM immune complexes had mesangial Ig or dextran deposits, compared with 100% of saline-treated controls (P less than 0.01). There was no significant difference in mice given only protease. In the active model, dextranase and protease separately each reduced glomerular dextran and C3 deposits, and hematuria (P less than 0.01). Dextranase also reduced the glomerular IgA deposits (20 vs. 100% of saline-treated mice) and the frequency and severity of mesangial matrix expansion (both P less than 0.02), but did not reduce the modest IgG or IgM codeposits. Protease reduced IgG and IgM deposits, proteinuria and mesangial hypercellularity compared with saline (P less than 0.02), but did not diminish IgA, and had no effect on mesangial matrix expansion. The combination of dextranase plus protease attenuated all components of glomerular injury as judged by clinical and pathological parameters, but inactivated dextranase plus inactivated protease had no effect on any parameter. We conclude that enzymatic digestion of antigen and antibody can reduce immune deposits, mesangial proliferation, proteinuria, and hematuria in experimental glomerulonephritis.
Animals↗
Some properties of an endodextranase inhibitor from continuous cultures of Streptococcus sobrinus.
Cell-free filtrates of Streptococcus sobrinus, cultured at low growth rate in the chemostat, contain a dextranase inhibitor that can completely inhibit the activity of S. sobrinus endodextranase. The range of conditions under which inhibition occurs, and the situations in which enzyme activity can reappear, have been examined in continuous cultures of strain 6715-13WT and the dextranase-deficient mutant 6715-13-201. A purified preparation of the inhibitor was specific for S. sobrinus dextranase, having no action on dextranases from other oral streptococci. The percentage inhibition of S. sobrinus dextranase varied with the enzyme concentration, and the complete inhibition of low amounts of enzyme indicated a very tight bond between the inhibitor and the enzyme.
Chromatography↗
Molecular cloning of the extracellular endodextranase of Streptococcus salivarius.
We report the cloning in Escherichia coli of the gene encoding an extracellular endodextranase (alpha-1,6-glucanhydrolase, EC 3.2.1.11) from Streptococcus salivarius PC-1. Recombinants from a S. salivarius PC-1-Lambda ZAP II genomic library specifying dextranase activity were identified as plaques surrounded by zones of clearing on blue dextran agar. One such clone, PD1, had a 6.3-kb EcoRI fragment insert which encoded a 190-kDa protein with dextranase activity. The recombinant strain also produced two lower-molecular-mass polypeptides (90 and 70 kDa) that had dextranase activity. Native dextranase was recovered from concentrated culture fluids of S. salivarius as a single 110-kDa polypeptide. PD1 phage lysate and PC-1 culture supernatant fluid extract were used to measure substrate specificity of the recombinant and native forms of dextranase, respectively. Analysis of these reaction products by thin-layer chromatography revealed the expected isomaltosaccharide products yielded by the recombinant-specified enzyme but was unable to resolve the larger polysaccharide products of the native enzyme. Furthermore, S. salivarius utilized neither the substrates nor the products of dextran hydrolysis for growth.
Blotting, Southern↗
Identification of Streptococcus salivarius by PCR and DNA probe.
AIMS: To establish species-specific PCR and DNA probe methods for Streptococcus salivarius and to clarify the distribution of dextranase in oral isolates of Strep. salivarius. METHODS AND RESULTS: A pair of PCR primers and a DNA probe were designed based on the nucleotide sequence of the dextranase gene of Strep. salivarius JCM5707. Both the PCR primer and the DNA probe specifically detected Strep. salivarius but none of the other oral streptococci (23 strains of 13 species). The primer and the probe were capable of detecting 1 pg and 1 ng of the genomic DNA, respectively, purified from Strep. salivarius JCM5707. All oral isolates (130 strains from 12 subjects) of Strep. salivarius from human saliva were positive by both methods. CONCLUSION: The present PCR and DNA probe methods are highly specific to Strep. salivarius and are useful for the its detection and identification of this bacterium. The dextranase widely distributes among oral isolates of Strep. salivarius. SIGNIFICANCE AND IMPACT OF THE STUDY: The DNA sequence of a dextranase gene present in the genome of Strep. salivarius is useful as the target DNA of the species-specific PCR and DNA probe.
DNA Primers↗
Biochemical properties of Streptococcus sobrinus reisolates from the gastrointestinal tract of a gnotobiotic rat.
Streptococcus sobrinus strain 6715-13-201 was inoculated into the oral cavity of a gnotobiotic rat and then reisolated from different portions of the gastrointestinal tract. Fourteen isolates, selected on the basis of their colonial morphology, were then screened for their ability to adhere to saliva-coated hydroxyapatite (SHA) in vitro, and their ability to produce extracellular polysaccharide from sucrose, and low pH in glucose broth. Certain isolates were also tested for their cariogenic potential as monoinfectants in gnotobiotic rats. All isolates differed in their abilities to adhere to SHA, with most showing an increased level of adhesion in the presence of sucrose, but this did not correlate with their ability to be aggregated by dextran. Most isolates were capable of producing glucosyltransferases (with only one exception) and dextranases (also one exception). There was more variability in the production of dextranase inhibitor. No isolate was capable of producing dextranase inhibitor in the absence of dextranase production. There were no correlations between the ability of isolates to adhere in vitro or produce/utilize polysaccharides and their ability to produce caries in vivo. Due to the differences between strains in their abilities to adhere, produce polysaccharides, utilize polysaccharides or produce a low pH and the lack of correlation between any of these parameters and cariogenicity, the results suggest that the ability of strains to colonize and produce caries depends on a number of different characteristics, no one of which is essential.
Animals↗
Role of antigen in the regulation of the immune response to dextran B512.
Dextranase mixed with dextran in vitro or injected into mice before the antigen abolished the immunogenicity of both thymus-dependent and thymus-independent forms of dextran. Dextranase could degrade dextran in vivo even when given 6-15 days after the antigen. Dextranase injected after the antigen suppressed the immune response when given 24 h, but not 48 h, after the antigen, indicating that the antigen must interact with the immune system for 48 h to initiate a response. Thereafter , the B cells are independent of further antigen stimulation. The induction of autoanti -idiotypic anti-dextran antibodies was abolished if dextranase was given 24 h, but not 48 h, after the antigen, indicating that anti-idiotypic antibodies can be induced in the absence of antigen or immune complexes once anti-dextran antibodies have been synthesized.
Antibody Formation↗
Dextran-induced aggregation in a mutant of Streptococcus sobrinus 6715-13.
A mutant of wild-type Streptococcus sobrinus 6715-13 has been isolated which resists aggregation by exogenous dextran. This variant is able to form adherent plaque deposits in vitro when cultured in the presence of sucrose and has dextranase activity. In these respects it is the complement of previously described isolates which are plaque formation defective but aggregation normal. Measurements of the incorporation of glucose from glucosyl-labeled sucrose into glucan by cell-associated glucosyltransferase enzyme activity and the thermal labilities of catalytic and receptor functions, as well as the binding of labeled dextrans to the cells, provide evidence that neither dextranase nor glucosyltransferase is the receptor involved in dextran-induced aggregation. Blockage of such bacterial aggregation by anti-glucosyltransferase or anti-dextranase sera suggests cross-reactivity between the antigenic determinants of proteins which recognize alpha(1-6) glucan linkages. A model is proposed, consistent with these and previous findings, in which enzymatic function precedes dextran receptor activity in emergence from the cell. It is also proposed that dextran receptor components of the multireactive glucosyltransferase enzyme(s) and dextranase(s) are spatially separate from, although functionally and antigenically related to, the receptors on the bacterial surface involved in dextran-induced aggregation.
Agglutination↗
Insoluble glucan hydrolyzing enzymes from oral bacteroides.
Crude enzymes (En(C)) which hydrolyze insoluble glucan produced by Streptococcus mutans FA-1 were extracted from Bacteroides oralis obtained from human dental plaque. Extracellular insoluble glucan of S. mutans (IsG) and the one which was partially modified by Smith degradation (M-IsG) were used as substrates. Commercial dextran (M.W. 2,000,000) was used as control. Composition of the types of glucosidic linkages of the glucans was determined by methylation analysis. The ratio of the alpha-(1--6) linkage and alpha-(1--3) linkage was 96.3% and 0.5% for dextran, 29.2% and 55.1% for IsG and 11.9% and 84.9% for M-IsG. En(C) was extracted by salting out of the culture of B. oralis with 60% saturation of ammonium sulfate. En(C) hydrolyzed IsG, M-IsG and dextran, whereas commercial dextranase (alpha-1,6 glucanase) hydrolyzed only dextran. IsG was treated with the commercial dextranase until no glucose was detected in the medium, and the remaining material was used for the substrate of enzymes. Release of glucose was detected from the substrate by treatment with En(C), but not with commercial dextranase. These results indicated that En(C) of Bacteroides oralis contained at least two types of glucanase, one being dextranase which hydrolyzes the alpha-(1--6) linkage and the other the so-called mutanase which hydrolyzes the alpha-(1--3) linkage.
Bacteroides↗
Controlled release of a model protein from enzymatically degrading dextran microspheres.
Protein-loaded dextran microspheres were prepared by a water-in-water emulsion technique. With this technique, an aqueous solution of methacrylated dextran (dex-MA) is emulsified in an aqueous solution of poly(ethylene glycol) (PEG). Subsequently, the dispersed dex-MA phase is crosslinked by radical polymerization of the dextran-bound methacryloyl groups. This method renders microspheres with a hydrogel character of which the crosslink density can be controlled by the water content and the degree of substitution of the dex-MA (DS, the number of methacrylates per 100 glucopyranose residues). If an IgG solution was added to the dex-MA/PEG aqueous system prior to the polymerization reaction, the protein could be encapsulated in the dextran microspheres with a high yield (88-98%). The release of IgG was studied as a function of the water content, the DS and the degradation rate of the microspheres. The microspheres were rendered degradable by co-encapsulation of an endo-dextranase. Non-degrading microspheres mainly showed a burst release, which decreased with increasing crosslink density. By either a low water content (50%, w/w, or lower) or a high DS (DS 13), it was possible to reduce the burst release to about 10%, meaning that almost complete entrapment of the protein could be achieved. The release of IgG from degrading microspheres was predominantly dependent on the DS and the amount of encapsulated dextranase. No differences in release of IgG from microspheres with and without dextranase were observed at high DS (DS 13). This was ascribed to the inability of the enzyme to degrade these microspheres. On the other hand, the entrapped protein was completely released from enzymatically degrading microspheres with a DS 4. Moreover, the release rate of IgG was proportional to the degradation rate of these microspheres (depending on the amount of co-encapsulated dextranase). Interestingly, an almost zero-order release was observed from these microspheres for periods up to 30 days.
Delayed-Action Preparations↗
Polysaccharide degrading enzymes of a toxigenic strain of Aspergillus clavatus from Nigerian poultry feeds.
The factors responsible for the production and activities of polysaccharide degrading enzymes of a toxigenic strain of Aspergillus clavatus (ASC - IB 202) on an oat meal chaff (OMC) medium were studied. The enzymes assayed for were amylases, CM-cellulases, filter paper (FP)-cellulases, xylanases and dextranases. Amylase production by A. clavatus (ASC - IB 202) reached a peak within 48 h of incubation at 30 degrees C while optimal production of CM-cellulases occurred on day 6. The FP-cellulase and xylanase production did not reach a peak within the 10 day incubation period. Determination of dextranase activity showed optimal production and activity between days 6 and 8. When grown on varying sources of carbon at 30 degrees C, CM-cellulase production was only recorded on cellobiose, CM-cellulose and on the OMC medium. FP-cellulase production occurred on the microcrystalline cellulose and on the OMC medium. The other enzymes were produced to various extents on the carbon sources used. The best mycelial growth was recorded at 30 degrees C and pH 6.0. So also were the peak production of CM-cellulases and FP-cellulases. The xylanase and dextranase activity of the culture filtrate were optimally produced at 30 degrees C and pH 5.4. The potential health hazards which the presence of this toxigenic strain of Aspergillus clavatus on poultry feeds poses to farm animals including man are discussed.
Amylases↗
Treatment of artificially induced storage disease with lysosomotropic microparticles.
An artificial storage disease has been induced in mice by intravenously injecting 14C-labelled crosslinked dextran. The dextran was essentially taken up in the spleen and liver by cells belonging to the reticuloendothelial system and stored there in the lysosomes. Soluble dextranase, when given intravenously, had no effect on the stored dextran as judged by the radioactivity content of the tissues. However, when the dextranase was immobilized in microparticles of polyacryldextran and injected intravenously, the clearance of the radiolabelled dextran was significantly catalyzed. The lysosomotropic microparticles carried the enzyme to the lysosomes, where the microparticles were destroyed by the dextranase, which thereafter digested the stored 14C-dextran. The results illustrate the use of "self-destroying" microparticles as a lysosome-directed carrier of enzymes.
Acrylic Resins↗
The use of titanium (IV) oxide for the immobilisation of carbohydrate-directed enzymes.
The use of particles of porous titanium (IV) oxide as a suitable matrix for enzyme immobilisation has been investigated with dextranase. Treatment of the particles with enzyme in the presence and absence of ammonium ions showed that the presence of ammonia induced a greater coupling of protein, whereas a greater retention of enzyme specific activity was achieved in the absence of ammonia. Properties of the immobilised enzyme include a pH-dependence and reversibility of the coupling between enzyme and matrix. The immobilised dextranase was most stable at pH 5.0. Automated analytical techniques for measuring the activity of dextranase and other polysaccharidases in soluble and insoluble forms are also reported.
Ammonia↗