Studies on dextranase. VIII. Some enzymatic properties of immobilized dextranase from Brevibacterium fuscum var. dextranlyticum.
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Brevibacterium↗
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Studies on mold dextranases. IV. Substrate specificity of Aspergillus carneus dextranase.
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Aspergillus↗
Studies on mold dextranases. I. Penicillium luteum dextranase: its production and some enzymatic properties.
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Carbohydrates↗
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.
Culture Media↗
Adsorption of Streptococcus sobrinus dextranase inhibitor to water-insoluble alpha-D-glucans of oral streptococci.
A low molecular weight dextranase inhibitor from Streptococcus sobrinus has previously been identified and purified. The range of conditions under which inhibition occurs, and the situations in which dextranase activity of S. sobrinus can reappear, have been examined in the chemostat. These studies have revealed that when dextranase production exceeds that of the inhibitor, all the inhibitor is tightly bound into enzyme-inhibitor complexes, and the excess enzyme remains active. Another factor that influences the activity of dextranase inhibitor has now been identified, namely the ability of the inhibitor to bind to water-insoluble glucans. Adsorption to water-insoluble alpha-D-glucans, produced by oral streptococci that were grown in batch culture, increased with their proportion of alpha-1,3-linked sequences of glucose residues. Studies with water-insoluble dextrans of Leuconostoc mesenteroides strains showed that alpha-1,6-linked sequences were also important for binding. The inhibitor was not active when adsorbed to glucan, but active inhibitor was released by incubation with soluble dextran. The interactions of sucrose, alpha-D-glucosyltransferases, alpha-D-glucans, dextranase and dextranase inhibitor are discussed in relation to the growth rate of S. sobrinus. At low growth rate in the chemostat the predominant alpha-D-glucosyltransferase (GTF) is a GTF-S that converts sucrose into soluble dextran, and the activity of free dextranase inhibitor in the culture filtrate is high. By contrast, at high growth rate the streptococci produce GTFs capable of synthesizing water-insoluble alpha-D-glucans, and no free inhibitor is found in culture filtrate. Thus the activity of free, extracellular dextranase inhibitor is controlled by (i) the extent of binding to dextranase and (ii) the extent of adsorption to water-insoluble alpha-D-glucan.
Adsorption↗
The influence of mutanase and dextranase on the production and structure of glucans synthesized by streptococcal glucosyltransferases.
Glucanohydrolases, especially mutanase [alpha-(1-->3) glucanase; EC 3.2.1.59] and dextranase [alpha-(1-->6) glucanase; EC 3.2.1.11], which are present in the biofilm known as dental plaque, may affect the synthesis and structure of glucans formed by glucosyltransferases (GTFs) from sucrose within dental plaque. We examined the production and the structure of glucans synthesized by GTFs B (synthesis of alpha-(1-->3)-linked glucans) or C [synthesis of alpha-(1-->6)- and alpha-(1-->3)-linked glucans] in the presence of mutanase and dextranase, alone or in combination, in solution phase and on saliva-coated hydroxyapatite beads (surface phase). The ability of Streptococcus sobrinus 6715 to adhere to the glucan, which was formed in the presence of the glucanohydrolases was also explored. The presence of mutanase and/or dextranase during the synthesis of glucans by GTF B and C altered the proportions of soluble to insoluble glucan. The presence of either dextranase or mutanase alone had a modest effect on total amount of glucan formed, especially in the surface phase; the glucanohydrolases in combination reduced the total amount of glucan. The amount of (1-->6)-linked glucan was reduced in presence of dextranase. In contrast, mutanase enhanced the formation of soluble glucan, and reduced the percentage of 3-linked glucose of GTF B and C glucans whereas dextranase was mostly without effect. Glucan formed in the presence of dextranase provided fewer binding sites for S. sobrinus; mutanase was devoid of any effect. We also noted that the GTFs bind to dextranase and mutanase. Glucanohydrolases, even in the presence of GTFs, influence glucan synthesis, linkage remodeling, and branching, which may have an impact on the formation, maturation, physical properties, and bacterial binding sites of the polysaccharide matrix in dental plaque. Our data have relevance for the formation of polysaccharide matrix of other biofilms.
Bacterial Adhesion↗
Molecular characterization of dextranase from Streptococcus rattus.
The complete nucleotide sequence of the dextranase gene of Streptococcus rattus ATCC19645 was determined. An open reading frame of the dextranase gene was 2,760 bp long and encoded a dextranase protein consisting of 920 amino acids with a molecular weight of 100,163 Da and an isoelectric point of 4.67. The S. rattus dextranase purified from recombinant Escherichia coli cells showed dextran-hydrolyzing activity with optimal pH (5.0) and temperature (40 C) similar to those of dextranases from Streptococcus mutans and Streptococcus sobrinus. The deduced amino acid sequence of the S. rattus dextranase revealed that the dextranase molecule consists of two variable regions and a conserved region. The variable regions contained an N-terminal signal peptide and a C-terminal cell wall sorting signal; the conserved region contained two functional domains, catalytic and dextran-binding sites. This structural feature of the S. rattus dextranase is quite similar to that of other cariogenic species such as S. mutans, S. sobrinus, and Streptococcus downei.
Amino Acid Sequence↗
Biodegradable microspheres. I. Duration of action of dextranase entrapped in polyacrylstarch microparticles in vivo.
Dextranase was entrapped in polyacryl starch microspheres of different compositions by emulsion polymerization. After i.v. injection in mice and rats, the particles were removed from the blood circulation by macrophages of the reticuloendothelial system. In these cells, the particles are accumulated in the lysosomes. The degradation of different 14C-labeled microparticles and their entrapped dextranase was followed in an isolated lysosomal fraction in vitro and in liver and spleen after i.v. injection in mice. The duration of entrapped dextranase in vivo was followed directly, i.e., by an enzyme assay, and indirectly by following the decrease of a stored material, [3H]dextran in the liver. The degradation of the entrapped enzyme was dependent on the composition of the particle matrix. More cross-linked spheres could better protect the entrapped enzyme in vitro and in vivo. The half-life of free dextranase in the lysosomal fraction was estimated to be about 4 hr, whereas the duration of entrapped dextranase in the liver was at least 48 hr, as measured with [3H]dextran. Finally, the effect of entrapped and free dextranase on an artificially induced storage disease was studied. The stored [3H]dextran was eliminated completely when dextranase was used in microparticles, whereas free dextranase had no effect in vivo.
Animals↗
Cloning and characterization of a dextranase gene from Lipomyces starkeyi and its expression in Saccharomyces cerevisiae.
A dextranase-encoding cDNA from L. starkeyi KSM22 was isolated and characterized. The 2052 bp cDNA fragment (lsd1) harbouring the dextranase gene exhibited one open reading frame (ORF) composed of 1824 bp flanked by a 41 bp 5'-UTR and a 184 bp 3'-UTR, including a 27 bp poly(A) tail. The lsd1 gene contains no introns. The open reading frame encodes a 608 amino acid polypeptide (LSD1) with a 67.6 kDa predicted molecular mass. There was a 77% deduced amino acid sequence identity between the LSD1 dextranase and the dextranase from Penicillium minioluteum. The primary structure of LSD1 dextranase exhibits distant similarity with the enzymes of the glycosyl hydrolase family 49 that comprises Penicillium dextranase. The optimum pH of LSD1 was 6.0 and the optimum temperature was 37 degrees C. LSD1 dextranase activity was substantially abolished by exposure to 1 mM Hg2+, Ag3+ and Mn2+. LSD1 exhibited high hydrolysing activity towards dextran (100%), soluble starch (22%) and mutan (8%).
Amino Acid Sequence↗
Molecular cloning and nucleotide sequencing of the Arthrobacter dextranase gene and its expression in Escherichia coli and Streptococcus sanguis.
A bacterial strain, which assimilated dextran and water-insoluble glucan produced by Streptococcus mutans, was isolated from soil. The bacterium produced and secreted potent dextranase activity, which was identified as Arthrobacter sp. and named CB-8. The dextranase was purified and some enzymatic properties were characterized. The enzyme efficiently decomposed the water-insoluble glucan as well as dextran. A gene library from the bacteria was constructed with Escherichia coli, using plasmid pUC19, and clones producing dextranase activity were selected. Based on the result of nucleotide sequencing analysis, it was deduced that the dextranase was synthesized in CB-8 cells as a polypeptide precursor consisting of 640 amino acid residues, including 49 N-terminal amino acid residues which could be regarded as a signal peptide. In the E. coli transformant, the dextranase activity was detected mostly in the periplasmic space. The gene for the dextranase was introduced into Streptococcus sanguis, using an E. coli-S. sanguis shuttle vector that contained the promoter sequence of a gene for glucosyltransferase derived from a strain of S. mutans. The active dextranase was also expressed and accumulated in S. sanguis cells.
Amino Acid Sequence↗
Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris.
The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicillium minioluteum by hybridization using the dextranase cDNA as a probe. Comparison of the gene and cDNA sequences revealed that the DEX gene does not contain introns. Amino acid sequences comparison of P. minioluteum dextranase with other reported dextranases reveals a significant homology (29% identity) with a dextranase from Arthrobacter sp. CB-8. The DEX gene fragment encoding a mature protein of 574 amino acids was expressed in the methylotrophic yeast Pichia pastoris by using the SUC2 gene signal sequence from Saccharomyces cerevisiae under control of the alcohol oxidase-1 (AOX1) promoter. Over 3.2 g/l of enzymatically active dextranase was secreted into the medium after induction by methanol. The yeast product was indistinguishable from the native enzyme in specific activity and the N-terminus of both proteins were identical.
Amino Acid Sequence↗
Cloning and expression of Penicillium minioluteum dextranase in Saccharomyces cerevisiae and its exploitation as a reporter in the detection of mycotoxins.
A dextranase gene from Penicillium minioluteum (strain IMI068219) has been cloned, sequenced and expressed in Saccharomyces cerevisiae via fusion of the DNA segment encoding the mature dextranase protein with alpha-factor signal sequence, and insertion into the GAL1-controlled expression vector pYES2/CT. Galactose-induced expression yielded extracellular dextranase activity of 0.63 units/ml and cell-associated dextranase activity of 0.48 units/ml, after 24 h incubation. The dextranase construct was introduced into a strain of S. cerevisiae expressing the human cytochrome P450 3A4 (CYP3A4) and the cognate reductase, which was then used to develop a microplate toxicity bioassay. Toxicity was signalled as inhibition of dextranase activity, assayed fluorimetrically. This novel bioassay was assessed using six economically significant mycotoxins.
Aflatoxin B1↗
Insertional inactivation of the Streptococcus mutans dexA (dextranase) gene results in altered adherence and dextran catabolism.
Streptococcus mutans is able to synthesize extracellular glucans from sucrose which contribute to adherence of these bacteria. Extracellular dextranase can partially degrade the glucans, and may therefore affect virulence of S. mutans. In order to isolate mutants unable to produce dextranase, a DNA library was constructed by inserting random Sau3AI-digested fragments of chromosomal DNA from S. mutans into the BamHI site of the streptococcal integration vector pVA891, which is able to replicate in Escherichia coli but does not possess a streptococcal origin of replication. The resultant plasmids were introduced into S. mutans LT11, allowing insertional inactivation through homologous recombination. Two transformants were identified which did not possess dextranase activity. Integration of a single copy of the plasmid into the chromosome of these transformants was confirmed by Southern hybridization analysis. Chromosomal DNA fragments flanking the plasmid were recovered using a marker rescue technique, and sequenced. Comparison with known sequences using the BLASTX program showed 56% homology at the amino acid level between the sequenced gene fragment and dextranase from Streptococcus sobrinus, strongly suggesting that the S. mutans dextranase gene (dexA) had been inactivated. The colony morphology of the dextranase mutants when grown on Todd-Hewitt agar containing sucrose was altered compared to the parent strain, with an apparent build-up of extracellular polymer. The mutants were also more adherent to a smooth surface than LT11 but there was no apparent difference in sucrose-dependent cell-cell aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Effects of dextranases on attachment of Streptococcus mutans to hydroxyapatite.
A Fusarium dextranase and a Penicillium dextranase were compared for their relative ability to quantitatively reduce the adsorption of (3)H-labeled Steptococcus mutans cells onto hydroxyapatite. Fusarium dextranase-treated hydroxyapatite disks caused a statistically significant decrease in the hydroxyapatite adsorption of both the OMZ 176 and NCTC 10449 strains of S. mutans relative to untreated control disks. The extent of initial bacterial adsorption was not promoted by sucrose-dependent glucan synthesis. Since the Fusarium dextranase has a much greater affinity for hydroxyapatite than the Penicillium dextranase, it could represent an enzyme with improved decay-preventive therapeutic properties. This was concluded because the Fusarium dextranase may interfere with both the initial attachment and later glucan-dependent accumulation of dental plaque microorganisms.
Adhesiveness↗
Dextranase enhances antibiotic efficacy in experimental viridans streptococcal endocarditis.
In endocarditis, exopolysaccharide production by viridans streptococci has been associated with delayed antimicrobial efficacy in cardiac vegetations. We compared the efficacies of temafloxacin alone and in combination with dextranase, an enzyme capable of hydrolyzing 20 to 90% of the bacterial glycocalyx, in a rabbit model of endocarditis. In in vivo experiments, rabbits were infected intravenously with 10(8) Streptococcus sanguis organisms and were treated 6 days later with temafloxacin (50 mg/kg of body weight intramuscularly twice a day) alone or combined with dextranase (1,000 U per rabbit per day intravenously). After 4 days of treatment (day 11), the animals were sacrificed and vegetations were quantitatively cultured. For ex vivo experiments, rabbits were infected as stated above and, on day 11, vegetations were excised aseptically and incubated in vitro in rabbit serum alone (control) or with temafloxacin or temafloxacin plus dextranase at concentrations similar to peak levels in plasma. In vitro, dextranase alone had no antimicrobial effect. In vivo and ex vivo, temafloxacin combined with dextranase was more effective than temafloxacin alone (P < 0.05). Our results suggest that dextranase is able to increase the effects of temafloxacin by reducing the amount of bacterial glycocalyx in infected vegetations, as confirmed in vitro by electron microscopy showing a markedly reduced amount of glycocalyx and a more clearly visible fibrin matrix.
Animals↗
Characterization of an extracellular dextranase from Fusarium moniliforme.
An extracellular dextranase (EC 3.2.1.11) was purified approximately 75-fold from cell-free culture filtrates of Fusarium moniliforme. The purified dextranase was of the endo type, and isomaltose was identified as the primary end product of dextran hydrolysis. The molecular weight of the dextranase was determined to be 39,000 by gel permeation chromatography. The enzyme was most active at pH 5.5, and the temperature optimum was near 55 C. Activity was not inhibited by either ethylenediaminetetraacetic acid or iodoacetate. The Km for dextran with an average molecular weight of 10,000 was estimated to be 1.1 X 10(-4) M. The electrophoretic mobility of the dextranase was distinctly different from that of a Penicillium-derived commercial dextranase. The F. moniliforme dextranase was also found to differ from the commercial preparation by its greater relative activity against glucans isolated from Streptococcus mutans.
Cell-Free System↗
Dextranases from oral bacteria: inhibition of water-insoluble glucan production and adherence to smooth surfaces by Streptococcus mutans.
The effect of dextranases (EC 3.2.1.11) from the oral isolates Actinomyces israelii and Bacteroides ochraceus on water-insoluble glucan production by the Streptococcus mutans dextransucrase (EC 2.4.1.5) and sucrose-dependent adherence to smooth glass surfaces by S. mutans was studied. Collection on membrane filters of water-insoluble polysaccharides synthesized from radioactive sucrose was used to demonstrate the marked sensitivity of insoluble glucan formation to the presence of dextranase. Concentrations of A. israelii dextranase as low as 0.002 U/ml inhibited insoluble glucan formation by 60%. Similar results were obtained the the B. ochraceus enzyme. An assay for sucrose-stimulated adherence of S. mutans to smooth surfaces involved attachment of radioactively labeled nongrowing cells to the bottom of glass scintillation vials. This facile and sensitive assay was utilized to demonstrate that sucrose-dependent adherence was affected by low levels of dextranase from either A. israelii or B. ochraceus. Enzyme at 0.005 U/ml reduced adherence of S. mutans by 80%. Treatment of S. mutans cells previously attached to glass with low concentrations of the dextranases resulted in removal of 50% to 60% of the bacteria. The results indicate that dextranase-producing oral bacteria may affect sucrose-dependent colonization of S. mutans on the tooth surface and offer a possible explanation for both the difficulties involved in implanting this bacterium into the human mouth and the limited intraoral transmission of S. mutans from one tooth surface to another.
Actinomyces↗
Overproduction of a dextranase inhibitor by Streptococcus sobrinus mutants.
An inhibitor of Streptococcus sobrinus endodextranase was detected in the extracellular fractions of UAB66 mutants identified following ethyl methanesulfonate mutagenesis as either devoid of dextranase activity (Dex-) or overproducing water-soluble glucan. The two groups of mutants had the same phenotype and displayed no dextranase activity in assays of extracellular fractions (H. Murchison, S. Larrimore, and R. Curtiss III, Infect. Immun. 34:1044-1055, 1981) and had been shown to be defective in adherence (Adh-) and capable of inhibiting adherence of wild-type strains during cocultivation in vitro (H. Murchison, S. Larrimore, and R. Curtiss III, Infect. Immun. 50:826-832, 1985) and in vivo in gnotobiotic rats (K. Takada, T. Shiota, R. Curtiss III, and S. M. Michalek, Infect. Immun. 50:833-843, 1985). By analysis of proteins in Western blots (immunoblots) and following blue dextran-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BD-SDS-PAGE), it was demonstrated that these Dex- mutants did synthesize enzymatically active dextranase. From the results of mixing experiments, it was determined that these Dex- Adh- mutants produced enhanced amounts of a cell surface-localized or a cell-associated dextranase inhibitor (Dei). Dei was heat stable but trypsin sensitive. By adding excess dextranase following BD-SDS-PAGE, Dei was detected as blue bands with apparent molecular masses of 43, 40, 37, 27, and 23 kDa. Dei competitively inhibits dextranase activity and is synthesized by wild-type S. sobrinus strains, with the amount varying depending upon growth medium and stage in the growth cycle. R. M. Hamelik and M. M. McCabe (Biochem. Biophys. Res. Commun. 106:875-880, 1982) previously described a Dei in a wild-type S. sobrinus strain.
Bacterial Adhesion↗