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[Formation of dextranases by mycelial fungi and actinomycetes].

The dextranase activity of cultures of mycelial fungi of different genera and actinomycetes from the Chromogenes species of the Actinomyces genus was studied. About one third of the mycelial fungi and 8% of actinomycetes showed dextranase activity. The resulting extracellular dextranases demonstrated on endotypic pattern of action on the substrate. Actinomycete dextranases were several times more active than fungal dextranases and exhibited a significant activity in the neutral and weakly alkaline medium. Highly productive strains that are promising as dextranase producers were isolated.

Actinomyces↗
PubMed1998

Purification and characterization of a dextranase from Sporothrix schenckii.

A dextranase (EC 3.2.1.11) was purified and characterized from the IP-29 strain of Sporothrix schenckii, a dimorphic pathogenic fungus. Growing cells secreted the enzyme into a standard culture medium (20 degrees C) that supports the mycelial phase. Soluble bacterial dextrans substituted for glucose as substrate with a small decrease in cellular yield but a tenfold increase in the production of dextranase. This enzyme is a monomeric protein with a molecular mass of 79 kDa, a pH optimum of 5.0, and an action pattern against a soluble 170-kDa bacterial dextran that leads to a final mixture of glucose (38%), isomaltose (38%), and branched oligosaccharides (24%). In the presence of 200 mM sodium acetate buffer (pH 5.0), the Km for soluble dextran was 0.067 +/- 0.003% (w/v). Salts of Hg2+, (UO2)2+, Pb2+, Cu2+, and Zn2+ inhibited by affecting both Vmax and Km. The enzyme was most stable between pH values of 4.50 and 4.75, where the half-life at 55 degrees C was 18 min and the energy of activation for heat denaturation was 99 kcal/mol. S. schenckii dextranase catalyzed the degradation of cross-linked dextran chains in Sephadex G-50 to G-200, and the latter was a good substrate for cell growth at 20 degrees C. Highly cross-linked grades (i.e., G-10 and G-25) were refractory to hydrolysis. Most strains of S. schenckii from Europe and North America tested positive for dextranase when grown at 20 degrees C. All of these isolates grew on glucose at 35 degrees C, a condition that is typically associated with the yeast phase, but they did not express dextranase and were incapable of using dextran as a carbon source at the higher temperature.

Dextranase↗
PubMed2003

Purification and properties of extracellular dextranase from a Bacillus sp.

Bacterial strains in the genus Bacillus were isolated from natural soil samples and screened for production of extracellular dextranases (E.C.3.2.1.11). One strain, determined by 16sRNA analysis as Paenibacillus illinoisensis exhibiting stable dextranase activity, was chosen for further analysis, and the dextranase from it was purified 733-fold using salt and PEG precipitations, two-phase extraction and DEAE-Sepharose chromatography with a total yield of 19%. The purified enzyme had three isoforms, with molecular masses of 76, 89 and 110kDa and isoelectric points of 4.95, 4.2 and 4.0, respectively. The mixture of the three dextranase isoforms has a broad pH optimum around pH 6.8 and a temperature optimum at 50 degrees C. The N-terminal sequence (Ala-Ser-Thr-Gly-Lys) was identical between the isoforms. No sequence homology with the known dextranases in the protein databanks was found.

Amino Acid Sequence↗
PubMed1996

Cloning and sequencing of a dextranase-encoding cDNA from Penicillium minioluteum.

A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-alpha-glucan hydrolase, EC 3.2.1.11) was isolated and characterized. cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization. Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs. The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system. The expression of a 2 kb cDNA fragment in E. coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody. The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide. There was 29% sequence identity between the P. minioluteum dextranase and the dextranase from Arthrobacter sp. CB-8.

Amino Acid Sequence↗
PubMed1975

Characterization of a dextranase produced by an oral strain of Actinomyces israelii.

A dextranase-producing, gram-positive, anaerobic, rod-shaped bacterium isolated from human dental plaque was identified as Actinomyces israeli. Although the extracellular dextranase (EC 3.2.1.11) formed by this microbe appeared to be constitutively produced, the bacterium did not utilize the reaction products as a carbon source during growth. A striking feature of the dextranase was the formation of two distinct groups of oligosaccharide end products. The two groups presumably correspond to the limit dextran and the released reaction product which appeared to be cleaved from the end(s) of larger dextran molecules. Low levels of dextranase activity were measured by [3H]NaBH4 reduction and alcohol fixation of the large, tritiated end products on filter paper disks. Of the carbohydrate substrates tested, only alpha-1,6-linked glucans were cleaved. The enzyme did not exhibit any metal ion requirements, and its pH optimum was 6.3. It is suggested that the A. israelii dextranase may function as a regulatory factor during extracellular in vivo glucan synthesis from sucrose by various plaque microbes.

Actinomyces↗
PubMed1986

Tight genetic linkage of a glucosyltransferase and dextranase of Streptococcus mutans GS-5.

A genetic library consisting of over 5000 clones with an average insert size of 6.9 kilobasepairs (kbp) of Streptococcus mutans GS-5 has been constructed in a bivalent plasmid vector pMK3, which is capable of replicating in Escherichia coli and Bacillus subtilis. The recombinant plasmid pSUCRI, containing a 6.0 kbp fragment of S. mutans GS-5 DNA, was the focus of this study. Using Southern hybridization, in vitro and in vivo gene expression techniques, and biochemical analysis, this clone was shown to encode the 55 kiloDalton (kDal) GS-5 gtfA gene product, as well as a 38 and a 66 kDal polypeptide. In addition to the gtfA gene, pSUCRI encodes a dextranase activity with specificity for alpha(1----6)-linked glucans, and with no detectable activity on mutan. The dextranase enzyme had an apparent molecular weight of 66 kDal as demonstrated by SDS-PAGE analysis of the proteins produced by a dextranase-negative deletion derivative. The pH optimum of the enzyme was approximately 6.0, and there was no detectable activity below pH 5.0. By subcloning various combinations of DNA fragments from pSUCRI, it was demonstrated that the dextranase gene (designated dexB) can be separated from the gtfA gene and still be efficiently expressed in both E. coli and B. subtilis. The dexB gene contained its own promoter and ribosome-binding site. The genetic linkage of the gtfA and dexB genes in the S. mutans GS-5 chromosome was confirmed by Southern hybridization and by the independent isolation of four distinct clones containing the gtfA gene and common flanking sequences. In addition to a glucosyltransferase and dextranase, an invertase-like activity is also encoded on pSUCRI, indicating that there is a cluster of genes on the S. mutans GS-5 chromosome which is devoted to the dissimilation of sucrose and concomitant synthesis or modification of glucans into a water-insoluble form, perhaps constituting an operon for glucan modification which can be coordinately regulated in response to environmental alterations.

Bacillus subtilis↗
PubMed1992

[Conditions for dextranase formation by Paecilomyces lilacinus].

Induced formation conditions of dextranase by Paecilomyces lilacinus were investigated. Effect of various carbohydrates on dextranase formation was examined, dextran was the best C-source and as an inducer. The effect of dextran with different molecular weight (from 17.2 to 1000 kD) on dextranase formation was compared, productivity of dextranase increased with increase of dextran molecular weight. When dextran of 1000 kD was used as C-source. The enzyme formation was 40% higher than that 17.2 kD dextran. When other sugars were separately added to the medium with dextran, the enzyme formation was repressed. Besides C-source, the other optimum conditions of dextranase formation were as follows: N-source, beef peptone; medium initial pH, 6.0-7.0; culture temperature, 28 degrees C; inoculum amount about 10%, and the organism was cultivated for 6 days on 200 r/min shaker in 250 ml flasker with 50 ml medium.

Carbohydrates↗
PubMed1992

Macromolecular prodrugs. XX. Factors influencing model dextranase-mediated depolymerization of dextran derivatives in vitro.

Endo-dextranase-mediated depolymerization of dextran and dextran derivatives under various experimental conditions in vitro was determined. By a simultaneous determination of Mn and MW of dextrans treated with the enzyme in aqueous buffer, an initial increase of the polydispersity of the polysaccharide sample was observed, indicating that dextranase cleaved the dextran molecules into chains which differed significantly in length. A pH optimum of 5 for the enzyme action was found. However, in the pH range 5-8, which prevails in the colon, the initial depolymerization rates differed by a factor of less than 2. Dextranase treatment of a dextran sample resulted in a constant increase of the concentration of terminal reducing glucose residues per time unit suggesting, that the initial depolymerization reaction followed zero-order kinetics. For degrees of substitution below 12 the efficacy of dextranase fragmentation of dextran conjugates decreased almost linearly with increasing DS. The chemical nature of the attached drug did not significantly affect the depolymerization rates. Maximally depolymerized dextran derivatives were obtained by the combined action of dextranase and various alpha-glucosidases. Treatment of such solutions with: a) model esterases b) 80% plasma and c) 20% liver homogenate did not give rise to an acceleration of the initial drug regeneration, as compared to identical experiments carried out in pure buffer solution (pH 7.4 and 37 degrees C).

Animals↗
PubMed1988

Purification and characterization of intracellular dextranase of Bacteroides oralis Ig4a.

Multiple forms of dextranase were detected in both intra- and extracellular fractions of Bacteroides oralis Ig4a. The molecular weights of these enzymes varied from 52,000 to 260,000 by sodium dodecyl sulfate-polyacrylamide-blue dextran gel electrophoresis. The intracellular dextranases were fractionated by chromatography and gel filtration steps, and the dextranases IV and V were obtained. The former was only partially pure. The molecular weights of the dextranases IV and V were estimated to be 120,000 and 105,000, respectively, by SDS-PAGE. The dextranase V was further characterized and it was revealed that the pH- and temperature optima were 5.0, and 55 degrees C, respectively. The Km value was 6.7 x 10(-2) mM for dextran T-70. The enzyme did not exhibit any metal ion requirements, but was inhibited by CoCl2 and HgCl2; lysine and alanine contents were especially high; it hydrolyzed the alpha-1,6-glucan by an exo-type mechanism, and was inactive toward glucans containing alpha-1,3-, alpha-1,4-, and beta-1,4-linkages.

Bacteroides↗
PubMed2004

Synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase.

A recycle ultrafiltration membrane reactor was used to develop a continuous synthesis process for the production of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase. A variety of membranes were tested and the parameters affecting reactor stability, productivity, and product molecular weight distribution were investigated. Enzyme inactivation in the reactor was reduced with the use of a non-ionic surfactant but its use had severe adverse effects on the membrane pore size and porosity. During continuous isomaltooligosaccharide synthesis, dextransucrase inactivation was shown to occur as a result of the dextranase activity and it was dependent mainly on the substrate availability in the reactor and the hydrolytic activity of dextranase. Substrate and dextranase concentrations (50-200 mg/mL(-1) and 10-30 U/mL(-1), respectively) affected permeate fluxes, reactor productivity, and product average molecular weight. The oligodextrans and isomaltooligosaccharides formed had molecular weights lower than in batch synthesis reactions but they largely consisted of oligosaccharides with a degree of polymerization (DP) greater than 5, depending on the synthesis conditions. No significant rejection of the sugars formed was shown by the membranes and permeate flux was dependent on tangential flow velocity.

Bioreactors↗
PubMed1994

Enhanced intracellular stability and efficacy of PEG modified dextranase in the treatment of a model storage disorder.

A model for storage disorders was produced in the livers of mice by the administration of liposomally encapsulated FITC-dextran. Liposomally delivered dextranase was found to be more efficient in degrading the accumulated substrate as compared to the free enzyme. Dextranase was covalently modified with PEG, and liposomes were used as carriers for delivering the free and the modified enzyme to the liver at similar rates. The PEG-dextranase conjugate showed greater intracellular stability as compared to the native enzyme. Liposomally delivered PEG-dextranase, by virtue of its enhanced intracellular stability, could not only degrade the accumulated FITC-dextran, but could also prevent its further accumulation over a period of time. This enhanced intracellular stability of enzymes would be of importance in extending the catalytic life of therapeutically active enzymes and thereby improve their therapeutic potential for the treatment of intracellular storage disorders.

Animals↗
PubMed1995

Sequence analysis of the Streptococcus mutans Ingbritt dexA gene encoding extracellular dextranase.

The complete nucleotide sequence (3,747 bp) of the dextranase gene (dexA) and flanking regions of the chromosome of Streptococcus mutans Ingbritt (serotype c) were determined. The open reading frame for dexA was 2,550 bp, ending with a stop codon TGA. A putative ribosome-binding site, promoter preceding the start codon, and potential stem-loop structure were identified. The presumed dextranase protein (DexA) consisting of 850 amino acids was estimated to have a molecular size of 94,536 Da and a pI of 4.79. The nucleotide sequence and the deduced amino acid sequences of S. mutans dexA exhibited homologies of 57.8% and 47.0%, respectively, to those of Streptococcus sobrinus dex. The homologous region of dex of S. sobrinus was in the N-terminal half. The C terminus of DexA consisted of a hexapeptide LPQTGD, followed by 7 charged amino acids, 21 amino acids with a strongly hydrophobic character, and a charged hexapeptide tail, which have been reported as a common structure of C termini of not only the surface-associated proteins of Gram-positive cocci but also the extracellular enzymes such as beta-fructosidase of S. mutans and dextranase of S. sobrinus. The DexA protein had no significant homology with the glucosyltransferases, the glucan-binding protein, or the dextranase inhibitor of mutans streptococci.

Amino Acid Sequence↗
PubMed2001

Nucleotide sequence and molecular characterization of a dextranase gene from Streptococcus downei.

DNA fragments encoding the Streptococcus downei dextranase were amplified by PCR and inverse PCR based on a comparison of the dextranase gene (dex) sequences from S. sobrinus, S. mutans, and S. salivarius, and the complete nucleotide sequence of the S. downei dex was determined. An open reading frame (ORF) of dex was 3,891 bp long. It encoded a dextranase protein (Dex) consisting of 1,297 amino acids with a molecular mass of 139,743 Da and an isoelectric point of 4.49. The deduced amino acid sequence of S. downei Dex had homology to those of S. sobrinus, S. mutans and S. salivanus Dex in the conserved region (made of about 540 amino acid residues). DNA hybridization analysis showed that a dex DNA probe of S. downei hybridized to the chromosomal DNA of S. sobrinus as well as that of S. downei, but did not to other species of mutans streptococci. The C terminus of the S. downei Dex had a membrane-anchor region which has been reported as a common structure of C termini of both the S. mutans and S. sobrinus Dex. The recombinant plasmid which harbored the dex ORF of S. downei produced a recombinant Dex enzyme in Escherichia coli cells. The analysis of the recombinant enzyme on SDS-PAGE containing blue dextran showed multiple active forms as well as dextranases of S. mutans, S. sobrinus and S. salivarius.

Amino Acid Sequence↗
PubMed1998

Detection of dextranase-producing gram-negative oral bacteria.

Thirty-one strains of 23 gram-negative oral bacterial species were examined for dextran-degrading activity on agar plates containing blue dextran. One strain each of Capnocytophaga ochracea, Capnocytophaga sputigena, Prevotella loescheii, Prevotella melaninogenica and Prevotella oralis had detectable dextranase activity. The culture supernatants of P. melaninogenica and P. oralis cells contained dextranases of multiple sizes, but those of the other three species had a single size of enzyme. A 56-kDa dextranase was purified from the culture supernatant of P. oralis and the antiserum against the enzyme was prepared with a rabbit. The Ouchterlony test showed that the antibody reacted with the supernatants of both P. melaninogenica and P. oralis but not with the others. Dot-blot hybridization using the dextranase gene of Streptococcus mutans as a probe revealed that there was no significantly homologous sequence in the chromosomal DNA of the five species.

Animals↗
PubMed1989

The purification and characterization of a dextranase from Lipomyces starkeyi.

Dextranase produced by Lipomyces starkeyi was purified 43-fold, by carboxymethyl-Sepharose chromatography followed by agarose gel-filtration chromatography. The purified enzyme showed four bands by SDS/polyacrylamide gel electrophoresis with estimated mass 74 kDa, 71 kDa, 68 kDa and 65 kDa. This preparation exhibited multiple isoelectric points between 5.6 and 6.1. All the isoelectric forms were active and catalytically similar. The dextranase contained a carbohydrate moiety (8%). The physical properties of the enzyme were pH and temperature optima of 5.0 and 55 degrees C, respectively. This dextranase was stable between pH 2.5 and 7.0 at temperatures below 40 degrees C. Lipomyces dextranase was a typical endodextranase with the final product of dextran hydrolysis being isomalto-oligosaccharides from glucose to isomaltotetrose.

Carbohydrates↗
PubMed1995

Isolation of a dextranase constitutive mutant of Lipomyces starkeyi and its use for the production of clinical size dextran.

A derepressed and partially constitutive mutant for dextranase of Lipomyces starkeyi was selected after ethyl methane sulphonate mutagenesis by zone clearance on blue dextran agar plates. The mutant produced dextranase when grown on glucose, fructose and sucrose as well as on dextran, and more enzyme was produced by the mutant than by the parental strain when grown on 1% dextran. The pH and temperature optima for the mutant dextranase were 5.5 and 55 degrees C, respectively. Dextranase produced on sucrose produced more isomaltose and less glucose after dextran hydrolysis than the equivalent enzyme produced on dextran. The clinical size dextran (average mol. wt of 75,000 +/- 25,000) yield of mixed culture fermentation with the mutant and Leuconostoc mesenteroides was 94% of the total dextran produced.

Dextranase↗
PubMed1977

Streptococcus mutans dextransucrase: functioning of primer dextran and endogenous dextranase in water-soluble and water-insoluble glucan synthesis.

The extracellular enzyme activities of Streptococcus mutans 6715 that synthesize glucans from sucrose were concentrated and partially purified by ammonium sulfate precipitation and gel permeation column chromatography. Polyacrylamide gel analysis demonstrated that all of the major proteins precipitated by ammonium sulfate were quantitatively recovered in the high-molecular-weight, enzyme-containing aggregates found in the void volume of the gel column. Anion-exchange column chromatography was used to fractionate the aggregates into preparations, alpha and beta, which produced water-insoluble and water-soluble glucans, respectively. Polyacrylamide gel analysis showed that alpha and beta contained unique proteins and dextransucrase (EC 2.4.1.5) activities. Studies on the time course of glucan synthesis by alpha demonstrated that this enzyme preparation contained dextranase activity, which partially degraded nascent alcohol-insoluble glucan into alcohol-soluble products that were subsequently reincorporated into insoluble product. The beta enzyme preparation contained no detectable dextranase activity. Mixing experiments in the absence of primer dextran demonstrated that the dextranase activity present in alpha could modify glucan production by beta. CsCl density gradient analysis of product glucans demonstrated that exogenous primer dextrans were used as acceptor molecules by both the alpha and beta enzyme preparations, and that water-soluble glucans synthesized by beta could be converted into water-insoluble glucans by alpha. It is proposed that the structural heterogeneity of the native glucans produced from sucrose by S. mutans is a result of the concerted action of glucan-forming dextransucrases and endohydrolytic dextranase activity.

Chromatography, Gel↗
PubMed1999

Molecular cloning of isomaltotrio-dextranase gene from Brevibacterium fuscum var. dextranlyticum strain 0407 and its expression in Escherichia coli.

The gene encoding an extracellular isomaltotrio-dextranase (IMTD), designed dexT, was cloned from the chromosomal DNA of Brevibacterium fuscum var. dextranlyticum strain 0407, and expressed in Escherichia coli. A single open reading frame consisting of 1923 base pairs that encoded a polypeptide composed of a signal peptide of 37 amino acids and a mature protein of 604 amino acids (M(r), 68,300) was found. The primary structure had no significant similarity with the structure of two other reported exo-type dextranases (glucodextranase and isomalto-dextranase), but had high similarity with that of an endo-dextranase isolated from Arthrobacter sp. Transformed E. coli cells carrying the gene encoding mature protein of IMTD overproduced IMTD under the control of the T7 phage promoter induced by IPTG. The purified recombinant enzyme showed the same optimum pH, lower specific activity, and similar hydrolytic pattern, as to those of native IMTD.

Amino Acid Sequence↗