Food-Grade Expression and Characterization of a Dextranase from Chaetomium gracile Suitable for Sugarcane Juice Clarification.

The microbial production of dextranase using cheap carbon sources is beneficial to solve the economic loss caused by the accumulation of dextran in syrup. A food-grade microbial cell factory was constructed by introducing the dextranase encoding gene DEX from Chaetomium gracile to the chromosome of Bacillus subtilis, and the antibiotic resistance marker gene was subsequently deleted via the Cre/loxP strategy. The dual-promoter system with a sequentially arranged constitutive P43 promoter resulted in an 85 % increase in DEX expression. Under the optimal fermentation conditions of 10 g/L maltose, 15 g/L casein, 1 g/L Na2 HPO4 , 1 g/L FeSO4 and 8 g/L NaCl, DEX activity was increased from 2.625 to 64.34 U/mL. Recombinant DEX was purified 5.98-fold with a recovery ratio of 26.67 % and specific activity of 3935.02 U/mg. Enzyme activity was optimal at 55 °C and pH 5.0 and remained 80.34 % and 71.36 % of the initial activity at 55 °C and pH 4.0 after 60 min, respectively. The enzyme possessed high activity in the presence of Co2+ , while Ag+ showed the strongest inhibition ability. The optimal substrate was 20 g/L dextran T-2000. The findings could facilitate the low-cost, large-scale production of food-grade DEX for use in the sugar industry.

Structural analysis of enzyme-resistant isomaltooligosaccharides reveals the elongation of α-(1→3)-linked branches in Weissella confusa dextran.

Weissella confusa VTT E-90392 is an efficient producer of a dextran that is mainly composed of α-(1→6)-linked D-glucosyl units and very few α-(1→3) branch linkages. A mixture of the Chaetomium erraticum endodextranase and the Aspergillus niger α-glucosidase was used to hydrolyze W. confusa dextran to glucose and a set of enzyme-resistant isomaltooligosaccharides. Two of the oligosaccharides (tetra- and hexasaccharide) were isolated in pure form and their structures elucidated. The tetrasaccharide had a nonreducing end terminal α-(1→3)-linked glucosyl unit (α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc), whereas the hexasaccharide had an α-(1→3)-linked isomaltosyl side group (α-D-Glcp-(1→6)[α-D-Glcp-(1→6)-α-D-Glcp-(1→3)]-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc). A mixture of two isomeric oligosaccharides was also obtained in the pentasaccharide fraction, which were identified as (α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc) and (α-D-Glcp-(1→6)[α-D-Glcp-(1→3)]-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc). The structures of the oligosaccharides indicated that W. confusa dextran contains both terminal and elongated α-(1→3)-branches. This is the first report evidencing the presence of elongated branches in W. confusa dextran. The (1)H and (13)C NMR spectroscopic data on the enzyme-resistant isomaltooligosaccharides with α-(1→3)-linked glucosyl and isomaltosyl groups are published here for the first time.

Molecular cloning and characterization of glucanase inhibitor proteins: coevolution of a counterdefense mechanism by plant pathogens.

A characteristic plant response to microbial attack is the production of endo-beta-1,3-glucanases, which are thought to play an important role in plant defense, either directly, through the degradation of beta-1,3/1,6-glucans in the pathogen cell wall, or indirectly, by releasing oligosaccharide elicitors that induce additional plant defenses. We report the sequencing and characterization of a class of proteins, termed glucanase inhibitor proteins (GIPs), that are secreted by the oomycete Phytophthora sojae, a pathogen of soybean, and that specifically inhibit the endoglucanase activity of their plant host. GIPs are homologous with the trypsin class of Ser proteases but are proteolytically nonfunctional because one or more residues of the essential catalytic triad is absent. However, specific structural features are conserved that are characteristic of protein-protein interactions, suggesting a mechanism of action that has not been described previously in plant pathogen studies. We also report the identification of two soybean endoglucanases: EGaseA, which acts as a high-affinity ligand for GIP1; and EGaseB, with which GIP1 does not show any association. In vitro, GIP1 inhibits the EGaseA-mediated release of elicitor-active glucan oligosaccharides from P. sojae cell walls. Furthermore, GIPs and soybean endoglucanases interact in vivo during pathogenesis in soybean roots. GIPs represent a novel counterdefensive weapon used by plant pathogens to suppress a plant defense response and potentially function as important pathogenicity determinants.

Purification and characterization of an isomaltotriose-producing endo-dextranase from a Fusarium sp.

An isomaltotriose-producing endo-dextranase was simply purified from cell-free culture broth of a Fusarium sp. by ethanol fractionation and consecutive column chromatographies using DEAE-Toyopearl and Bio-Gel P-100. The purified enzyme was judged to be homogeneous on PAGE and SDS-PAGE as well as isoelectric focusing. The molecular mass of the enzyme was estimated to be about 69 kDa by SDS-PAGE. The enzyme is an acidic protein with a pI of 4.6. The optimum pH and temperature were pH 6.5 and 35 degrees C, respectively. The enzyme was completely stable over the range of pH 4.5-11.8 at 4 degrees C for 24 h and at temperatures below 45 degrees C. Inactivation of the enzyme was found to be partial with 5 mM Cu2+, being about 70% inhibition and complete with 5 mM of Fe3+, Hg2+, Ag+ or NBS. The enzyme split dextran in an endo-lytic action to produce a large amount of isomaltotriose and a slight amount of isomaltose and glucose. The anomeric configurations of the reaction products formed by the enzyme were alpha-form, indicating that the alpha-glycoside linkages in the substrate are retained. The final yield of isomaltotriose from dextran T-2000 was about 62%.

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.

Purification, characterization, and specificity of dextranase inhibitor (Dei) expressed from Streptococcus sobrinus UAB108 gene cloned in Escherichia coli.

The dextranase inhibitor gene (dei) from Streptococcus sobrinus UAB108 was previously cloned, expressed, and sequenced. Its gene product (Dei) has now been purified as a single band with apparent molecular mass of 43 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity of Dei increased 121-fold upon purification. Most Dei activity (91.2%) was located in the periplasmic fraction from recombinant Escherichia coli cells. Dei competitively inhibits dextranase (Dex). This competitive inhibition mechanism has been further shown by detection and recovery of the intermediate enzyme-inhibitor (Dex-Dei) complex by gel filtration technology using fast protein liquid chromatography. Calibration of their molecular masses indicated that native Dei exists as a tetramer, Dex exists as dimer, and the Dex-Dei complex consists of two Dex molecules with two Dei molecules. Deletion analysis indicates that the intact Dei molecule is essential for Dei activity but not for glucan binding and immune cross-reaction. Dei is a special kind of glucan-binding protein with ability to inhibit Dex with high specificity. It can inhibit endogenous Dex, which can make more branches in glucan with the cooperation of the glucosyltransferase GTF-I. This inhibition cause the accumulation of water-soluble glucan. The latter reaction product can inhibit plaque formation and adherence of the mutans group of streptococcal cells. Dei derived from S. sobrinus UAB108 can inhibit only Dex from S. sobrinus (serotypes d and g), S. downei (previously S. sobrinus, serotype h), and S. macacae (serotype h). This finding suggests that Dei is another important protein existing in some serotypes of the mutans group of streptococci which participates in sucrose metabolism through its interaction with Dex.

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.

Influence of growth rate on the relative activities of free and bound dextranase and dextranase inhibitor in continuous cultures of Streptococcus sobrinus.

The rate of growth of Streptococcus sobrinus was a major factor governing the activity of free dextranase and free dextranase inhibitor in continuous culture filtrates. Depending on the growth conditions, a variable proportion of dextranase and dextranase inhibitor was combined in a tightly bound enzyme-inhibitor (EI) complex. Dissociation of the EI complexes revealed that the total productivity (free + bound) of both the enzyme and the inhibitor increased with growth rate, and that the activities of the enzyme and inhibitor released from the EI complex greatly exceeded their free activities, when the dilution rate (D) was high (D, 0.45 h-1). At low growth rate (D, 0.05 h-1), all the enzyme was bound to the inhibitor, and no free dextranase could be determined in culture filtrates; by contrast, at high growth rate (D, 0.45 h-1), all the inhibitor was bound to dextranase in the active EI complex, leaving active dextranase but no free inhibitor.

Dissociation and electrophoretic separation of dextranase and dextranase inhibitor from a tightly bound enzyme-inhibitor complex of Streptococcus sobrinus.

Endodextranase was separated from dextranase inhibitor in culture filtrates of Streptococcus sobrinus by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in gel slabs containing blue dextran. Sample preparation included dissociation of the enzyme from its inhibitor by boiling for 1 min in SDS. During subsequent incubation of the gel, dextranase was located as clear bands on a blue background, and dextranase inhibitor appeared as blue zones on a clear background following incubation in dextranase solution. The enzyme and the inhibitor existed in multiple forms, and the range of molecular masses for dextranase (223-132 kDa) permitted an excellent separation from dextranase inhibitor (49-25 kDa). Although dextranase-negative mutants, and wild type strains grown at low dilution rate in the chemostat, were devoid of free dextranase activity, the enzyme was easily located by analytical SDS-PAGE. Likewise, analysis of filtrates from wild type strains, which contained no free inhibitor activity when growth occurred at high dilution rate, revealed dextranase inhibitor activity on the gels. The total production (free + combined) of dextranase and inhibitor by S. sobrinus was determined by dissociation of enzyme-inhibitor complexes in concentrated cell-free filtrates, their separation by preparative SDS-PAGE and electroelution from the gels, followed by renaturation of protein activity. From a comparison of activity tests of free dextranase and free inhibitor in untreated filtrates with the results of similar tests on renatured electroeluates, the proportion of each constituent bound into a complex under each growth condition could be deduced.

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.

Characterization of the dextranase purified from Streptococcus mutans Ingbritt.

We purified dextranase from the culture supernatant of Streptococcus mutans Ingbritt by procedures including ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of the enzyme was estimated as 78 kDa by SDS-PAGE. The enzyme degraded dextran at the optimum pH of 5.5, but not other glucans and fructans at all. Paper chromatographic analysis revealed that the enzyme cleaved dextran by an endo-type mechanism. The enzyme was inhibited by Hg2+, Fe3+, Zn2+, and anionic detergents SDS and deoxycholic acid, but not inhibited by non-ionic detergents Triton X-100, Lubrol PX, Nonidet P-40, and Tween 80. SDS-blue dextran-PAGE analysis of the culture supernatant revealed that the enzyme activity detected in the 96 kDa band shifted gradually to the 78 kDa band during handling the supernatant. This shift was inhibited by phenylmethylsulfonyl fluoride, suggesting that the shift of the molecular size is due to proteolytic degradation of the enzyme by serine protease.

Effect of variation in growth conditions on the activity of dextranase inhibitor in continuous cultures of Streptococcus sobrinus.

The activity of free extracellular dextranase inhibitor was determined in strains of Streptococcus sobrinus which were grown in a chemostat under a variety of defined conditions. Maximum release of dextranase inhibitor occurred at low growth rate in glucose-limited medium at pH 6.5. Free inhibitor could not be detected when the strains were grown at high growth rate or in batch culture.

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.

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.

Antigens of Streptococcus mutans implicated in virulence--production of antibodies.

The present studies assayed antibody activities in serum and saliva of animals immunized by different routes, with cells of S. mutans or cell-free preparations containing GTF, FTF, LTA and/or dextranase synthesized by S. mutans. The results show that the type of immunogenic preparation and the route of its administration can elicit different antibody response and may in part explain the disparity of results achieved by different investigators. The results further emphasize the need to use standardized preparations and carefully described protocols for vaccination.

Analysis of the dextranase activity produced by an oral strain of Bacteroides ochraceus.

An anaerobic, gram-negative, dextranase-producing filamentous bacterium isolated from human dental plaque has been identified as a strain of Bacteroides ochraceus. The inducible intracellular dextran-degrading activities produced by this microoranism can be fractionated into endohydrolytic and exohydrolytic enzymes with distinct pH optima. These enzymes reduce the apparent rate of glucan production from sucrose by the dextransucrase produced by Streptococcus mutans and consequently may influence the in vivo production of polysaccharides involved in plaque accumulation and metabolism.

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.