Co-immobilization of dextransucrase and dextranase in alginate

Dextransucrase from Leuconostoc mesenteroides and dextranase from Penicillium lilacinum were co-immobilized and used to produce isomaltooligosaccharides from sucrose. The enzymes were co-immobilized by encapsulating soluble dextransucrase and dextranase covalently attached to Eupergit C in alginate (beads, fibers, and capsules). The alginate capsule co-immobilization was done in the presence of soluble starch and resulted in a high immobilization yield (71%), and the enzymes retained their activities during 20 repeated batch reactions and for a month in storage at 4 °C. The presence of starch was essential for the stability of dextransucrase in alginate capsules. Furthermore, it is important that the dextranase be pre-immobilized prior to alginate capsule co-immobilization to prevent dextranase leakage and inactivation of dextransucrase. The co-immobilized enzymes formed oligosaccharides from sucrose, which can be used as prebiotics. In addition, the oligosaccharides that were produced after the addition of sucrose reacted with the alginate fiber-encapsulted dextransucrase, thus increasing the amount of prebiotics. Co-immobilization in alginate fiber and beads also resulted in high yields (70 and 64%), but enzymatic activities decreased by 74 and 99%, respectively, after a month in storage at 4 °C. The newly developed alginate capsule method for co-immobilization of dextransucrase and dextranase is simple yet effective and has the potential for industrial-scale production of isomaltooligosaccharides.     

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.     

Construction of a fusion enzyme of dextransucrase and dextranase: Application for one-step synthesis of isomalto-oligosaccharides

The linear isomalto-oligosaccharides (IMO) with DP2–DP10 were produced by one-step process using engineered fusion enzyme (DXSR) of endo-dextranase and only α-(1–6) glucan synthesizing dextransucrase. The fusion enzyme was successfully expressed in Escherichia coli and characterized. Compared to individual enzymes, DXSR had 150% increased endo-dextranase activity and 98% decreased dextransucrase activity. The partially purified DXSR displayed molecular mass of 240 kDa as analyzed by SDS–PAGE. It showed both enzyme activities on analysis by zymogram. The thermal- and pH-stability of DXSR was around 28 °C and pH at 5.0–6.4, respectively. IMOs production by DXSR was increased by the addition of metal ions such as Fe²⁺, Li⁺, K⁺ and Ni²⁺, but the enzyme was strongly inhibited by Hg²⁺ and Ag⁺. DXSR produced linear IMO with DP2–DP10 using sucrose as a sole substrate. The molecular weight and amount of IMO could be controlled by the sucrose concentration. DXSR gave 30-fold higher production of IMO than that of an equal activity mixture of the two enzymes such as dextranase and dextransucrase.     

Volume changes during enzyme reactions. The influence of pressure on the action of invertase, dextranase and dextransucrase.

The pressure dependence of the maximum velocities and the Michaelis constants for the enzymes invertase and dextranase was measured up to 1400 bar. The corresponding activation volumes deltaV not equal to c and deltaV not equal to Km proved to be independent of pressure. Together with data from other sources the meaning of deltaV not equal to c and deltaV not equal to Km is established and the volume profiles of the reactions are constructed. These profiles are similar in contour to the volume profile of the dextran formation catalyzed by the enzyme dextransucrase, but the amount of the volume changes is very much larger for dextransucrase. The evaluation of salt effects shows, that for all three enzymes solvent interactions are not important in explaining the results. The reaction mechanisms seem to be governed by conformation changes of the enzymes. The larger effects in dextransucrase are explained by the produced dextran chain remaining tightly bound to the enzyme and being transported relative to the enzymes position in each reaction cycle.     

Immobilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F on Eupergit C supports

Dextransucrase from Leuconostoc mesenteroides B-512F was immobilized on epoxy-activated acrylic polymers with different textural properties (Eupergit C and Eupergit C 250L). Prior to immobilization, dextransucrase was treated with dextranase to remove the dextran layer covering the enzyme surface, thus increasing the accessibility of its reactive groups to the epoxide centers of the support. Elimination of 99% of the initial carbohydrate content was determined by the anthrone method. To prevent enzyme inactivation, the immobilization was carried out at pH 5.4, at which the coupling to the support took place through the carboxylic groups of the enzyme. The effects of the amount (mg) of dextransucrase added per gram of support (from 0.2:1 to 30:1), temperature and contact time were studied. Maximum activity recovery of 22% was achieved using Eupergit C 250L. Using this macroporous support, the maximum specific activity (710 U/g biocatalyst) was significantly higher than that obtained with the less porous Eupergit C (226 U/g biocatalyst). The dextransucrase immobilized on Eupergit C 250L showed similar optimal temperature (30 degrees C) and pH (5-6) compared with the native enzyme. In contrast, a notable stabilization effect at 30 degrees C was observed as a consequence of immobilization. After a fast partial inactivation, the dextransucrase immobilized on Eupergit C 250L maintained more than 40% of the initial activity over the following 2 days. The features of this immobilized system are very attractive for its application in batch and fixed-bed bioreactors.     

A facile purification of Leuconostoc mesenteroides B-512FM dextransucrase

Leuconostoc mesenteroides NRRL B-512F has been mutated by treatment with N-nitrosoguanidine. The resulting mutant (designated as B-512FM) produces 300 times as much enzyme as the parent strain. B-512FM dextransucrase was treated extensively with Sigma crude dextranase, followed by column chromatography on Bio-Gel A-5m. The purified dextransucrase had a specific activity of 84 IU/mg, a 100-fold purification with 42% yield, and was shown by SDS-PAGE to have a single protein of molecular weight of 158,000 with dextransucrase activity. The procedure has been used to produce purified enzyme for sequencing. The molecular weight of 158,000 agrees with that calculated from its amino acid sequence.     

Production,purification, and properties of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

The production of dextransucrase fromLeuconostoc mesenteroides NRRL B-512F was stimulated 2-fold by the addition of 0.005% of calcium chloride to the medium; levansucrase levels were unaffected. Dextransucrase was purified by concentration and dialysis of the culture supernatant with a Bio-Fiber 80 miniplant, and by treatment with dextranase followed by chromatography on Bio-Gel A-5m. A 240-fold purification, with a specific activity of 53 U/mg, was obtained. Contaminating enzyme activities of levansucrase, invertase, dextranase, glucosidase, and sucrose phosphorylase were decreased to non-detectable levels. Poly(acrylamide)-gel electrophoresis of the purified enzyme showed only two protein bands, both of which had dextransucrase activity. These bands also gave a carbohydrate stain, indicating that the dextransucrase could be a glycoprotein. Acid hydrolysis, followed by paper chromatography, of the purified enzyme showed that the major carbohydrate was mannose. ConcanavaIin A completely removed dextransucrase activity from solution, confirming the mannoglycoprotein character of the enzyme. Dextransucrase activity was not altered by the addition of 0.008?4 mg/ml of dextran, but its storage stability was increased by the addition of 4 mg/ml of dextran. As previously shown by others, the activity of dextransucrase was decreased by EDTA, and was restored by the addition of calcium ions. Zinc, cadmium, lead, mercury, and copper ions were inhibitory to various degrees.     

Immobilization of dextranase from Chaetomium erraticum

In order to facilitate the Co-Immobilization of dextransucrase and dextranase, various techniques for the immobilization of industrial endo-dextranase from Chaetomium erraticum (Novozymes A/S) were researched. Adsorption isotherms at various pH-values have been determined for bentonite (Montmorillonite), hydroxyapatite and Streamline DEAE. Using bentonite and hydroxyapatite, highest activity loads (12,000 Ug(-1); 2900 Ug(-1), respectively) can be achieved without a significant change of the apparent Michaelis-Menten constant K(M). For successful adsorption, enzyme to bentonite ratios greater than 0.4 (w/w) have to be used as lower ratios lead to 90% enzyme inactivation due to bentonite contact. In addition, covalent linkage using the activated oxiran carriers Eupergit C and Eupergit C250L as well as linkage with aminopropyl silica via metaperiodate activation of glycosyl moiety of dextranase are discussed. This is also the first report probing the structure of a matrix containing dextranase by use of substrate species with different molecular weights. From this we can observe a relationship between the porosity of Eupergit and dextran dependent activity. For the reactor concept using Co-Immobilisates, hydroxyapatite will be preferred to Eupergit because of its higher specific activity and dispersity.     

Alginate–pectin co-encapsulation of dextransucrase and dextranase for oligosaccharide production from sucrose feedstocks

Bioprocess and Biosystems Engineering - The genes for dextransucrase and dextranase were cloned from the genomic regions of Leuconostoc mesenteroides MTCC 10508 and Streptococcus mutans MTCC 497,...     

Milligram to gram scale purification and characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

A sequence of dextranase treatment, DEAE-cellulose chromatography, affinity chromatography on Sephadex G-200, and chromatography on DEAE-Trisacryl M has been optimized to give a dextransucrase preparation with low carbohydrate content (1-100 micrograms/mg protein) and high specific activity (90-170 U/mg protein) relative to previous procedures, in 30-50% yield. Levansucrase was absent after DEAE-cellulose chromatography, and dextranase was undetectable after Sephadex G-200 chromatography. The method could be scaled up to produce gram quantities of purified enzyme. The purified dextransucrase had a pH optimum of 5.0-5.5, a Km of 12-16 mM, and produced the same lightly branched dextran as before purification. The purified enzyme was not activated by added dextran, but the rate of dextran synthesis increased abruptly during dextran synthesis at a dextran concentration of approximately 0.1 mg/mL. The enzyme had two major forms, of molecular weight 177,000 and 158,000. The 177,000 form predominated in fresh preparations of culture supernatant or purified enzyme, whereas the amount of the 158,000 form increased at the expense of the 177,000 form during storage of either preparation.     

Production,characterization and (co-)immobilization of dextranase from <Emphasis Type="Italic">Penicillium aculeatum</Emphasis>

Fermentation kinetics of Penicillium aculeatum ATCC 10409 demonstrated that fungal growth and dextranase release are decoupled. Inoculation by conidia or mycelia resulted in identical kinetics. Two new isoenzymes of the dextranase were characterized regarding their kinetic constants, pI, MW, activation energy and stabilities. The larger enzyme was 3-fold more active (turnover number: 2,230 ± 97 s⁻¹). Pre-treatment of bentonite with H₂O₂ did not affect adsorption characteristics of dextranase. Enzyme to bentonite ratios above 0.5:1 (w/w) resulted in a high conservation of activity upon adsorption. Furthermore, dextranase could be used in co-immobilizates for the direct conversion of sucrose into isomalto-oligosaccharides (e.g. isomaltose). Yields of co-immobilizates were 2–20 times that of basic immobilizates, which consist of dextransucrase without dextranase.     

Effect of dextran and ammonium sulphate on the reaction catalysed by a glucosyltransferase complex from Streptococcus mutans

The highly aggregated proteins precipitated by (NH4)2SO4 from the culture fluid of three strains of Streptococcus mutans gradually released less aggregated glucosyltransferase activities - dextransucrase and mutansucrase - which catalysed the synthesis of water-soluble and insoluble glucans from sucrose. Mutansucrase was eluted from a column of Sepharose 6B before dextransucrase. This activity was lost during subsequent dialysis and gel filtration, but there was a corresponding increase in dextransucrase activity which catalysed the formation of soluble glucan when incubated with sucrose alone, and insoluble glucan when incubated with sucrose and 1.55 M-(NH4)2SO4. Relative rates of synthesis of soluble and insoluble glucan in the presence of 1.55 M-(MH4)2SO4 were dependent upon the enzyme concentration: high concentrations favoured insoluble glucan synthesis. Insoluble glucans synthesized by mutansucrase or by dextransucrase in the presence of 1.55 M-(NH4)2SO4 were more sensitive to hydrolysis by mutanase than by dextranse, but soluble glucans were more extensively hydrolysed by dextranase than by mutanase. Partially purified dextransucrase sedimented through glycerol density gradients as a single symmetrical peak with an apparent molecular weight in the range 100000 to 110000. In the presence of 1.55 M-(NH4)2SO4, part of the activity sedimented rapidly as a high molecular weight aggregate. The results strongly suggest that soluble and insoluble glucans are synthesized by interconvertible forms of the same glucosyltransferase. The aggregated form, mutansucrase, preferentially catalyses (1 leads to 3)-alpha bond formation but dissociates during gel filtration to the dextransucrase form which catalyses (1 leads to 6)-alpha bond formation.     

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.     

Acceleration of Dextransucrase Activity of Streptococcus mutans by Secretory Immunoglobulin A

The effect of immunoglobulins on the activity of dextransucrase purified from Streptococcus mutans strain HS-6 is described. When human salivary immunoglobulin A (IgA) or colostral IgA, either natured or denatured, was incubated with dextransucrase, the rate of the dextran synthesis was markedly accelerated, whereas human serum IgA or IgG neither accelerated nor inhibited the enzyme activity. The results suggest that a portion unique for secretory IgA, the secretory component, might be related to the enzyme acceleration. On the other hand, specific rabbit antiserum against the dextransucrase inhibited completely dextran synthesis by the enzyme.     

Purification of dextran-binding protein from cariogenic Streptococcus mutans

An extracellular protein produced by $\text{Streptococcus mutans}$ was purified to electrophoretic homogeneity by affinity chromatography on Sephadex G50 followed by gel filtration. The protein is devoid of both dextransucrase and dextranase activity but binds dextran and therefore probably is implicated in the adherence of $\text{S. mutans}$ cells to the host tooth surface. The presence of the dextran-binding protein may be a determinant of the pathogenicity of such cariogenic micro-organisms.     

Stabilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F by nonionic detergents, poly(ethylene glycol) and high-molecular-weight dextran

Dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase, EC 2.4.1.5) (3 IU/ml culture supernatant) was obtained by a modification of the method of Robyt and Walseth (Robyt, J.F. and Walseth, T.F. (1979) Carbohydr. Res. 68, 95-111) from a nitrosoguanidine mutant of Leuconostoc mesenteroides NRRL B-512F selected for high dextransucrase production. Dialyzed, concentrated culture supernatant (crude enzyme) was treated with immobilized dextranase (EC 3.2.1.11) and chromatographed on a column of Bio-Gel A-5m. The resulting, purified enzyme lost activity rapidly at 25 degrees C or on manipulation, as did the crude enzyme when diluted below 1 U/ml. Both enzyme preparations could be stabilized by low levels of high-molecular-weight dextran (2 micrograms/ml), poly(ethylene glycol) (e.g., 10 micrograms/ml PEG 20 000), or nonionic detergents (e.g., 10 micrograms/ml Tween 80). The stabilizing capacity of poly(ethylene glycol) and of dextran increased with molecular weight. Calcium had no stabilizing action in the absence of other additions, but reduced the inactivation that occurred in the presence of 0.5% bovine serum albumin or high concentrations (greater than 0.1%) of Triton X-100. In summary, dextransucrase could be stabilized against activity losses caused by heating or by dilution through the addition of low concentrations of nonionic polymers (dextran, PEG 20000, methyl cellulose) or of nonionic detergents at or slightly below their critical micelle concentrations.     

Characterization of the multiple forms and main component of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

Multiple forms of dextransucrase (sucrose:1.6-alpha-D-glucan 6-alpha-D-glucosyltransferae EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F strain were shown by gel filtraton and electrophoretic analyses. Two components of enzyme, having different affinities for dextran gel, were separated by a column of Sephadex G-100. The major component voided from the Sephadex column was treated with dextranase and purified to an electrophoretically homogeneous state. The ]urified enzyme had a molecular weight of 64 000-65 000, pI value of 4.1, and 17% of carbohydrate in a molecule. EDTA showed a characteristic inhibition on the enzyme while stimulative effects were observed by the addition of exogenous dextran to the incubation mixture. The enzyme activity was stimulated by various dextrans and its Km value was decreased with increasing concentration of dextran. The purified enzyme showed no affinity for a Sephadex G-100 gel, and readily aggregated after the preservation at 4 degrees C in a concentrated solution.     

Co-immobilization of dextransucrase and dextranase for the facilitated synthesis of isomalto-oligosaccharides: Preparation, characterization and modeling

Co-Immobilization of dextransucrase (DS) and dextranase (DN) into calcium alginate includes the co-entrapment of soluble DS and adsorbed DN. DS converts sucrose into dextran, which is the substrate for DN, so that isomalto-oligosaccharides (IMOs) are follow-up products of dextran hydrolysis. The boundary conditions for the successful preparation are investigated with respect to choice of DN adsorbate, surface modifications using blotting agents and optimal enzyme activity ratios. Further, repetitive batch experiments suggest the selection of medium activity ratios for continuous use (0.3 U(DN)U(-1) (DS), e.g.). Product formation at various cosubstrate:substrate concentrations as well as at different DN:DS ratios are discussed. Moreover, the complexity of the bi-enzymatic system can be reduced considering the molar ratios of cosubstrate:substrate (glucose:sucrose). Based on these factors, a mechanistic kinetic model is developed, which distinguishes the corresponding contributions of the two enzymes upon overall product formation. In general, at low glucose:sucrose ratios isomaltose synthesis is featured primarily by DN action. Yet with increasing amounts of glucose both the quantity and quality of DN substrate changes, so that its contribution to product formation decreases in an exponential manner; still the overall product yield continuously increases due to enhanced DS contribution.     

The Role of Zinc Atoms in Nuclease P1

A dextransucrase inhibitor, ribocitrin, was purified about 580-fold from a culture broth of Streptomyces neyagawaensis MF980-CF1 by combination chromatography.

Ribocitrin is a previously unknown type of oligosaccharide composed of ribose. It inhibited the crude preparation of dextransucrase noncompetitively with regard to sucrose and the inhibitory activity (Inh%) was sensitive to pH. It was very stable at alkaline pH values, but gradually lost its inhibitory activity as the acidity increased. It had no antimicrobial activity. It did not inhibit glycoside hydrolases and was not susceptible to the above enzymes. It had a low toxicity.

Plaque formation of Streptococcus mutans E49 on a smooth glass surface was diminished by ribocitrin and the colony form of this microorganism on Mitis Salivarius agar was evidently affected by ribocitrin.     

Dextransucrase secretion in Leuconostoc mesenteroides depends on the presence of a transmembrane proton gradient.

The relationship between proton motive force and the secretion of dextransucrase in Leuconostoc mesenteroides was investigated. L. mesenteroides was able to maintain a constant proton motive force of -130 mV when grown in batch fermentors at pH values 5.8 to 7.0. The contribution of the membrane potential and the transmembrane pH gradient varied depending on the pH of the growth medium. The differential rate of dextransucrase secretion was relatively constant at 1,040 delta mU/delta mg (dry weight) when cells were grown at pH 6.0 to 6.7. Over this pH range, the internal pH was alkaline with respect to the external pH. When cells were grown at alkaline pH values, dextransucrase secretion was severely inhibited. This inhibition was accompanied by an inversion of the pH gradient as the internal pH became more acidic than the external pH. Addition of nigericin to cells at alkaline pH partially dissipated the inverted pH gradient and produced a fourfold stimulation of dextransucrase secretion. Treatment of cells with the lipophilic cation methyltriphenylphosphonium had no effect on the rate of dextransucrase secretion at pH 5.5 but inhibited secretion by 95% at pH 7.0. The reduced rate of secretion correlated with the dissipation of the proton motive force by this compound. Values of proton motive force greater than -90 mV were required for maximal rates of dextransucrase secretion. The results of this study indicate that dextransucrase secretion in L. mesenteroides is dependent on the presence of a proton gradient across the cytoplasmic membrane that is directed into the cell.