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.     

Characterization of dextransucrase immobilized on calcium alginate beads from Leuconostoc mesenteroides PCSIR-4

Immobilization of dextransucrase from Leuconostoc mesenteroides PCSIR-4 on alginate is optimized for application in the production of dextran from sucrose. Dextransucrase was partially purified by ethanol upto 2.5 fold. Properties of dextransucrase were less affected by immobilization on alginate beads from soluble enzyme. Highest activities of both soluble and immobilized dextransucrase found to be at 35 degrees C and optimum pH for activity remain 5.00. Substrate maxima for immobilized enzyme changed from 125 mg/ml to 200 mg/ml. Incubation time for enzyme-substrate reaction for maximum enzyme activity was increased from 15 minutes to 60 minutes in case of immobilized enzyme. Maximum stability of immobilized dextransucrase was achieved at 25 degrees C with respect to time.     

Properties of Leuconostoc mesenteroides B-512FMC constitutive dextransucrase

Leuconostoc mesenteroides B-512FMC, a constitutive mutant for dextransucrase, was grown on glucose, fructose, or sucrose. The amount of cell-associated dextransucrase was about the same for the three sugars at different concentrations (0.6% and 3%). Enzyme produced in glucose medium was adsorbed on Sephadex G-100 and G-200, but much less enzyme was adsorbed when it was produced in sucrose medium. Sephadex adsorption decreased when the glucose-produced enzyme was preincubated with dextrans of molecular size greater than 10 kDa. The release of dextransucrase activity from Sephadex by buffer (20 mM acetate, pH 5.2) was the highest at 28°–30°C. The addition of dextran to the enzyme stimulated dextran synthesis but had very little effect on the temperature or pH stability. Dextransucrase purified by ammonium sulfate precipitation, hydroxyapatite chromatography, and Sephadex G-200 adsorption did not contain any carbohydrate, and it synthesized dextran, showing that primers are not necessary to initiate dextran synthesis. The purified enzyme had a molecular size of 184 kDa on SDS-PAGE. On standing at 4°C for 30 days, the native enzyme was dissociated into three inactive proteins of 65, 62, and 57 kDa. However, two protein bands of 63 and 59 kDa were obtained on SDS-PAGE after heat denaturation of the 184-kDa active enzyme at 100°C. The amount of 63-kDa protein was about twice that of 59-kDa protein. The native enzyme is believed to be a trimer of two 63-kDa and one 59-kDa monomers.     

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.     

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.     

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.     

On the production of dextran by free and immobilized dextransucrase

Dextransucrase from Leuconostoc mesenteroides was produced in a semicontinuous culture with slow addition of a concentrated sucrose solution. The resulting high activity of the fermentation broth allowed a one-step purification method, by gel permeation chromatography (GPC) in 96.4% yield. This procedure resulted in 140-fold purification, with specific activity of 122 U/mg. The enzyme was immobilized onto an amino-Spherosil support activated with glutaraldehyde. Preparations with dextransucrase activities as high as 40.5 U/g of support were obtained, when low specific area supports were used and maltose was added during the enzyme coupling. Diffusional limitations were found during enzyme reaction, as shown by a kinetic study. As a consequence of immobilization, the average molecular weight of dextrans seems to increase. Immobilized dextransucrase looks promising for low-molecular-weight dextran production. Clinical dextran was synthesized when the polysaccharides produced in the presence of maltose were used as acceptor of a second synthesis reaction. The molecular weight distribution of the resulting production was less disperse than when clinical dextran was produced by acid hydrolysis of high-molecular-weight dextran.     

Purification and characterization of a glycosyltransferase complex from the culture broth of Streptococcus mutans FA1.

The extracellular glycosyltransferases from Streptococcus mutans FA1 were purified by using the following procedures: ammonium sulfate precipitation, poly-(acrylamide) gel filtration, DEAE-cellulose chromatography, and agarose-gel filtration. The dextransucrase and levansucrase activities were purified 350- and 500-fold, repsectively, and the ratio of the two activities remained almost constant throughout the purification. Both enzymes have a pH optimum of 6.0, a Km for sucrose of 55mM, and isoelectric points of 3.7 and 4.6. The enzymes are inactivated by repeated freezing and thawing, but retain partial activity even after heating at 100 degrees. The enzyme preparation contains a carbohydrate moiety which does not appear to be either bound levan or dextran.     

Interactions of dextransucrase purified from Streptococcus mutans 890 with plant polyphenols

Plant polyphenols have been extensively studied for their chemopreventive properties for human health. Dextransucrase plays an essential role in synthesizing exopolysaccharides from its exclusive substrate sucrose in Streptococcus mutans. In the present study, the effect of polyphenols gallic acid and tannic acid was investigated on the dextransucrase activity. The enzyme was purified by ethanol precipitation followed by column chromatography by Sephadex G-200 gel chromatography, followed by PEG-400 treatment. The purified enzyme exhibited 52 fold enrichment with 17.5% yield and specific activity of 3.54 Units/mg protein. On SDS-PAGE enzyme protein gave a single band with a molecular weight of 160 kDa. Dextransucrase activity was inhibited 80–90% by 0.04 mM tannic acid (TA) or 0.4 mM gallic acid (GA) suggesting that tannic acid has 10- fold more inhibitory potential than gallic acid on the activity of dextransucrase. CD/ORD studies revealed modifications in the tertiary structure of enzyme protein in presence of tannic acid and gallic acid, which were further confirmed by fluorescence spectra of the protein in presence of tannic acid. These results suggest that inhibition of dextransucrase activity in S. mutans by polyphenols may have potential applications in the prevention and control of dental caries.     

The mechanism of dextransucrase action: Activation of dextransucrase from Streptococcus mutans OMZ 176 by dextran and modified dextran and the nonexistence of the primer requirement for the synthesis of dextran

Streptococcus mutans OMZ 176 was grown in a sucrose-free medium containing fructose as a carbohydrate source. Dextransucrase was precipitated from the culture supernatant with 40% saturated ammonium sulfate. The activity of dextransucrase was shown to be stimulated by exogenous dextran. Maximum activity was reached when the concentration of exogenous dextran was 2 mg/ml. Dextrans modified at the nonreducing ends by reaction with tripsyl chloride and/or by hydrolysis with an exodextranase also activated dextransucrase four to six times over that of a control. The exodextranasemodified dextrans have nonreducing chains that are very short in comparison with unmodified dextran and the tripsyl-modified dextrans have chains that are blocked at the nonreducing ends with a triisopropylbenzenesulfonyl group on the C₆ hydroxyl group. Because the nonreducing ends of the modified dextrans are not available for reaction, the activation of dextransucrase by these modified dextrans cannot be due to primer reactions with the nonreducing ends. The activation of dextransucrase, thus, must be by an alternate mechanism. Two alternative mechanisms discussed are an allosteric effect and nucleophilic displacement reactions by the added dextran. It was also found that the addition of increasing amounts of dextran shifted the synthesis from an insoluble dextran to a soluble dextran.     

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.     

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.     

Purification and immobilization of dextranase

An enzymic characteristic of Novo dextranase was presented. In addition to a high dextranolytic activity (7,200 U/ml), the crude enzyme also contained small amounts of protease, glucoamylase, polygalacturonase, carboxymethylcellulase, laminarinase and chitinase. A highly purified dextranase was then simply separated from a commercial preparation by column chromatographies on DEAE-Sepharose, CM-Sepharose, and by chromatofocussing on Polybuffer Exchanger PBE-94. The enzyme was recovered with an over 200-fold increase in specific activity and a yield of 84%. The final preparation was homogeneous, as observed during high performance liquid chromatography (HPLC). Size-exclusion HPLC indicated that dextranase had a molecular mass of 35 kDa and its isoelectric point, established by chromatofocussing, was 4.85. Analysis of the dextran break-down products indicated that purified dextranase represents an endolytic mode of action, and isomaltose and isomaltotriose were identified as the main reducing sugars of dextran hydrolysis. The enzyme was then covalently coupled to the silanized porous glass beads modified by glutaraldehyde (Carrier I) or carbodiimide (Carrier II). It was shown that immobilization of dextranase gave optimum pH and temperature ranges from 5.4 to 5.7 and from 50°C to 60°C, respectively. The affinity of the enzyme to the substrate decreased by a factor of more than 13 for dextranase immobilized on Carrier I and increased slightly (about 1.4-times) for the enzyme bound to Carrier II.     

Detection of dextransucrase and levansucrase on polyacrylamide gels by the periodic acid-Schiff stain: staining artifacts and their prevention

One use of the periodic acid-Schiff (PAS) stain is to detect dextransucrase and levansucrase activities on polyacrylamide gels by staining their polysaccharide products, dextran and levan. When gels with heavy dextran or levan bands were PAS stained, proteins other than dextransucrase and levansucrase also were stained, and a high background developed during storage. The staining of proteins other than dextransucrase and levansucrase is caused by the diffusion of the periodate-oxidized carbohydrate before and after staining. This diffusion could be greatly slowed, and the staining artifact decreased, by following the PAS stain by a crosslinking treatment of the carbohydrate-dye complex. Protein staining artifacts could be prevented by using chymotrypsin to remove the protein from the gel at the stage after polysaccharide synthesis but before the PAS stain.     

Preparation and characterization of a dextran-amylase conjugate

Bacillus amyloliquefaciens alpha-amylase was attached to dextran after activation of the polysaccharide by using a modification of the cyanogen bromide method. The soluble dextran-amylase conjugate was purified by molecular-sieve chromatography. The conjugated enzyme has greater stability than the unmodified enzyme at low pH values, during heat treatment, and on removal of calcium ions with a chelating agent. Attachment of dextran to alpha-amylase did not alter the Michaelis constant of the enzyme acting on starch. The polysaccharide-enzyme conjugate probably consists of a cross-linked aggregate of many dextran and many enzyme molecules, in which a proportion of the enzyme molecules, although not inactivated, are unable to express their activity, except after dextranase treatment.     

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.     

Origin and function of the multiple extracellular glucosyltransferase species from cultures of a serotype c strain of Streptococcus mutans

Two methods were used to purify the bifunctional extracellular enzyme sucrose: (1-6)- and (1-3)-alpha-D-glucan-6-alpha-D-glucosyltransferase (EC 2.4.1.5; dextransucrase) from continuous cultures of a serotype c strain of Streptococcus mutans. The first method, based on a previously published report, involved Sepharose 6B gel filtration and DEAE cellulose anion exchange chromatography. This resulted in a dextransucrase preparation with an apparent molecular mass of 162 kDa and a specific activity of 125 mg of glucan formed from sucrose h-1 (mg of protein)-1, at 37 degrees C. It was almost homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The ratio of carbohydrate to protein was 0.14 and the recovery was 14% relative to the total glucosyltransferase activity in the original culture fluid. In the subsequently preferred method, hydroxyapatite-Ultrogel was used to purify dextransucrase with a 24% yield. The specific activity, 197 mg of glucan formed h-1 (mg of protein)-1, was the highest yet reported and this preparation contained less than 0.5 glucose-equivalent per subunit of molecular mass 162 kDa. Dextransucrase is therefore not a glycoprotein. Exogenous dextran stimulated activity, but was not essential for activity. The purified protein slowly degraded to multiple lower molecular mass forms during storage at 4 degrees C and 87% of the activity was lost after 20 days. The molecular mass of the most prominent, active degradation product was 140 kDa, similar to that of one of the multiple forms of dextransucrase detected in other laboratories. Preparations in which either the 140-kDa or the 162-kDa species predominated catalyzed the synthesis of a water-soluble glucan with sucrose alone, but catalyzed that of an insoluble glucan with sucrose and a high concentration of either (NH4)2SO4 or polyethylene glycol. The water-insoluble glucan was shown to lack sequences of 1,3-alpha-linked glycosyl residues typical of the insoluble glucan, mutan, which has been implicated in dental caries. We conclude that mutan is synthesized by the concerted action of two independent glucosyltransferases rather than by interconvertible forms of a single enzyme, as was proposed previously.     

Dextransucrase as an Enzyme Associating with Alkaline Earth Metal Ions

Dextransucrase (EC. 2.4.1.5) of Leuconostoc mesenteroides was purified from the culture filtrate by precipitation with solid ammonium sulfate in the presence of egg white albumin followed by successively treating with columns of DEAE-cellulose and Bio Gel P-150.

The purified enzyme lost the activity upon dialysis against EDTA, and was reactivated by the addition of alkaline arth metal ions. The best reactivation was brought about by calcium ion. The enzyme inactivated by EDTA was unstable and readily denatured irreversibly. Several other properties of the purified enzyme were also investigated and discussed.     

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.     

Identification, effective purification and functional characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-640

The extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-640 was purified using polyethylene glycol fractionation (PEG) and gel-filtration. The cell free extract was subjected to fractionation by PEG-200, 400 and 1500. The 10% (w/v) PEG-1500 gave dextransucrase with maximum specific activity of 23 with 40 fold purification in a single step. The purified enzyme showed multiple molecular forms on SDS-PAGE, however the same sample showed a single band on non-denaturing native-PAGE. The purified dextransucrase fractions obtained from PEG-1500, confirmed the presence of dextran, when run on SDS-PAGE under non-denaturing gels for in situ activity detection by Periodic Acid Schiff's staining. The activity bands corresponded to the native and active form of the purified dextransucrase of approximately, 180kDa molecular size, that appeared on the denaturing gels stained with Coomassie Brilliant Blue. No bands appeared after staining the activity of dextransucrase on non denaturing SDS-PAGE gels with raffinose, which excluded the presence of fructosyltransferases. Further purification of 10% PEG-1500 purified dextransucrase by gel-filtration gave dextransucrase with specific activity of 35 with 61 fold purification.