No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Inhibition of dextransucrase by Zn2+, Ni2+, Co2+, and Tris(hydroxymethyl)aminomethane (Tris)
Abstract
Initial rate kinetics of polysaccharide formation indicate that Zn2+, Ni2+, and Co2+ inhibit dextransucrase [sucrose: 1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase, EC 2.4.1.5] by binding to two types of metal ion sites. One type consists of a single site and has a low apparent affinity for Ca2+. At the remaining site(s), Ca2+ has a much higher apparent affinity than Zn2+, Ni2+, or Co2+, and prevents inhibition by these metal ions. These findings are consistent with a two-site model previously proposed from studies with Ca2+ and EDTA. Initial rate kinetics also show that Tris is competitive with sucrose, but that, unlike Zn2+, Tris does not bind with significant affinity to a second site. This argues that there is a site which is both the sucrose binding site and a general cation site.
... A wide range of different substances has been found to inhibit GTF or the closely related dextransucrase of Leuconostoc mesenteroides [Ciardi et al., 1978;Vacca-Smith and Bowen, 1996;Wunder and Bowen, 1999]. These inhibitors may be broadly grouped into: ions such as Zn 2+ or small charged molecules like pyridine and amino sug-354 Caries Res 2002;36:353-359 Wright/Thelwell/Svensson/Russell ars [Thaniyavarn et al., 1981[Thaniyavarn et al., , 1982Miller and Robyt, 1986]; derivatives of sucrose, including a wide range of chemically synthesised deoxy-, thio-, amino-, methyl-or fluoro-sucroses [Binder and Robyt, 1986;Bombard et al., 1995;Simand et al., 1995]; sugar or pseudo-sugar analogues known to inhibit other glycosidases [Newbrun et al., 1983;Kim et al., 1998;Devulapalle and Mooser, 2000]; anionic and cationic detergents [Ciardi et al., 1978;Kawabata et al., 1993;Shani et al., 2000], and natural products such as polyphenols found in tea, cocoa or fruits [Nakahara et al., 1993;Matsumoto et al., 1999;Ooshima et al., 2000;Yanagida et al., 2000;Hamilton-Miller, 2001]. Amongst the wide range of substances tested, none has yet been identified that can be regarded as being both a potent inhibitor and highly specific for GTF: properties that are desirable if a substance is to suppress GTF sufficiently and to have a beneficial effect while having no adverse side effects by inhibiting other enzymes. ...
... Kinetic analysis of the inhibition of GTF-I by Tris revealed that it acts as a competitive inhibitor with a K i of 4.5 mM. Similar observations have been made with other GTF [Ciardi et al., 1978;Miller and Robyt, 1986]. Tris has been found to competitively inhibit a number of other glycosidases with a 1:1 stoichiometry of the enzyme-Tris complex and is tightly bound at the active site [Aghajari et al., 1998]. ...
The ability of a range of potential inhibitors to affect the catalytic activity or binding of dextran by a glucosyltransferase (GTF-I) that synthesises insoluble alpha1,3-linked glucan was tested. Acarbose, deoxynojirimycin, N-dodecyldeoxynojirimycin and Tris, which are thought to interfere with the active site of the enzyme of GTF and related glycosidases, inhibited glucan synthesis but not glucan binding. Tris was found to act as a competitive inhibitor of GTF-I. The effectiveness of the active site inhibitors was not altered by immobilisation of GTF-I on saliva-coated hydroxyapatite. In contrast, three amine hydrofluorides were markedly less effective against immobilised GTF than soluble GTF. The pH of the reaction mixture was found to have a strong influence on inhibition by acarbose, Tris and amine hydrofluorides, a finding that is of direct relevance to use of inhibitors in vivo.
... Te mixture was incubated for 30 minutes at 27°C. Phosphate bufer was used instead of Tris-HCl bufer due to the inhibitory efect of the latter on α-amylase [49,50] and other enzymes from the glycoside hydrolase family [51,52]. Te incubation was stopped by adding 500 μl of DNS reagent and boiling it for 10 minutes. ...
This work aimed to evaluate the effects of dietary phytic acid on the enzymatic activities of the hepatopancreas of the redclaw crayfish, Cherax quadricarinatus. For this purpose, a completely randomized in vitro trial was conducted with three phytic acid levels (0.56, 1.68, and 2.80%) and three phytase doses (0, 250, and 500 PU/kg DM). Solubilized protein, reducing sugars, and soluble phosphorus showed significant responses to the interaction between phytic acid and phytase p < 0.001 . Only the main effects were detected on the released amino acids, in keeping with the main effects of alkaline protease activity, which are negatively affected by phytic acid p < 0.001 and improved by phytase inclusion p < 0.001 . Differences in released reducing sugars were attributed to a reduction in amylase activity by increased levels of phytic acid and not to cellulase activity, where only a negative trend of phytic acid was found p = 0.068 . Phytic acid depresses calcium availability, which would explain the decrease in amylase activity. A 500 PU/kg DM dose improved amino acid, reduced sugars, and phosphorus release. These in vitro results might have in vivo implications for the digestibility of proteins, minerals, and energy. Further investigations are required to determine the chelated calcium effect on redclaw amylase activity, molting, and survival.
... The activation of dextransucrases by 1 mM Ca 2þ has already been reported by many sources, that is, Miller and Robyt. [24] But the Cu 2þ ions significantly inhibited enzyme activity, which has also been reported by Kralj et al. [25] for L. mesenteroides and L. reuteri Gtf. Figure S12 illustrated the glucansucrase production by DRP2-19 under conditions of different pH values. The glucansucrase production reached to 2.66 ± 0.12 U/mL after 30 hr at pH 6.0. ...
Strain DRP2-19 was detected to produce high yield of glucansucrase in MRS broth, which was identified to be Leuconostoc mesenteroides. In order for industrial glucansucrase production of L. mesenteroides DRP2-19, a one-factor test was conducted, then response surface method was applied to optimize its yield and discover the best production condition. Based on Plackett–Burman (PB) experiment, sucrose, Ca²⁺, and initial pH were found to be the most significant factors for glucansucrase production. Afterwards, effects of the three main factors on glucansucrase activity were further investigated by central composite design and the optimum composition was sucrose 35.87 g/L, Ca²⁺ 0.21 mmol/L, and initial pH 5.56. Optimum results showed that glucansucrase activity was increased to 3.94 ± 0.43 U/mL in 24 hr fermentation, 2.66-fold higher than before. In addition, the crude enzyme was purified using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of glucansucrase was determined as approximately 170 kDa by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified 15.77-fold and showed a final specific activity of 338.56 U/mg protein.
... The strain showed maximum glucansucrase production (4.70 AE 0.25 U mL À1 ) at 0.1 mM Zn 2+ (Fig. S15) (P < 0.05). The activation of dextransucrases by 1 mM Ca 2+ has already been reported by many sources, for example Miller and Robyt (1986). Cu 2+ ions significantly inhibited enzyme activity, which has also been reported by (Robyt and Walseth, 1979) for L. mesenteroides. ...
... The Eadie-Hofstee plots in Fig. 3 are typical of activity plots for metal-requiring enzymes, thus indicating that B-1118 DsrI is probably a calcium metalloenzyme. Many papers have appeared over the years describing the effect of calcium on the activity of dextransucrases from L. mesenteroides (e.g., Brock Neely and Hallmark 1961;Itaya and Yamamoto 1975;Lawford et al. 1979;Kobayashi et al. 1985;Miller and Robyt 1986;Malten et al. 2004;Naessens et al. 2005;Yi et al. 2009). Recently, two glucansucrases, one from Lactobacillus reuteri (Vujicic-Zagar et al. 2010) and one from Streptococcus mutans (Ito et al. 2011) have been crystallized, and both shown to contain a bound calcium atom near the active site. ...
We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K
M 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0–6.2. The enzyme was quickly inactivated at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-d-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked d-glucopyranosyl units and a low degree of branching.
... bitory effect on dextransucrase (Thaniyavarn et al., 1981; Received for publication January 20, 1992 Accepted for publication May 19, 1992 This investigation was supported in part by USPHS Research Grant DE07907 from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, and the Fogarty Fellowship F05 TW04389. Miller and Robyt, 1986). Delmopinol hydrochloride (Decapinol) is an example of a surface-active morpholinoethanol derivative [3-(4- propylheptyl)-4-morpholinoethanol hydrochloride] which reduces plaque formation (Simonsson et al., 1991b) and reduces gingivitis in human volunteers (Collaertet al., 1992), though itwas found to have alowin vivo antimicrobial act ...
The prolonged retention of an effective chemotherapeutic agent on oral surfaces and in dental plaque aids in plaque control. The objective of this study was to investigate interactions between delmopinol, a morpholinoethanol derivative, and experimental pellicle. Hydroxyapatite beads were coated with different constituents of pellicle (e.g., saliva, carbohydrates, cell-free enzymes, and bacteria). Delmopinol demonstrated a higher affinity for saliva-coated hydroxyapatite (sHA) and for experimental pellicle coated with in situ-synthesized glucans than for untreated hydroxyapatite. High-molecular-weight (MW) dextran but not low-MW dextran interfered with the adsorption of delmopinol to sHA. Delmopinol did not compete with dextran for the same binding sites on sHA, nor did it compete with saliva for the same binding sites on untreated hydroxyapatite. Delmopinol inhibited the activity of cell-free fructosyltransferase adsorbed onto sHA. In addition, synthesis of glucans by Streptococcus mutans adsorbed onto sHA was significantly reduced in the presence of delmopinol.
Influence of temperature, metal ions and organic solvents on the behavior of extracellular glucansucrase, produced by Leuconostoc mesenteroides AA1 was studied. Glucansucrase is stable at 30°C and 35°C for 1 hour, whereas loss in enzyme activity was observed at 40°C and 45°C 67 % and 93 % respectively. The enzyme is strongly inhibited by Hg 2+ while Cd 2+, Fe 2+, Ba 2+, Na +, K + and Mg 2+ caused moderate inhibition at low concentration but complete inhibition was observed at concentration higher than 5mM. Mn 2+ and Co 2+ less affected the enzyme and more than 90 % of its activity was detected even at 10mM. In the presence of Ca 2+ and Ni 2+, the enzyme also retained activity at 1mM and 5mM concentration respectively. It was observed that at different solvent concentrations glucansucrase showed different % relative activity. 50 % relative glucansucrase activity was detected in 20 % DMSO, 50 % 2-propanol, 10 % ethanol and 20 % acetone where as complete loss of activity was detected in ethanolamine.
Recently, it was affirmed that the exopolysaccharides (EPSs) of Lactobacillus curvatus TMW 1.624, Lactobacillus reuteri TMW 1.106 and Lactobacillus animalis TMW 1.971 improve the quality of gluten-free breads and that they can be produced in situ to levels enabling baking applications. In this study we provide insight into the molecular and biochemical background of EPS production of these three strains. EPS formation strongly correlated with growth and took place during the exponential phase. Gtf genes were heterologously expressed, purified and their enzymatic properties as well as the structures of the EPSs formed were compared. Structural comparison of EPS formed by heterologously expressed glucosyltransferases (Gtfs) and of those formed by the wildtype lactobacilli confirmed that the respective genes/enzymes were identified and examined. The glucan formed by L. animalis Gtf was identified as a linear low molecular weight dextran. Optimal enzymatic conditions were pH 4.4 and 45 °C for the L. reuteri Gtf and pH 4.4 and 31 °C for L. curvatus Gtf. The Gtf from L. animalis had an optimal pH of 5.8 and displayed more than 50% of activity over a broad temperature profile (22-59 °C). The three Gtfs were stimulated by various mono- and divalent metal ions, dextran, as well as levan to different extents.
A new genetically derived variant of the glucosyltransferase from Streptococcus oralis has been characterized physicochemically and kinetically. Compared with the industrially used glucosyltransferase from Leuconostoc mesenteroides, the enzyme variant GTF-R S628D possesses 25 times higher affinity for the specific glucosylation of glucose. For a concept of integrated reaction and product isolation, a fluidized bed reactor with in situ product removal was applied. The technical feasibility and the applicability of the kinetic models for reaction and adsorption could be demonstrated. The immobilized enzyme was stable (20% activity loss after 192 h) and product could be obtained with 90% purity. A bioprocess model was generated which allowed the integral assessment of the enzymatic synthesis and in situ product adsorption. The model is a powerful tool which assists with the localization of optimal process parameters. It was applied for the process evaluation of other glucosyltransferases and demonstrated key characteristics of each enzymatic system.
Mukerjea and Robyt [Carbohydr. Res. 2012, 352, 137-142] showed that a primer-free potato starch-synthase synthesized starch chains de novo, without the addition of a primer. A dichotomy arises as to why 61 studies from 1964 to the present have had to add a carbohydrate primer to obtain starch-synthase activity. All of these studies used 25-100 mM Tris, Bicine, or Tricine buffers. We have found that the Tris-type buffers completely inhibit starch-synthase at these concentrations. The addition of 10 mg/mL of the putative primers, glycogen and maltotetraose, gave a partial reversal of the inhibition, with glycogen being better than maltotetraose. It has been found that the Tris-type buffers form a complex with the ADPGlc substrate, removing it from the digest, causing the inhibition. The addition of the putative primers releases some of the ADPGlc from the complex, permitting it to act as a substrate for starch-synthase. The study definitively shows that the need for primers for starch-synthase by many investigators from 1964 to the present has been caused by Tris-type buffer inhibition that was partially reversed by putative primers. This has led to the perpetuation of the primer myth for the biosynthesis of starch chains by starch-synthase.
The aim was to explore the effects of delmopinol, a substituted amino-alcohol compound recently reported as a potential antiplaque agent, on GTF activity in solution and when adsorbed on to sHA. Delmopinol was without a significant effect on GTF activity in solution. In contrast, a reduction in the bound glucans synthesized by the adsorbed GTF was found in the presence of delmopinol. Delmopinol did not displace the adsorbed GTF from the sHA, nor was there significant desorption of glucans from sHA. The total glucan synthesis (bound and unbound) was reduced in the presence of delmopinol. Inhibition of GTF was not reversed by sucrose. Inhibition of GTF activity by delmopinol apparently results from drug-enzyme interaction on the surface of sHA beads. These observations provide further support for the important differences in the properties of adsorbed GTF and GTF in solution, illustrating that GTF-drug interaction differs between enzyme adsorbed to surfaces and enzyme in solution.
Treatment of Leuconostoc mesenteroides B-512F dextransucrase with diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees or photo-oxidation in the presence of Rose Bengal or Methylene Blue at pH 6.0 and 25 degrees, caused a rapid decrease of enzyme activity. Both types of inactivation followed pseudo-first-order kinetics. Enzyme partially inactivated by DEP could be completely reactivated by treatment with 100 mM hydroxylamine at pH 7 and 4 degrees. The presence of dextran partially protected the enzyme from inactivation. At pH 7 or below, DEP is relatively specific for the modification of histidine. DEP-modified enzyme showed an increased absorbance at 240 nm, indicating the presence of (ethoxyformyl)ated histidine residues. DEP modification of the sulfhydryl group of cysteine and of the phenolic group of tyrosine was ruled out by showing that native and DEP-modified enzyme had the same number of sulfhydryl and phenolic groups. DEP modification of the epsilon-amino group of lysine was ruled out by reaction at pH 6 and reactivation with hydroxylamine, which has no effect on DEP-modified epsilon-amino groups. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm, also indicating that histidine had been oxidized, and no decrease in the absorbance at 280 nm, indicating that tyrosine and tryptophan were not oxidized. A statistical, kinetic analysis of the data on inactivation by DEP showed that two histidine residues are essential for the enzyme activity. Previously, it was proposed that two nucleophiles at the active site attack bound sucrose, to give two covalent D-glucosyl-enzyme intermediates. We now propose that in addition, two imidazolium groups of histidine at the active site donate protons to the leaving, D-fructosyl moieties. The resulting imidazole groups then facilitate the formation of the alpha-(1----6)-glycosidic linkage by abstracting protons from the C-6-OH groups, and become reprotonated for the next series of reactions.
The kinetic mechanism of dextransucrase was studied using the Streptococcus mutans enzyme purified by affinity chromatography to a specific activity of 36.9 mumol/min/mg of enzyme. In addition to dextran synthesis, the enzyme catalyzed sucrose hydrolysis and isotope exchange between fructose and sucrose. The rates of sucrose hydrolysis and dextran synthesis were partitioned as a function of dextran concentration such that exclusive sucrose hydrolysis was observed in the absence of dextran and exclusive dextran synthesis at high dextran concentrations. An analogous situation was observed with fructose-dependent partitioning of sucrose hydrolysis and fructose exchange. Steady state dextran synthesis and fructose isotope exchange kinetics were simplified by assay at dextran or fructose concentrations high enough to eliminate significant contributions from sucrose hydrolysis. This limited dextran synthesis assays to dextran concentrations above apparent saturation. The limitation was diminished by establishing conditions in which the enzyme does not distinguish between dextran as a substrate and product which allowed initial discrimination among mechanisms on the basis of the presence or absence of dextran substrate inhibition. No inhibition was observed, which excluded ping-pong and all but three common sequential mechanisms. Patterns of initial velocity fructose production inhibition and fructose isotope exchange at equilibrium were consistent with dextran synthesis proceeding by a rapid equilibrium random mechanism. A nonsequential segment was apparent in the exchange reaction between fructose and sucrose assayed in the absence of dextran. However, the absence of detectable glucosyl exchange between dextrans and the lack of steady state dextran substrate inhibition indicate that glucosyl transfer to dextran must occur almost exclusively through the sequential route. A review of the kinetic constants from steady state dextran synthesis, fructose product inhibition, and fructose isotope exchange showed a consistency in constants derived from each reaction and revealed that dextran binding increases the affinity of sucrose and fructose for dextransucrase.
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.
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 irreversity. Several other properties of the purified enzyme were also investigated and discussed.
Dextransucrase (Sucrose: 1, 6-α-D-glucan 6-α-glucosyltransferase, EC 2. 4. 1. 5) produced by Leuconostoc mesenteroides IAM 1046 was highly purified, its properties examined, and the structure of dextrans formed from sucrose by the enzyme was studied. The periodate oxidation of enzymatically synthesized dextrans showed that the content of α-1, 6-glucosidic bonds in dextran was increased as the purification of the enzyme proceeded. Moreover, it was found that dextrans with higher proportion of non-α-l, 6-glucosidic bonds were obtained when some fraction from culture broth was added to purified enzyme on enzymatic synthesis. Results indicated that some factor (s) tentatively referred to as “branching factor” was involved in the synthesis of non-1, 6-links. Nature of the factor was examined, and it was found that calcium and other divalent metal ions had the effect as the branching factor.
Basic N-substituted β-d-galactosylamines are bound to the active site of β-d-galactosidase from E. coli > 102–104-fold more tightly than non-basic, neutral or positively charged β-d-galactosyl derivatives of closely related structure. Acid catalysis of O-galactoside hydrolysis and tight binding of basic inhibitors is observed only when Mg2+ is bound to the enzyme. Formation of an ion pair by proton transfer from the acidic group required for catalysis to the bound galactosylamine is therefore proposed as the cause of the enhanced affinity. The magnitude of the observed effects indicates strong shielding of the active site from the aqueous environment. The pH-dependence of Ki for 1-d-galactosylpiperidine and d-galactosyl-benzene shows that no deprotonation from the active site occurs up to pH 10. The hydrophobic nature of the aglycon site inferred from earlier studies is confirmed by the high affinity of N-benzyl- (Ki 9 × 10−9M) and N-heptyl-β-d-galactosylamine (Ki 0.6 × 10−9M). A model for the active site is discussed, in which the charge of an essential carboxylate group is neutralised by an adjacent cationic group.
Dextransucrase was shown to catalyze the hydrolysis of sucrose. The hydrolytic activity was found to be directly correlatable with dextransucrase activity on poly-(acrylamide) disc-gel electrophoresis. In studies on the hydrolysis of sucrose and formation of dextran as a function of time and substrate concentration, the two activities were found to be competitive with each other. Competition was also observed between hydrolysis and the transfer of d-glucosyl groups to added acceptors. The results suggest that the three activities, namely, polymerization, d-glucosyl transfer, and hydrolysis, compete for a form of the enzyme that is common to all three reactions. It is proposed that this form may be a d-glucosylated derivative of the enzyme.
Streptococcus mutants 6715 was grown in trypticase soy broth and chemically defined media. When compared by cellular mass, DNA content, acid production, or glucosyltransferase (GTase) production, the growth parameters were nearly identical. The doubling time for the organism grown in either medium was approximately 75 min. The extracellular glucosyltransferase produced byS. mutans 6715 grown in both media was purified from the culture supernatant with nearly total recovery and a degree of purification approximately 74-fold. Apparent proteolytic degradation of the enzyme was prevented by nitriloacetate. The temperature effects showed typical loss of enzymatic activity from 37 to 60C. When the GTase was heated above 60C there was partial restoration of activity. Immunological studies were used to establish the relationship between the enzymatically active proteins separated by gel filtration chromatography.
Dextransucrase [EC 2.4.1.5] activity from cell-free culture supernatant of Leuconostoc mesenteroides NRRL B-1299 was purified by (NH4)2SO4 fractionation, adsorption on hydroxyapatite, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The extracellular enzyme was separated into two principal forms, enzymes I and N, and the latter was shown to be an aggregated form of the protomer, enzyme I. Enzymes I and N were both electrophoretically homogeneous and their relative activities reached 820 and 647 times that of the culture supernatant, respectively. On sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, enzyme N dissociated into the protomer enzyme I, with a molecular weight of 48,000. Enzyme I was gradually converted into enzyme N upon aging, and this conversion was stimulated in the presence of NaCl. The optimum pH and temperature of enzyme I activity were pH 6.0 and 40 degrees, respectively, while those of enzyme N were pH 5.5 and 35 degrees. The Km values of enzymes I and N were 13.9 and 13.1 mM, respectively. Ca2+, Mg2+, Fe2+, and Co2+ stimulated the activity of enzyme N, and EDTA showed a potent inhibitory effect on this enzyme. Moreover, the activity of enzyme N was more effectively stimulated by exogenous dextrans as compared with enzyme I.
The intensity of staining of protein zones in polyacrylamide gels by Coomassie brilliant blue G250 in perchloric acid solution was increased by a factor of 3 when a wash of 5% acetic acid followed staining. Concentrations as low as 5 ng of human serum albumin could be detected in the gels.
The production of dextransucrase from Leuconostoc 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-Fm. 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. Concanavalin 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.
The glycosyltransferases of S. mutans strain Ingbritt have been resolved by SDS-polyacrylamide gel electrophoresis, followed by incubation in the presence of non-ionic detergent to restore enzyme activity. A group of high molecular weight proteins synthesizing glucans has been identified, as well as three distinct fructan-synthesizing activities. The glucan-forming enzymes have been purified by affinity chromatography on insoluble glucan, followed by gel chromatography in SDS, and antiserum to the purified enzymes has shown that they are antigenically identical within serotypes c, e and f, and cross-react strongly with serotype b.
Dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-glucosyltransferase EC 2.4.1.5) activity of Leuconostoc mesenteroides NRRL B-1299 cells has been purified by adsorption on hydroxyapatite, followed by chromatographies on DEAE-cellulose and DEAE-Sephadex. The enzyme activity was readily separated into two principal forms of the enzyme, I and II, by DEAE-cellulose chromatography. Both enzymes I and II were purified to an electrophoretically homogeneous state in which the relative enzyme activities reached 32-and 14-fold of the original specimen, respectively. Molecular weights were 69 000 for the enzyme I and 79 000 for the enzyme II as determined by electrophoresis in sodium dodecyl sulphate-polyacrylamide, and no subunit structure was observed. Enzyme I had an optimum pH at 6.3-6.5 and exhibited a maximal activity at 45 degrees C, while the optimum pH and temperature of enzyme II were pH 5.5-5.9 and 35-40 degrees C. The Km values of enzymes I and II were 10.7 and 250 mM, respectively. The effects of several metal ions, chemical reagents, and addition of various dextrans were also examined. Beside linear alpha-1,6-linkages the polymer synthesized by the enzyme II contained lesser amount of alpha-1,2-and alpha-1,3-linkages, which seems to be a primary characteristic of the B-1299 dextran.
The experimental variance of enzymic steady-state kinetic experiments depends on velocity as approximated by a power function (Var(v) = K1 . valpha (Askelöf, P., Korsfeldt, M. and Mannervik, B. (1976) Eur. J. Biochem. 69, 61--67). The values of the constants (K1, alpha) can be estimated by making replicate measurements of velocity, and the inverse of the function can then be used as a weighting factor. In order to avoid measurement of a large number of replicates to establish the error structure of a kinetic data set, a different approach was tested. After a preliminary regression using a 'good model', which satisfies reasonable goodness-of-fit criteria, the residuals were taken to represent the experimental error. The neighbouring residuals were grouped together and the sum of their mean squared values was used as a measure of the variance in the neighbourhood of the corresponding measurements. The values of the constants obtained in this way agreed with those obtained by replicates.
The formation of water-insoluble glucan by extracellular glucosyltransferase from Streptococcus mutans 6715 found to be greatly stimulated by various mono- or divalent cations. An enzyme preparation, obtained by ethanol fractionation, was able to catalyze the formation of water-insoluble glucan from sucrose in the presence of monovalent cations above 100mM or divalent cations above 20 mM at neutral pH. As the concentration of monovalent and divalent cations was reduced to below 10 mM and 1 mM, respectively, the formation of insoluble glucan decreased to a negligible amount. High concentrations of these cations were found to stimulate the formation of insoluble glucan in the following ways: (i) it increased the activity of total glucosyltransferase up to 1.6- and 2.7-fold in the absence and presence of a primer dextran, respectively, and (ii) it changed the formation of soluble glucan to insoluble. It was postulated that one of the essential factors for the formation of insoluble glucan would be to keep more than two water-soluble glucan chains close to enzyme aggregates and that such interaction could be enhanced by the presence of high cation concentrations.
Dextrans were first used in clinical practice in 1947. Raw dextran is produced by bacterial conversion of sucrose to a branched chain structure which is heterogeneous in composition. Further refinement is carried out by hydrolysis and fractionation. Clinically useful dextrans form only 1 percent of the total molecular spectrum of raw dextran. The two most frequently used dextrans are Dextran 40 and Dextran 70. These have molecular weights of 40,000 and 70,000 respectively. The use of average molecular weight to describe dextrans is generally unsatisfactory since an infinite variety of dextrans could meet a particular weight specification. Categorization of dextrans will be discussed in detail. Clinical uses include plasma expansion, antithrombogenesis and haemodilution. Major efforts to reduce the antigenically active components of clinically used dextrans have been made and manufacturers have identified the principal factors responsible for allergic responses. However, major allergic responses to dextran still cause clinicians great concern and the problem is by no means resolved.
A procedure has been developed whereby native and proteolyzed forms of dextransucrase have been purified; it involves gel filtration, and hydroxylapatite chromatography in the presence of 0.10% sodium dodecyl sulfate. This procedure is highly reproducible, and permits approximately 30% recovery of high purity (94% homogeneous) enzyme as an inactive, SDS complex that can be reactivated by the addition of Triton X-100. The purified enzymes have been compared with regard to amino acid compositions, and isoelectric and catalytic properties. An analysis of the structure of their product D-glucans was also made. Although the structural characteristics of the enzyme forms differ, proteolysis does not cause alterations in their catalytic properties.
Initial rate kinetics of dextran synthesis by dextransucrase (sucrose:1,6-alpha-D-glucan-6-alpha-D-glucosyltransferase, EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F showed that below 1 mM, Ca2+ activated the enzyme by increasing Vmax and decreasing the Km for sucrose. Above 1 mM, Ca2+ was a weak competitive inhibitor (Ki = 59 mM). Although it was an activator at low concentration, Ca2+ was not required for dextran synthesis, either of main chain or branch linkages. Neither was it required for sucrose hydrolysis, acceptor reactions, or enzyme renaturation after SDS-polyacrylamide gel electrophoresis. A model for dextran synthesis is proposed in which dextransucrase has two Ca2+ sites, one activating and one inhibitory. Ca2+ at the inhibitory site prevents the binding of sucrose.
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.
Robyt et al. have proposed a mechanism for dextransucrase in which dextran is synthesized by the cooperative action of two equivalent nucleophiles (Robyt, J.F., Kimble, B.K. and Walseth, T.F. (1974) Arch. Biochem. Biophys. 165, 634-640). To distinguish between the possibilities that the enzyme is a monomer bearing both nucleophiles, or a dimer with each subunit bearing one nucleophile, the molecular weight of the enzyme was determined by SDS-polyacrylamide gel electrophoresis and by radiation inactivation. Two major forms of dextransucrase from Leuconostoc mesenteroides NRRL B-512F were found on SDS-polyacrylamide gel electrophoresis, with Mr 177 000 and 158 000, and sometimes a minor form with Mr 168 000. No form of dextransucrase smaller than Mr 158 000 was found, either in the presence or absence of dextran T10, although levansucrase was detected at Mr 92 000 and 116 000. On irradiation with 60Co, dextransucrase behaved as a single species with a maximum size of Mr 201 000. Because Mr 201 000 is much smaller than the minimum dimer size of Mr 316 000 (= 2 X 158 000), it is concluded that both nucleophiles are probably located on the same peptide, rather than one on each subunit of a dimer, and that peptide association is probably not required for dextran synthesis.
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.
The mechanism of inhibition of the two glucoamylases from a Rhizopus sp. and Aspergillus saitoi by aminoalcohol derivatives was investigated.
1. Hydrolysis of maltose by the glucoamylases was inhibited competitively by aminoalcohols at pH 5.0, and tris(hydroxymethyl)aminomethane, 2-amino-2-ethyl-1,3-propanediol and 2-aminocyclohexanol were relatively good inhibitors of the glucoamylases among the aminoalcohol derivatives tested.
2. One hydroxyl group and an amino group in these inhibitors were indispensable for the inhibitory action, and the addition of other hydroxyl, amino or ethyl groups was enhancing.
3. With an increase in pH from 4.0 to 6.0, the K1 values of the aminoalcohols decreased. This result suggested the participation of a carboxyl group, which was related to the glucoamylase activity and had a pKa of 5.7, in the binding of aminoalcohols.
4. The UV difference spectra induced on binding of the aminoalcohol analogues with the glucoamylases may indicate a change of the environment of tryptophan residues to a slughtly higher pH on inhibitor binding.
5. The influence of aminoalcohols on the fluorescence intensity due to tryptophan residues and the CD-spectra of the glucoamylases was less than that of maltitol. Thus, the interaction of aminoalcohols with tryptophan residues in the glucoamylases might be less pronounced than that in the case of substrate analogues.
6. The modes of binding of the aminoalcohols with the two glucoamylases were very similar. Therefore, the phenomenon might be a common feature of glucoamylases in general.
1-Aminoglycosides represent a new class of specific and relatively potent inhibitors of glycosidases. These compounds are specific against those enzymes which act upon glycosides that correspond to glycone of the inhibitor. Thus α- and β-- are inhibited by -glucosidases but not by -galactosylamine and -mannosylamine. α- and -galactosidases are inhibited by -galactosylamine but not by the other two glycosylamines. -Mannosylamine inhibits mannosidase.
Steady‐state analysis of product inhibition shows that the most probable kinetic mechanism of intestinal sucrase is ping‐pong bi‐bi (ordered uni‐bi for hydrolysis alone).
The site of activation by Na ⁺ was tentatively localized at a level prior to the further transformation of the “glucose”· enzyme complex.
Tris(hydroxymethyl)aminomethane competes with the substrate for the glucose subsite.
In the appendix a simple kinetic text is described for detecting or ruling out a mutual competition between inhibitors of different or of identical kinetic type.
The dextransucrase (EC 2.4.1.5) activity from cell-free culture supernatants of Streptococcus mutans strain 6715 has been purified approximately 1,500-fold by ammonium sulfate precipitation, hydroxylapatite chromatography, and isoelectric focusing. The enzyme was eluted as a single peak of activity from hydroxylapatite, and isoelectric focusing of the resulting preparation gave a single band of dextransucrase activity which focused at a pH of 4.0. The final enzyme preparation contained two distinct, enzymatically active proteins as judged by assay in situ after polyacrylamide gel electrophoresis. One of the proteins represented 90% of the total dextransucrase activity and 53% of the total protein. The molecular weight of the enzyme was estimated by gel filtration to be 94,000. The temperature optimum of the enzyme was broad (34 to 42 C) and its pH range was rather narrow, with optimal activity at pH 5.5. The K(m) for sucrose was 3 mM, and fructose competitively inhibited the enzyme reaction with a K(i) of 27 mM.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
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.
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.
The acceptor reaction of dextransucrase consists of the transfer of D-glucosyl groups from sucrose to other carbohydrates, and occurs at the expense of dextran synthesis. In the present study, solutions of [14C]sucrose and of each of seventeen acceptor sugars were digested with highly purified Leuconostoc mesenteroides B-512F dextransucrase. The products were separated by paper chromatography, and quantitated by liquid scintillation counting. Maltose was the most effective acceptor; its products, members of an isomaltodextrinyl-maltose series (d.p. 3 to 6), accounted for greater than 75% of the D-glucosyl groups of sucrose. Other acceptors giving rise to a similar series of oligosaccharide products were (in order of decreasing effectiveness): isomaltose, nigerose, methyl alpha-D-glucoside, 1,5-anhydro-D-glucitol, D-glucose, turanose, methyl beta-D-glucoside, cellobiose, and L-sorbose. Lactose, raffinose, melibiose, D-galactose, and D-xylose each gave a single, mono-D-glucosylated product; D-fructose and D-mannose each gave a pair of mono-D-glucosylated (disaccharide) products. Another series of digests contained sucrose and various proportions of maltose. As the level of maltose increased, the size of the largest oligosaccharide acceptor-product decreased, and less dextran was produced. The virtual absence of high-d.p. (8 to 13) oligosaccharide products in all acceptor digests is interpreted as evidence against a role for acceptors as primers of dextran synthesis.
Multiple forms of purified dextransucrase have been observed in the presence of low detergent concentrations ( Luzio , G.A., Grahame , D. A. and Mayer, R.M. (1982) Arch. Biochem. Biophys. 216, 751-757). We now show these forms to arise partly as a result of proteolysis, and partly due to incomplete dissociation of the enzyme. Upon 25 degrees C incubation of the crude enzyme, several new bands appeared with little or no change in total activity. The electrophoretic pattern of aged, crude enzyme was similar to that of partially purified enzyme. Specific detection of dextransucrase on SDS gels revealed a single polypeptide of 174 kDa, which is converted to a 156 kDa protein during the aging process. The observation indicates the occurrence of proteolysis. The polypeptide composition of several of the enzyme forms was determined by two-dimensional electrophoresis. Forms Ia and IIa are composed exclusively of 174 kDa polypeptides. Forms III and IVa consist of 156 kDa units, as does the newly observed form Ic. It is likely that form Ib contains both 174 and 156 kDa polypeptides. The results indicate that incomplete dissociation of aggregates of the 174 kDa unit accounts for all of the bands observed on native gels run on fresh culture extracts. Additional enzyme forms result from aggregation of the 156 kDa proteolysis product alone, and from aggregation with unproteolyzed units to form hybrid aggregates.
DEXTRANSUCRASE is a representative member of the general class of enzymes known as transglycosidases. In this particular example, the glucosyl group from sucrose is transferred to a suitable acceptor, which causes chain initiation, and dextran is formed. Polymer-formation is governed by many factors such as the nature of the acceptor molecule, pH and temperature1.