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J F Robyt

Publications and source records attributed to J F Robyt.

At least 55 records · Page 3Linked to original sources
PubMed1985

13C-N.M.R. study of the conformation of helical complexes of amylodextrin and of amylose in solution.

Amylose (average d.p. 1000) and amylodextrin (average d.p. 25) have identical 13C-n.m.r. spectra, except for some minor signals from the small amount of alpha-1----6 branch linkages present in amylodextrin. Amylodextrin can be obtained as stable solutions in much higher concentrations than amylose and so requires only 1/100th as many scans to obtain a spectrum comparable to that of amylose. 13C-N.m.r. spectroscopy has been used to study the formation of amylodextrin complexes with organic complexing agents in aqueous solution. A control study using dextran, which does not form helical complexes, showed that, when complexing agents are added, the signals from all of the carbons show a slight downfield shift due to a general solvent effect. In the case of amylodextrin, the addition of increasing concentrations of complexing agent also produced a downfield shift of the signals of all the carbons, but there was a greater shift of the signals for carbons 1 and 4 than for carbons 2, 3, and 6, indicating that something more than a solvent effect was occurring. The cycloamyloses (cyclic alpha-1----4 linked D-glucose oligosaccharides which may be considered as model for an amylose helix) in water have chemical shifts for carbons 1 and 4 that are comparable to those shown by the amylodextrin complexes. It is thus proposed that the formation of a helical complex with amylodextrin results in a change in the conformation of the glycosidic linkage, which is reflected by greater downfield shifts of the signals for carbons 1 and 4, relative to those for carbons 2, 3, and 6. It was observed that differences in the ratio of the downfield shifts of C-1 and C-4 of the different amylodextrin complexes indicate differences in the degree of compactness of the helical structures. A comparison of the 13C chemical shifts of methyl alpha-D-glucoside and methyl alpha-maltoside showed that, for a molecule as small as a disaccharide, there is a conformational change about the glycosidic linkage when complexing agents are added.

Amylose↗
PubMed1985

The effect of substrate modification on porcine pancreatic alpha-amylase subsite binding: hydrolysis of substrates containing 2-deoxy-D-glucose and 2-amino-2-deoxy-D-glucose.

Modified alpha-D-(1----4)-glucans containing a small proportion of 14C-labeled 2-deoxy-D-glucose or 2-amino-2-deoxy-D-glucose were examined as substrates for porcine pancreatic alpha-amylase (PPA). Cyclomaltoheptaose containing single 2-deoxy-D-glucose residues, synthesized by incubation of 2-deoxyglucosylglycogen with cyclomaltodextrin glucanotransferase in the presence of Triton X-100, was hydrolyzed by PPA to produce 2-deoxy-D-glucose; two isomers of 2-deoxymaltose, and a mixture of modified maltotrioses. These results indicate that 2-deoxymaltose, and a mixture of modified maltotrioses. These results indicate that 2-deoxy-D-glucose may be productively bound at all five subsites of the PPA active site. Reaction kinetics and the distribution of products formed suggest, however, that productive binding of the modified residue does not occur readily at the point of catalytic attack (subsite 3) and that the preferred position of hydrolysis of modified substrates may be different from that of unmodified substrates. Results of PPA hydrolysis of glycogen containing [14C]-2-amino-2-deoxy-D-glucose showed that a modified trisaccharide and a modified disaccharide were the smallest substituted products formed. Analysis of these products indicated that they did not contain modified residues at their reducing ends. Formation of the observed 2-amino-2-deoxy-maltooligosaccharides is consistent with a scheme where productive binding of 2-amino-2-deoxy-D-glucose is allowed at subsites 1, 2, 4, and 5, but not at subsite 3, the subsite at which hydrolysis occurs.

Animals↗
PubMed1984

Porcine pancreatic alpha-amylase hydrolysis of hydroxyethylated amylose and specificity of subsite binding.

Hydrolysis of partially hydroxyethylated amylose by porcine pancreatic alpha-amylase gives rise to a number of hydroxyethylated di-, tri-, and tetrasaccharides, as well as larger products. No modified monosaccharides were detected. The structures of the products containing two to four D-glucose residues have been analyzed by chromatographic and enzymatic techniques. In no instance were these oligosaccharides modified in the reducing-end residue. The location of hydroxyethylated glucose residues within the oligosaccharides has been interpreted in terms of the ability of that (hydroxyethyl)glucose to bind productively at each of the five subsites of the enzyme active site. Results indicate that subsite 3, the subsite at which catalytic attack occurs, is especially sensitive to changes in the substrate and that unmodified glucose is required for productive binding at this subsite. Other subsites specifically allow binding of some (hydroxyethyl)glucose isomers, but not others. Hydroxyethylation is permitted at C-2, C-3, and C-6 for residues bound at subsite 1 and is permitted at C-6 and possibly at C-2 and C-3 for residues bound at subsite 5. However, substitution is permitted only at C-3 and C-6 for binding at subsite 2 and at C-2 and C-3 for binding at subsite 4.

Amylose↗
PubMed1984

Structure studies of amylose-V complexes and retrograded amylose by action of alpha amylases, and a new method for preparing amylodextrins.

Human-salivary, porcine-pancreatic, and Bacillus subtilis alpha amylases were used to study the structure of amylose-V complexes with butyl alcohol, tert-butyl alcohol, 1,1,2,2-tetrachloroethane, and 1-naphthol, and of retrograded amylose. Alpha amylase hydrolyzes the amorphous, folding areas on the surfaces of the lamella of packed helices, with the formation of resistant, amylodextrin fragments. Their degree of polymerization (d.p.) corresponds to the diameter of the helices and the folding length of the chain. The resistant fragments were fractionated on a column of Bio-Gel A-0.5m. Gel filtration of human-salivary and porcine-pancreatic alpha amylase hydrolyzates gave resistant fragments whose peak fractions, i.e., the three pooled fractions from the gel-filtration column with the highest amount of carbohydrate, had a d.p. of 75 +/- 4 for the amylose complex with butyl alcohol, 90 +/- 3 for those with tert-butyl alcohol and tetrachloroethane, and 123 +/- 2 for that with 1-naphthol. These d.p. values correspond to helices of six residues per turn with a folding length of 10 nm, seven residues per turn with a folding length of 10 nm, and eight residues per turn with a folding length of 12 nm (or nine residues per turn with a folding length of 10 nm), respectively. Acid hydrolysis of retrograded amylose gave a resistant fragment having an average d.p. of 32, human-salivary and porcine-pancreatic alpha amylases gave a resistant fragment of d.p. 43, and Bacillus subtilis alpha amylase gave a resistant fragment of d.p. 50. A structure for retrograded amylose is proposed in which there are crystalline, double-helical regions that are 10 nm long, interspersed with amorphous regions. The amorphous regions are hydrolyzed by acid and by alpha amylases, leaving the crystalline regions intact. The differences in the sizes of the resistant amylodextrins depend on the differences in the specificities of the hydrolyzing agents: acid hydrolyzes right up to the edge of the crystalline region, whereas the alpha amylases hydrolyze up to some point several D-glucosyl residues away from the crystalline region, leaving "stubs" on the ends of the amylodextrins whose sizes are dependent on the sizes of the binding sites of the individual alpha amylases.(ABSTRACT TRUNCATED AT 400 WORDS)

Alcohols↗
PubMed1984

The formation of alpha-D-(1----3) branch linkages by a D-glucansucrase from Streptococcus mutans 6715 producing a soluble D-glucan.

An exocellular D- glucansucrase that synthesizes a water-soluble, alpha-D-(1----6)-linked D-glucan having a high proportion of alpha-D-(1----3) branches was purified from the culture broth of Streptococcus mutans 6715. The rate of incorporation of D-[14C]glucose from [14C]sucrose into D-glucan of high molecular weight by this enzyme was increased (stimulated) by the presence of exogenous Leuconostoc mesenteroides B- 512F dextran, and it was found that this dextran could act as an acceptor. A highly branched dextran, containing 45-50% of alpha-D-(1----3) branch linkages, did not stimulate the enzyme nearly so much as B- 512F dextran, which has a low degree (5%) of alpha-D-(1----3) branches. We interpret this as evidence that the stimulating effects of dextran are not due to priming. If they were, the more highly branched dextran should have produced the greatest stimulation per unit weight, because a much greater number of nonreducing-end, priming sites would be available. We show that the D- glucansucrase was capable of transferring D-glucosyl groups from sucrose to B- 512F dextran to form alpha-D-(1----3) branches, thereby rendering the dextran more resistant to hydrolysis by endodextranase . The presence of 1.6M ammonium sulfate caused the enzyme to synthesize a D-glucan having a much higher percentage of alpha-D-(1----3) linkages.

Dextrans↗
PubMed1984

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.

Calcium↗
PubMed1983

Disproportionation reactions catalyzed by Leuconostoc and Streptococcus glucansucrases.

Glucansucrases from Leuconostoc mesenteroides NRRL B-512F and Streptococcus mutans 6715 were found to utilize a number of D-gluco-oligosaccharides as D-glucosyl donors and as acceptors. These donors included isomaltotriose and its homologs, panose, maltotriose, and dextran. In each case, D-glucosyl groups were transferred from the donor to an acceptor sugar. When the donor sugar also acted as an acceptor, disproportionation reactions occurred. Isomaltotriose, for example, gave rise to isomaltose and isomaltotetraose initially, and to a series of isomalto-oligosaccharides eventually. In addition to forming alpha-D-(1----6) linkages in the reactions, dextransucrase from S. mutans 6715 was capable of forming alpha-D-(1----3)-linked products.

Carbohydrate Conformation↗
PubMed1983

p-Nitrophenyl alpha-D-glucopyranoside, a new substrate for glucansucrases.

p-Nitrophenyl alpha-D-glucopyranoside has been shown to be a substrate for the glucansucrases of various strains of Leuconostoc mesenteroides and Streptococcus mutans. The products from a digest of p-nitrophenyl alpha-D-glucopyranoside with L. mesenteroides B-512F dextransucrase were found to include dextran, a series of p-nitrophenyl isomaltodextrin glycosides, and p-nitrophenyl nigeroside. The kinetics of the reaction were non-Michaelis-Menten, possibly because p-nitrophenyl alpha-D-glucopyranoside has a dual role in the reaction as both a D-glucosyl donor and acceptor.

Chromatography, Thin Layer↗
PubMed1983

Relative, quantitative effects of acceptors in the reaction of Leuconostoc mesenteroides B-512F dextransucrase.

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.

Glucose↗
PubMed1983

Mechanism of synthesis of D-glucans by D-glucosyltransferases from Streptococcus mutans 6715.

Two glucosyltransferases from Streptococcus mutans 6715 were purified and separated. One of the glucosyltransferases synthesized an insoluble glucan, and the other, a soluble glucan. The enzymes were immobilized on Bio-Gel P-2 beads, and the mechanism of glucan synthesis was studied by pulse and chase techniques with 14C-sucrose. Label was associated with the immobilized enzymes. The label could be quantitatively released by heating at pH 2. Analysis of the labeled products from the pulse experiment showed labeled glucose and labeled glucan; the chase experiment showed labeled glucan and a significant decrease in labeled glucose. The glucans from the pulse and the chase experiments were separated from glucose by chromatography on Bio-Gel P-6. They were reduced with sodium borohydride, and the products hydrolyzed with acid. Analysis of the labeled products from the reduced and hydrolyzed, pulsed glucans showed labeled glucose and labeled glucitol; label in the glucitol was greatly decreased in the chase experiment. These experiments showed that glucose and glucan were covalently attached to the active site of the enzymes during synthesis, and that the glucose was being transferred to the reducing end of the glucan chain. A mechanism for the synthesis of the glucans is proposed in which there are two catalytic groups on each enzyme that holds glucosyl and glucanosyl units. During synthesis, the glucosyl and glucanosyl units alternate between the two sites, giving elongation of the glucans from the reducing end. The addition of increasing amounts of B-512F dextran to the insoluble-glucan-forming glucosyltransferase produced a decrease in the proportion of insoluble glucan formed and a concomitant increase in a soluble glucan. The total amount of glucan synthesized (soluble plus insoluble) was increased 1.6 times over the amount of insoluble glucan formed when no exogenous dextran was added. It is shown that the addition of B-512F dextran affects the solubility of the synthesized alpha-(1 to 3)-glucan by accepting alpha-(1-3)-glucan chains at various positions along the dextran chain, to give a soluble, graft polymer.

Enzymes, Immobilized↗
PubMed1982

Isolation and partial characterization of an extracellular glucansucrase from Leuconostoc mesenteroides NRRL B-1355 that synthesizes an alternating (1 goes to 6), (1 goes to 3)-alpha-D-glucan.

Leuconostoc mesenteroides NRRL B-1355 grows on sucrose to produce two extracellular alpha-D-glucans. Although both are termed dextrans, they are chemically and physically distinct, and can be separated by fractional ethanol precipitation into fractions designated L and S. Fraction L is similar to B-512F dextran, having 95% alpha-(1 goes to 6) linkages and 5% alpha-(1 goes to 3) branch linkages, but fraction S has an alternating sequence of alpha-(1 goes to 6) and alpha-(1 goes to 3) linkages. Because of its structural differences from dextran, its different physical characteristics, and its resistance to hydrolysis by endodextranase, we have named glucan S, alternan, and the enzyme that synthesizes it from sucrose, alternansucrase. Alternansucrase has been isolated by two different methods. The first involves removal of the fraction L glucan from the culture fluid via hydrolysis by an endodextranase, followed by chromatography on Bio-Gel A5m. The void-volume fraction synthesizes only alternan, whereas the slower-migrating, second fraction synthesizes mainly dextran, together with some alternan. The second method utilized hydrophobic chromatography on O-(phenoxyacetyl) cellulose; a portion of the alternansucrase did not bind, whereas the bound portion, removed by eluting with detergent, contained both alternansucrase and dextransucrase. The glucans were identified by physical appearance, the concentration of ethanol required for precipitation, periodate-oxidation behavior, and susceptibility to hydrolysis by endodextranase. Also studied was the inhibition of the enzymes by 3-deoxy-3-fluoro-alpha-D-glucopyranosyl fluoride, tris(hydroxymethyl)aminomethane, 2-aminoethanol, and octyl beta-D-glucopyranoside.

Carbohydrates↗
PubMed1979

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

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.

Calcium Chloride↗
PubMed1979

Nature of the fructan of Streptococcus mutans OMZ 176.

The fructan of Streptococcus mutans OMZ 176 was shown to have a levan structure by comparing the chromatographic mobilities of the saccharides produced by partial acid hydrolysis of ghe fructan and known levan and inulin. This was confirmed by using concanavalin A as a lectin in a double-diffusion gel technique.

Chemical Phenomena↗
PubMed1978

The mechanism of acceptor reactions of Leuconostoc mesenteroides B-512F dextransucrase.

Reactions of dextransucrase and sucrose in the presence of sugars (acceptors) of low molecular weight have been observed to give a dextran of low molecular weight and a series of oligosaccharides. The acceptor reaction of dextransucrase was examined in the absence and presence of sucrose by using D-[14C]glucose, D-[14C]fructose, and 14C-reducing-end labeled maltose as acceptors. A purified dextransucrase was preincubated with sucrose, and the resulting D-fructose and unreacted sucrose were removed from the enzyme by chromatography of columns of Bio-Gel P-6. The enzyme, which migrated at the void volume was collected and referred to as "charged enzyme". The charged enzyme was incubated with 14C-acceptor in the absence of sucrose. Each of the three acceptors gave two fractions of labeled products, a high molecular weight product, identified as dextran, and a product of low molecular weight that was an oligosaccharide. It was found that all three of the acceptors were incorporated into the products at the reducing end. Similar results were obtained when the reactions were performed in the presence of sucrose, but higher yields of labeled products were obtained and a series of homologous oligosaccharides was produced when D-glucose or maltose was the acceptor. We propose that the acceptor reaction proceeds by nucleophilic displacement of glucosyl and dextranosyl groups from a covalent enzyme-complex by a specific, acceptor hydroxyl group, and that this reaction effects a glycosidic linkage between the D-glucosyl and dextranosyl groups and the acceptor. We conclude that the acceptor reactions serve to terminate polymerization of dextran by displacing the growing dextran chain from the active site of the enzyme; the acceptors, thus, do not initiate dextran polymerization by acting as primers.

Chromatography, Paper↗