John F. Robyt

MINIATURIZATION OF THREE CARBOHYDRATE ANALYSES USING A MICROSAMPLE PLATE READER

Three carbohydrate analyses (reducing value by copper-bicinchoninate, total carbohydrate by phenol-sulfuric acid, and D-glucose by glucose oxidase) have been miniaturized using a microsample plate reader. The use of the reducing-value procedure to measure the hydrolysis of starch by alpha-amylase and the use of the glucose oxidase method to measure the hydrolysis of lactose by lactase are illustrated.

Multiple attack hypothesis of α-amylase action: Action of porcine pancreatic, human salivary, and Aspergillus oryzae α-amylases

Abstract Three conceptual action patterns of α-amylase hydrolysis of amylose have been considered: single chain, multichain, and multiple attack. To test these concepts, curves were obtained relating the drop in amylose-iodine color to the increase in reducing value for amylolysis by human salivary (HS), porcine pancreatic (PP), Aspergillus oryzae (AO) α-amylases, and 1 m H 2 SO 4 . The observed differences in the curves for the amylases could only be interpreted as due to differences in degree of multiple attack. To test these concepts further, amylase digests at various stages of hydrolysis were separated by ethanol precipitation into polysaccharide and oligosaccharide fractions. The degree of multiple attack was determined from the ratio of the reducing value of the oligosaccharide fraction to that of the polysaccharide fraction. Under optimal conditions of pH and temperature, PP had a degree of multiple attack of 6, three times that of HS or AO. At pH 4.5, the degree of multiple attack of PP did not change, although its activity was reduced 10-fold. At pH 10.5, however, the degree of multiple attack was reduced to 0.7, approaching a multichain pattern.

Action pattern and specificity of an amylase from Bacillus subtilis

Abstract The nature, amounts, and sequence of products formed from well-characterized substrates by the action of a crystalline α-type amylase from Bacillus subtilis were determined by qualitative and quantitative paper chromatography. The substrates studied were amylose, amylopectin, glycogen, β-amylase limit dextrins, pure individual maltodextrins, and the cyclic Schardinger dextrins. The amylase from B. subtilis showed a dual product specificity for the formation of maltotriose and maltohexaose. This dual specificity was quite pronounced when amylopectin was the substrate. The distributions of products from the interior segments of amylopectin and glycogen were completely different from the distribution of products obtained from amylose. The reactions of the maltodextrins were highly specific and dependent upon the molecular size of the dextrin. From these studies, a mechanism for the dual product specificity and action on branched substrates is proposed.

The mechanism of dextransucrase action: Direction of dextran biosynthesis

Abstract The mechanism for the biosynthesis of dextran by dextransucrase from Leuconostoc mesenteroides NRRL-B512F has been studied. Dextransucrase attached to cells and dextransucrase insolubilized on Bio-Gel beads have been obtained. It was found that these forms become labeled when incubated with a pulse of [ 14 C]sucrose. The label can be released from cells and from Bio-Gel by adjusting the pH to 2 and heating to 95 °C for 10 min. Two types of labeled carbohydrates were found to be released, glucose and dextran. The direction of the biosynthesis was determined by pulse and chase experiments with [ 14 C]sucrose. The labeled dextran was isolated, reduced, and acid hydrolyzed. The hydrolytic products, glucose and sorbitol, were separated by paper chromatography, and their radioactivities were determined. The ratio of the labeled sorbitol to labeled glucose for the pulse of Bio-Gel-dextransucrase was 1:1 and for the chase 1:100. It was concluded that dextran is biosynthesized by the transfer of glucose from sucrose to the reducing end of the growing dextran chain. This was confirmed by the hydrolysis of pulsed dextran by glycoamylase. The total radioactivity in the residual dextran did not change during the hydrolysis and labeled glucose could not be detected. It is proposed that dextransucrase forms an enzymatically active covalent complex with glucose and dextran and that the glucose is inserted between the enzyme and the dextran by a nucleophilic attack of the C 6 —OH of glucose onto C 1 of the dextran forming an α-1 → 6 glucosidic bond. This releases one of the nucleophilic groups at the active site which attacks sucrose to give an enzyme-glucosyl complex. The C 6 —OH of this glucose then repeats the process of attacking C 1 of dextran. Dextran is built up by extrusion from the enzyme when glucose units are transferred from sucrose to the active site and inserted between the enzyme and the reducing end of the dextran polymer.

Oxidation of Primary Alcohol Groups of Naturally Occurring Polysaccharides with 2,2,6,6-Tetramethyl-1-Piperidine Oxoammonium Ion

ABSTRACT The primary alcohol groups of ten polysaccharides, with widely different structures and water solubilities, were oxidized to carboxyl groups using 2,2,6,6-tetramethyl-1-piperidine oxoammonium ion (TEMPO) at pH 10.8 and 0°C. The yield and selectivity for the primary alcohol group were high for all ten of the polysaccharides. The oxidation greatly increased the water-solubility of the polysaccharides. Water-insoluble polysaccharides such as amylose, cellulose, and chitin became water-soluble to the extent of approximately 10% (w/v). The water-soluble polysaccharides had their degree of solubility doubled or tripled. The specific optical rotation, viscosity, and gelling properties with calcium ion were determined. The oxidized polysaccharides are new anionic polymers with unique structures that could have application as gums, gels, and films.

Isolation and partial characterization of an extracellular glucansucrase from Leuconostoc mesenteroides NRRL B-1355 that synthesizes an alternating (1→6), (1→3)-α-d-glucan

Abstract Leuconostoc mesenteroides NRRL B-1355 grows on sucrose to produce two extracellular α- 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% α-(1→6) linkages and 5% α-(1→3) branch linkages, but fraction S has an alternating sequence of α-(1→6) and α-(1→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-α- d -glucopyranosyl fluoride, tris(hydroxymethyl)aminomethane, 2-aminoethanol, and octyl β- d -glucopyranoside.

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.

Mechanisms in The Glucansucrase Synthesis of Polysaccharides and Oligosaccharides From Sucrose

Publisher Summary This chapter examines the mechanisms involved in the glucansucrase synthesis of polysaccharides and oligosaccharides from sucrose. The enzymes that catalyze the synthesis of glucans from sucrose are secreted into the culture medium. The enzymes produced by Leuc. mesenteroides strains are inducible by sucrose, which is absolutely required in the culture media for their production. The glucansucrases use sucrose as a high-energy glucosyl donor for polysaccharide and oligosaccharide syntheses. For glucan synthesis, the energy of the glucose glycosidic linkage of sucrose must be conserved until the glucose is incorporated into the polysaccharide. In addition, the glucose moiety must be manipulated so that the enzyme can perform its catalytic function of synthesizing a glycosidic bond. This can be achieved by the transfer of the glucose moiety of sucrose to the enzyme with the formation of a covalent, high-energy-enzyme intermediate. The two-site insertion mechanism for the de Novo synthesis of polysaccharide by the addition of glucose to the reducing end is also elaborated in the chapter.

Multiple attack and polarity of action of porcine pancreatic α-amylase ☆

Abstract The action pattern of porcine pancreatic α-amylase on maltooctaose (G8) has been examined at pH 6.9 (optimum) and pH 10.5 (unfavorable). At pH 6.9, 27% of the G8 molecules react by multiple attack, while at pH 10.5 multiple attack is negligible. By using G8 labeled with 14C in the reducing or nonreducing end, and comparing the radioactive product distributions at pH 6.9 and pH 10.5, it was established that the direction of multiple attack is toward the nonreducing end of the substrate. The results indicate that, after initial enzymic attack, the right-hand fragment of the substrate dissociates from the enzyme surface. However, the left-hand fragment remains at the enzyme active site long enough to permit it to become repositioned and undergo further attack.

Separation and quantitative determination of nanogram quantities of maltodextrins and isomaltodextrins by thin-layer chromatography

Abstract Relatively fast solvent-systems have been developed for TLC separation of maltodextrins and isomaltodextrins containing 1–20 glucose residues. The primary solvent contains methyl cyanide (acetonitrile)-ethyl acetate-1-propanol-water in volume proportions of 85:20:50:X, where the water component, X, is varied from 50–70 for maltodextrins and 90–100 for isomaltodextrins. Separation of the α-(1 → 6) branched maltodextrins, including the two branched hexasaccharide isomers, resulting from the hydrolysis of amylopectin by alpha amylase, was achieved using 3 ascents of the solvent with 50 parts water. Separation of the α-(1 → 3) branched isomaltodextrins, resulting from the hydrolysis of dextran by dextranase, was achieved using the solvent with 70 parts water. A detection technique in the nanogram range has been developed by dipping the plate into ethanol containing 0.5% α-naphthol and 5% sulfuric acid, followed by heating for 10 min at 120°C. Saccharides appear as brown to black spots on a white background. Densitometric scanning of the TLC plate gives a linear relationship for 50 to 2000 ng of glucose. Identical plots on a weight basis are obtained for glucose, maltodextrins, and isomaltodextrins, showing that the densitometric response is independent of the saccharide structure. Thus, the relative weight percent of a series of malto- or isomalto-dextrins may be obtained by dividing the density of each saccharide by the sum of the densities of the saccharides. Malto- and isomalto-dextrins, down to dp 15–16, can be quantitatively determined by densitometric scanning of the TLC plate. These results, along with the increased sensitivity of the detection method, greatly widens the scope of the type of quantitative experiments that can be performed, which heretofore have been dependent on the use of radioactive compounds.