Reducing values: dinitrosalicylate gives over-oxidation and invalid results whereas copper bicinchoninate gives no over-oxidation and valid results.

A comparative study was made between two carbohydrate reducing value methods, a relatively old, highly alkaline, 3,5-dinitrosalicylic acid (DNSA) method and a relatively newer, low alkaline (pH 10.5), copper bicinchoninate (CuBic) method. Reducing values for a series of equimolar amounts of maltose-maltohexaose, isomaltose-isomaltohexaose, and cellobiose-cellohexaose were compared by the two methods.

Tests for the mechanism of starch biosynthesis: de novo synthesis or an amylogenin primer synthesis.

Studies in 1940 on potato phosphorylase reaction with starch found that d-glucopyranose from α-d-glucopyranosyl-1-phosphate was added to the nonreducing-ends of starch chains. This led to the hypothesis that the biosynthesis of starch required a preformed primer. Later it was found that phosphorylase was exclusively a degradative enzyme in vivo and that starch-synthase was the enzyme that reacted with ADPGlc to biosynthesize starch.

Significant increases in potato Starch-Synthase and Starch-Branching-Enzyme activities by dilution with buffer containing dithiothreitol and polyvinyl alcohol 50 K.

We have recently found that the dilution of purified potato Starch-Synthases (SS) and Starch-Branching-Enzymes (SBEs), by a glycine buffer (pH 8.5), containing 1.0mM dithiothreitol (DTT) and 0.

Potent inhibition of starch-synthase by Tris-type buffers is responsible for the perpetuation of the primer myth for starch biosynthesis.

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.

De novo biosynthesis of starch chains without a primer and the mechanism for its biosynthesis by potato starch-synthase.

The starch-synthase enzymes used in this study were the second acetone precipitate and Fractions 21 and 23, Table 1, [Mukerjea, Ru.; Falconer, D. J.

Preparation and characterization of new and improved soluble-starches, -amylose, and -amylopectin by reaction with benzaldehyde/zinc chloride.

Seven different starches from potato, rice, maize, waxymaize, amylomaize-VII, shoti, and tapioca, and potato amylose and potato amylopectin have been reacted with benzaldehyde, catalyzed by ZnCl(2), to give new water-soluble starches and water soluble-amylose and soluble-amylopectin. In contrast to the native starches, aqueous solutions of the modified starches could not be precipitated with 2-, 3-, or 4-volumes of ethanol. β-Amylase gave no reaction with the modified starches, in contrast to the native starches, indicating that the modification occurred exclusively at the nonreducing-ends, giving 4,6-benzylidene-D-glucopyranose at the nonreducing-ends.

Comparative study of the efficacies of nine assay methods for the dextransucrase synthesis of dextran.

A comparative study of nine assay methods for dextransucrase and related enzymes has been made. A relatively widespread method for the reaction of dextransucrase with sucrose is the measurement of the reducing value of D-fructose by alkaline 3,5-dinitrosalicylate (DNS) and thereby the amount of D-glucose incorporated into dextran. Another method is the reaction with (14)C-sucrose with the addition of an aliquot to Whatman 3MM paper squares that are washed three times with methanol to remove (14)C-D-fructose and unreacted (14)C-sucrose, followed by counting of (14)C-dextran on the paper by liquid scintillation counting (LSC).

Biosynthesis of dextrans with different molecular weights by selecting the concentration of Leuconostoc mesenteroides B-512FMC dextransucrase, the sucrose concentration, and the temperature.

Leuconostoc mesenteroides B-512FMC dextransucrase was found to synthesize dextrans of varying molecular weights by selecting the concentrations of dextransucrase and sucrose, as well as the temperature. Four enzyme concentrations (50, 10, 1.0, and 0.

Large-scale isolation, fractionation, and purification of soluble starch-synthesizing enzymes: starch synthase and branching enzyme from potato tubers.

Soluble starch-synthesizing enzymes, starch synthase (SSS) and starch-branching enzyme (SBE), were isolated, fractionated, and purified from white potato tubers (Solanum tuberosum) on a large scale. Five steps were used: potato tuber extract from 2 kg of peeled potatoes, two acetone precipitations, and two fractionations on a large ultrafiltration polysulfone hollow fiber 100 kDa cartridge. Three kinds of fractions were obtained: (1) mixtures of SSS and SBE; (2) SSS, free of SBE; and (3) SBE, free of SSS.

Enzymatic synthesis of L-DOPA alpha-glycosides by reaction with sucrose catalyzed by four different glucansucrases from four strains of Leuconostoc mesenteroides.

L-DOPA alpha-glycosides were synthesized by reaction of L-DOPA with sucrose, catalyzed by four different glucansucrases from Leuconostoc mesenteroides B-512FMC, B-742CB, B-1299A, and B-1355C. The glucansucrases catalyzed the transfer of d-glucose from sucrose to the phenolic hydroxyl position-3 and -4 of L-DOPA. The glycosides were fractionated and purified by Bio-Gel P-2 column chromatography, and the structures were determined by (1)H NMR spectroscopy.

Controlled release of D-glucose from starch granules containing 29% free D-glucose and Eudragit L100-55 as a binding and coating agent.

Waxy maize starch (100% amylopectin) granules were modified by reaction of the granules with glucoamylase in a minimum amount of water to give 29% (w/w) D-glucose inside the granules [Kim, Y.-K.; Robyt, J.

Isolation, structure, and characterization of the putative soluble amyloses from potato, wheat, and rice starches.

Amylose, a putative linear alpha-(1-->4)-glucan and a component of most starches, was isolated from potato, rice, and wheat starches by forming the 1-butanol complex in a solution of the starches. It previously had been found that these amyloses were incompletely hydrolyzed by beta-amylase, indicating that it was partially branched. Solubilization of the butanol complex in water and steam distillation of the 1-butanol, followed by cooling to 4 degrees C gave precipitation of the double helical, linear, retrograded amylose over a 15 h period, leaving the soluble amylose in solution.

Synthesis of dopamine and L-DOPA-alpha-glycosides by reaction with cyclomaltohexaose catalyzed by cyclomaltodextrin glucanyltransferase.

Dopamine-HCl and L-DOPA-alpha-glycosides were prepared by reaction with cyclomaltohexaose, catalyzed by Bacillus macerans cyclomaltodextrin glucanyltransferase. The reaction gave maltodextrins attached to dopamine and L-DOPA; the maltodextrins were trimmed by reactions with glucoamylase and beta-amylase to produce alpha-glucosyl- and alpha-maltosyl-glycosides, respectively. The glucoamylase- or beta-amylase-treated dopamine- and L-DOPA-alpha-glycosides were fractionated and purified by BioGel P-2 gel-filtration column chromatography and preparative descending paper chromatography.

Starch biosynthesis: experiments on how starch granules grow in vivo.

Four varieties of starch granules from potato, wheat, maize, and rice were fractionated into homogeneous 10-microm-sized ranges. The size with the largest amount of granules was reacted with ADP-[(14)C]Glc, washed, and peeled into 7-9 layers, using a controlled peeling process, involving 90:10 volume proportions of Me(2)SO-H(2)O at 10 degrees C. All of the starches showed biosynthesis of starch throughout the granules.

Dextransucrase and the mechanism for dextran biosynthesis.

Remaud-Simeon and co-workers [Moulis, C.; Joucla, G.; Harrison, D.

Formation of covalent beta-linked carbohydrate-enzyme intermediates during the reactions catalyzed by alpha-amylases.

Porcine pancreatic and Bacillus amyloliquefaciens alpha-amylases were examined for the formation of covalent carbohydrate intermediates during reaction. The enzymes were precipitated and denatured by adding 10 volumes of acetone. When these denatured enzymes were mixed with methyl alpha-6-[(3)H]-maltooligosaccharide glycosides and chromatographed on BioGel P-2, no carbohydrate was found in the protein void volume peak.

Determination of the maximum water solubility of eight native starches and the solubility of their acidic-methanol and -ethanol modified analogues.

The maximum water solubilities of eight native starches from potato, shoti, tapioca, maize, waxy maize, amylomaize-7, wheat, and rice and their acid-methanol and acid-ethanol modified analogues have been determined. Maximum solubilities of 18.7 and 17.

Significant differences in the activities of alpha-amylases in the absence and presence of polyethylene glycol assayed on eight starches solubilized by two methods.

Starch is a reserve chemical source of the energy of the sun found in plants as a water-insoluble granule that differs in their chemical and physical properties, depending on the source. The granules can be solubilized by heating in water or by treatment with various reagents, such as 1M NaOH. alpha-Amylases are widely distributed enzymes that initiate the hydrolysis of starch into low molecular weight maltodextrins.

Cloning, expression and characterization of an extracellular enolase from Leuconostoc mesenteroides.

Enolase on the surface of streptococci putatively facilitates pathogenic invasion of the host organisms. The related Leuconostoc mesenteroides 512FMCM is nonpathogenic, but it too has an extracellular enolase. Purified isolates of extracellular dextransucrase from cultures of L.

Controlled peeling of the surfaces of starch granules by gelatinization in aqueous dimethyl sulfoxide at selected temperatures.

Microscopic examination of starch granules in 90:10 (v/v) Me(2)SO-H(2)O indicated that the granules were slowly being gelatinized from their surfaces. The rate of gelatinization was dependent on two variables: (1) the amount of water in Me(2)SO and (2) the temperature. An increase of water in Me(2)SO and/or an increase in temperature increased the rate of gelatinization and vice versa.

Optimized synthesis of specific sizes of maltodextrin glycosides by the coupling reactions of Bacillus macerans cyclomaltodextrin glucanyltransferase.

Bacillus macerans cyclomaltodextrin glucanyltransferase (CGTase, EC 2.4.1.

Starch biosynthesis: further evidence against the primer nonreducing-end mechanism and evidence for the reducing-end two-site insertion mechanism.

Two reactions were studied with three varieties of starch granules from maize, wheat, and rice. In Reaction-I, the granules were reacted with 1 mM ADP-[(14)C]Glc and in Reaction-II, a portion of the granules from Reaction-I was reacted with 1 mM ADP-Glc. The starch granules were solubilized and reacted with the exo-acting glucoamylase and beta-amylase to an extent of 50% or less of the (14)C-label.

Enzymatic synthesis and anti-coagulant effect of salicin analogs by using the Leuconostoc mesenteroides glucansucrase acceptor reaction.

Glucansucrases from Leuconostoc mesenteroides catalyze the transfer of glucosyl units from sucrose to other carbohydrates by acceptor reaction. We modified salicyl alcohol, phenol and salicin by using various glucansucrases and with sucrose as a donor of glucosyl residues. Salicin, phenyl glucose, isosalicin, isomaltosyl salicyl alcohol, and a homologous series of oligosaccharides, connected to the acceptors and differing from one another by one or more glucose residues, were produced as major reaction products.

Cloning and expression of levansucrase from Leuconostoc mesenteroides B-512 FMC in Escherichia coli.

Leuconostoc mesenteroides B-512 FMC produces dextran and levan using sucrose. Because of the industrial importance of dextrans and oligosaccharides synthesized by dextransucrase (one of glycansucrases from L. mesenteroides), much is known about the dextransucrase, including expression and regulation of gene.

Starch biosynthesis: the primer nonreducing-end mechanism versus the nonprimer reducing-end two-site insertion mechanism.

Two mechanisms are recognized for polysaccharide chain elongation: (a) the nonreducing-end, primer-dependent mechanism and (b) the reducing-end, two-site insertion mechanism. We recently demonstrated the latter mechanism for starch biosynthesis by pulsing starch granules with ADP-[14C]Glc and chasing with ADPGlc for eight varieties of starch granules. Others have reported the addition of glucose from ADPGlc to the nonreducing ends of maltose, maltotriose, and maltopentaose and a branched maltopentasaccharide.