Limit Dextrin Research Articles

Specific assay of α-dextrin 6-glucanohydrolase using labeled pullulan

A new method was developed for the assay of α-dextrin 6-glucanohydrolase (EC 3.2.1.41), which hydrolyzes the α- d (1 → 6) glucosidic bonds occurring in polyglucans such as pullulan and amylopectin limit dextrins. After enzymatic hydrolysis of a pullulan-dye conjugate, the remaining substrate is precipitated by adding ethanol. Colored reaction products are determined by measuring the supernatant absorbance at 534 nm. The assay is simple, specific, and suitable for both plant and bacterial enzymes.

Characterization of salivary alpha-amylase binding to Streptococcus sanguis.

The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase (EC 3.2.1.1) was the prominent salivary component eluted from S. sanguis. Studies with 125I-labeled HSMSL or 125I-labeled HPS also demonstrated a component with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of [125I]alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch.

Immobilization of Amylases on Silica Support to Study Breakdown Products of Potato Starch by HPLC

Immobilization of α- and β-amylases on epoxypropylsilanized PartiSphere-5 was achieved. Hydrolysis of 2% potato starch solution yielded limit dextrin on α-amylase bound column while a mixture of li...

Partial characterisation of amylopectin alpha-dextrins

Alpha-dextrins, obtained by fractional precipitation with methanol from hydrolysates of waxy-maize amylopectin after the action of Bacillus subtilis alpha-amylase, were analysed using sweet-potato beta-amylase and rabbit muscle phosphorylase. Those obtained after alpha-amylolysis for 60 min had similar phosphorolysis (39.5%) and beta-amylolysis limits (48.4%), and those obtained after 210 min had much lower limits. The limit values were higher for the smaller dextrins. The mean chain-length in the alpha-dextrins obtained after alpha-amylolysis for 60 and 210 min were 14.9 and 11.1, respectively, regardless of the d.p. The smaller chain-length in the latter alpha-dextrins was due to a shortening of the external chains. It is suggested ( a) that the alpha-amylolysis involves two independent processes, namely, formation of maltohexaose from the external chains and of branched intermediate alpha-dextrins by fission of longer internal chains; and ( b) that the phosphorolysis limit dextrins had three d-glucosyl residues on the external B-chains and four residues on the A-chains.

Investigation of the fine structure of amylopectin using alpha- and beta-amylase

The hydrolysis of waxy-maize amylopectin and its beta-amylolysis limit dextrin by the alpha-amylase of Bacillus subtilis was followed by gel filtration on Sepharose CL-6B. The mixture of alpha-dextrins was also treated with beta-amylase. The d.p. of these β-limit dextrins was similar to those of the products obtained when the amylopectin β-limit dextrin was the substrate. It is suggested that amylopectin and its β-limit dextrin are hydrolysed by the alpha-amylase by fission of longer internal chains between unit clusters. A model is presented which shows how these unit clusters can be connected to build up the amylopectin molecule. Maltohexaose was produced from the amylopectin, but not from the amylopectin β-limit dextrin, and it is concluded that the latter is produced solely from the external chains.

Structural and immunochemical analysis of three α-limit dextrin oligosaccharides

Complete structures are described for three urinary oligodextrins from one patient with type II and one patient with type III glycogen storage disease. GLC-MS, direct probe MS, and 1H NMR demonstrate two heptasaccharides and one hexasaccharide containing only α1–4 and α1–6 linkages. The observation that all three oligosaccharides were present in urine of both patients and the occurrence of α1–4 and α1–6 linkages in characteristic sequences indicates that the oligodextrins are limit dextrins derived from α-amylolytic degradation of glycogen. The binding affinities of the oligodextrins for a monoclonal antibody ( 401 6 ) raised against Glcα1-6Glcα1-4Glcα1-4Glc, were determined by frontal analysis. The highest affinity was exhibited by Glcα1-6Glcα1-4Glcα1-4Glc followed by the two heptasaccharides and the hexasaccharide. The results from quantitative affinity measurements agree with results of structural analysis by physical methods in that all oligodextrins containing the nonreducing terminal sequence, Glcα1-6Glcα1-4Glc …, are specifically bound by the antibody with similar affinities, but the affinity is somewhat higher for chains containing the tetrasaccharide sequence Glcα1-6Glcα1-4Glcα1-4Glc at the nonreducing terminal. Utilization of affinity methods offers clear advantages for isolation and characterization of oligosaccharides with very similar structures.

遺伝的要因が澱粉の特性に及ぼす影響

1. Starch granules were prepared from mature kernels of several single mutants, double- and triple-mutant combinations of waxy (wx) gene with other endosperm genes, and their normal counterpart in the inbred 0h43 maize (Zea mays L.) background. Starches of the mutants containing the wx gene comprised almost 100% amylopectin. jS-Amylase limit dextrins prepared from the starches were successively debranched by isoamylase and pullulanase, and followed by quantitative gel-filtration on Toyopearl HW54SF. The unit chain-length distribution of amylopectins of the normal and wx starches were similar. Amylopectin of the amylose-extender ; waxy (ae ; wx) mutant had an increased proportion of long B chains and a decreased proportion of short B chains, compared with wx amylopectin, whereas amylopectin of dull ; waxy (du ; wx) mutant had a decreased proportion of long B chains and an increased proportion of short B chains. Therefore, the ae ; wx and du ; wx amylopectins were novel. Amylopectin of the ae ; du; wx mutant belonged to the du ; wx type instead of the ae ; wx type. 2. Starch granules were prepared from rice (Oryza sativa L.) endosperms of dull (du), sugary (sug), and high-amylose (ae) mutants. The amylose content of the du starch was half as low as that of the nonmutant (normal) one. The sug starch contained more amylose than the normal starch. The starch in the ae mutants was characterized by a higher content of amylose and loosely branched amylopectin with longer chains compared with the normal starch.

Purification and characterization of a novel thermostable β-amylase from Clostridium thermosulphurogenes

An extracellular beta-amylase from Clostridium thermosulphurogenes was purified 811-fold to homogeneity, and its general molecular, physico-chemical and catalytic properties were determined. The native enzyme was a tetramer of 210 kDa composed of a single type subunit; its 20 amino acid N-terminus displayed 45% homology with Bacillus polymyxa beta-amylase. The beta-amylase was enriched in both acidic and hydrophobic amino acids. The pure enzyme displayed an isoelectric point of 5.1 and a pH activity optimum of 5.5. The optimum temperature for beta-amylase activity was 75 degrees C, and enzyme thermostability at 80 degrees C was enhanced by substrate and Ca2+ addition. The beta-amylase hydrolysed amylose to maltose and amylopectin and glycogen to maltose and limit dextrins, and it was inhibited by alpha- and beta-cyclodextrins. The enzyme displayed kcat. and Km values for boiled soluble starch of 400,000 min-1 per mol and 1.68 mg/ml, respectively. The enzyme was antigenically distinct from plant beta-amylases.

The iodine-staining properties and fine structure of some mammalian and invertebrate glycogens

Glycogens from mammalian and invertebrate sources have been compared by measuring the iodine-staining spectra of the debranched polymers and the debranched beta-amylase limit dextrins. From the results, it is concluded that, whereas the interior chains of each group of glycogen are very similar, the exterior chains of the mammalian glycogens generally contain a small number of longer chains which are not found in the invertebrate glycogens.

Fermentation of corn starch to ethanol with genetically engineered yeast

Expression of the glucoamylase gene from Aspergillus awamori by laboratory and distiller's strains of Saccharomyces cerevisiae allowed them to ferment soluble starch. Approximately 95% of the carbohydrates in the starch were utilized. Glycerol production was significantly decreased when soluble starch was used instead of glucose. Ethanol yield on soluble starch was higher than that on glucose. The rate of starch fermentation was directly related to the level of glucoamylase activity. Strains with higher levels of glucoamylase expression fermented starch faster. The decline in starch fermentation rates toward the end of the fermentation was associated with accumulation of disaccharides and limit dextrins, poor substrates for glucoamylase. The buildup of these products in continuous fermentations inhibited glucoamylase activity and complete utilization of the starch. Under these conditions maltose-fermenting strains had a significant advantage over nonfermenting strains. The synthesis and secretion of glucoamylase showed no deleterious effects on cell growth rates, fermetation rates, and fermentation products.

Binding of glycogen, oligosaccharides, and glucose to glycogen debranching enzyme.

The binding of glucose and a series of oligosaccharides to glycogen debranching enzyme was determined by the ability of the saccharides to decrease the rate of reaction of sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoate) (DTNB). At pH 7.2, the strength of binding increases with chain length from glucose to maltotriose to maltopentaose but not to maltohexaose, and the free energies for binding of the oligosaccharides suggest subsites of equivalent affinities for the four glucose units following the initial reducing moiety. The rate of reaction of DTNB with enzyme saturated with saccharide is the same for all compounds, suggesting that all the saccharides, including glucose, induce the same conformational state. The site of binding may be that which binds the alpha-1,6-linked side chain of the natural limit dextrin substrate. At pH 8.0, this site exhibits similar characteristics, but an additional site, which may bind the four terminal glucose units of the main chain of the natural substrate, is manifested and exhibits different characteristics, including a very low affinity for glucose itself. The binding of glycogen to the debranching enzyme was monitored by centrifugal separation from the protein and exhibits a much lower dissociation constant than that for the oligomers, suggesting that branched polymers have more than one set of subsites.

グルコアミラーゼの生澱粉分解作用と基質特異性

The raw-starch-digesting activities of five glucoamylases, one each from Aspergillus niger, and Aspergillus sp. K-27, and three forms from Rhizopus delemar, GI, GII and GIII, were discussed as to the kinetic parameters (Km and Vmax) for maltooligosaccharides, amylopectin and glycogen, and their molecular structures. The R. delemar GIII and Aspergillus sp. K-27 enzymes, which showed strong raw starch digesting activity, showed much lower Km values for amylopectin and glycogen than the A. niger and R. delemar GI and GII enzymes, which lack raw-starchdigesting activity. The R. delemar GI and GII enzymes were suggested to be the products of limited proteolysis of the GIII enzyme after biosynthesis from the results of amino acid and carbohydrate analyses of these three forms and the partial proteolysis of the GIII enzyme with chymotrypsin. From these results and other lines of evidence, “starch binding site” other than the active site on the raw-starch-digesting enzyme was postulated. It is considered that this site shows high affinity to high molecular substrates and raw starch, and is involved in the hydrolyses of raw starch granules, amylopectin and glycogen, and the absorption on starch granules. The amino acid composition of this site of the GIII enzyme was suggested to be rich in serine, tyrosine and aspartic acid (including asparagine).All glucoamylases hydrolyze starch from its non-reducing end and their action stops at the C-6-phosphorylated residues and the residue just before the C-3-phosphorylated residue. Therefore, they are unable to completely hydrolyze potato starch, -20%, depending on the amount of the phosphate, remaining as limit dextrin. It is concluded that the cooperative action of α-amylase and phosphatase is essential for the complete degradation of starch.Aspergillus sp. K-27 produces a strong raw starch degrading enzyme, which is a mixture of glucoamylase and α-amylase, with an activity ratio of 7:3. The glucoamylase alone showed strong activity toward various raw starches, including potato starch, but the activity was enhanced synergistically with the addition of a small amount (1%) of the α-amylase. However, this synergistic action was observed also more or less with other α-amylases, Taka-amylase, and Bacillus subtilis liquefying and saccharogenic α-amylases.The relationship between the structure and function of the “starch binding site” and the mechanism of the synergistic action of glucoamylase and α-amylase as to raw starch degradation is an interesting problem for future studies.

Chain Length Distribution of Amylopectins of Several Single Mutants and the Normal Counterpart, and Sugary‐1 Phytoglycogen in Maize (Zea mays L.)

Abstractβ‐Limit dextrins of starches of normal (nonmutant), amylose‐extender (ae), dull (du), sugary‐2 (su2) and waxy (wx) maize, and phytoglycogen of sugar‐1 (su1) maize were prepared. The β‐limit dextrins were successively debranched by isoamylase and pullulanase, and followed by quantitative gel‐filtration.The ratio of A to B chains for the ae starch appeared to be high and that for su1 phytoglycogen was low. The ratio of short B to long B chains for the du starch was high and that for the ae starch appeared to be low. The phytoglycogen did not contain long B chains. The unit chain‐length distribution of amylopectins of the normal, wx, and su2 starches were similar. Fine structures of maize amylopectins and su1 phytoglycogen were modeled and described.

カツオ筋肉の脱分枝酵素に関する研究

In order to obtain more information of glycogen degradating metabolism in bonito skeletal muscle (red muscle), the glycogen debranching system (EC 2.4.1.25+EC 3.2.1.33) was purified and its properties were investigated. SDS electrophoresis showed that the purified enzyme appears to be made up of a single poly-peptide of molecular weight=165000. With a substrate, phosphorylase limit dextrin as the best substrate and α-1, 6-glucosyl cyclodextrin was hydrolyzed by the enzyme, but glycogen and other related polysaccharides were not hydrolyzed, The enzyme have two different activities containing both α-1, 6-glucosidase and glycosyltransferase. Sulfhydryl group reactivity of the enzyme and the inactivation by pH and temperature were reduced by the exogenous addition of glycogen or soluble starch. The debranching system appears to play no rate-limiting role in the process of glycogen degradation by phosphorylase.

64 ASSAY OF AMYLO-1,6-GLUCOSIDASE-TRANSFERASE ACTIVITY BY RELEASE OF GLUCOSE FROM PHOSPHORYLASE LIMIT DEXTRIN - A REASSESSMENT

The assay of glycogen debranching activity by measurement of the release of glucose from phosphorylase limit dextrin (PLD), was originally used for assay of enzyme activity in muscle and liver tissue. Results are corrected for the presence of nonspecific glucosidases by subtraction of the activity obtained with glycogen as substrate. When assays are performed on muscle or liver, the activity obtained with glycogen is very low and its exact nature is not of practical importance. The widespread use of blood cells and fibroblasts, in which the activity on glycogen is considerable, has prompted an examination of the apparent kinetic constants of this reaction. In liver and muscle the apparent Km for PLD ranged from 0.5-2.0 mg/ml, whereas that for glycogen was lower by an order of magnitude. The Vmax with glycogen as substrate did not exceed 20% of that with PLD. In leukocytes and platelets the Km for glycogen was higher than for PLD (0.1 and 0.5 mg/ml for PLD, 0.4 and 0.8 mg/ml for glycogen, in platelets and leukocytes, respectively), and the Vmax for PLD exceeded that for glycogen by 80%, In fibroblasts the Km for both PLD and glycogen was 1.5-3 mg/ml and the differences in Vmax were small. These results indicate that substrate concentration should be varied according to the kinetic constants of each cell type and point to the importance of distinguishing between the (low?) activity of the debranching enzyme on glycogen and nonspecific hydrolysis.

A new non-equilibrium enzyme linked immunosorbent assay for a glycogen-derived urinary tetrasaccharide

The tetrasaccharide, Glcα1-6Glcα1-4Glcα1-4Glc, denoted (Glc)4, is a limit dextrin formed by amylolytic degradation of glycogen. In order to evaluate the possible clinical importance of (Glc)4 excretion as an indicator of certain physiological and pathological conditions, we have developed a new rapid and inexpensive immunoassay using a monoclonal antibody raised against (Glc)4 glycosidically-linked to a carrier protein. As the antibody is highly specific, it can be used to measure native (Glc)4 directly, without the chemical reduction step required in previous methods. A new type of non-equilibrium ELISA inhibition test was developed based on the capacity of free (Glc)4 to decrease initial rates of antibody binding to (Glc)4-coated microtiter wells. The method is highly reproducible and is as sensitive and accurate as the gas chromatography method or radioimmunoassay used previously.

Debranching enzyme in fibroblasts, amniotic fluid cells and chorionic villi: pre- and postnatal diagnosis of glycogenosis type III

Glycogenosis type III is characterized by a deficiency of debranching enzyme in most tissues, and it can be detected by the inability to liberate glucose from limit dextrin. However, using this assay, the deficiency is not expressed in cultured fibroblasts from patients with glycogenosis type III. We have demonstrated that the failure to detect debranching enzyme deficiency in fibroblasts is entirely due to interference of acid α-glucosidase, which can also hydrolyse limit dextrin. A method is described to remove specifically acid α-glucosidase allowing clear discrimination between fibroblasts from patients and controls, whereas heterozygotes showed intermediate values. The results with amniotic fluid cells and chorionic villi suggest the feasibility of first- and second-trimester prenatal diagnosis of glycogenosis III.

Structural characteristics of water-soluble dextran from Leuconostoc mesenteroides NRRL B-1298.

A structural study of the water-soluble dextran made by Leuconostoc mesenteroides strain C (NRRL B-1298) was conducted by enzymic degradation and subsequent 13C-NMR analysis of the native dextran and its limit dextrins. The α-1, 2-debranching enzyme removed almost all of the branched D-glucose residues, and gave a limit dextrin having a much longer sequence of the internal chain length (degree of linearity: n=24.5 compared with the value of n=3.3 for the native dextran). The degree of hydrolysis with debranching enzyme corresponded to the content of α-1, 2-linkages determined by chemical methods, which suggested that most of the α-1, 2-linkages in the dextran B-1298 constituted branch points of a single D-glucose residue. A synergistic increase of susceptibility of the dextran B-1299 was observed by simultaneous use of debranching enzyme and endodextranase. 13C-NMR spectral analysis indicated the similarity of structure of dextran B-1298 to that of B-1396, rather than that of B-1299. Occurrence of α-1, 3-linkages in the limit dextrin was supported by a newly visualized chemical shift at 83.7 ppm.

Does pyridoxal 5'-phosphate function in glycogen phosphorylase as an electrophilic or a general acid catalyst?

alpha-D-Glucose 1-diphosphate interacts with pyridoxal-reconstituted rabbit muscle phosphorylase b activated by AMP (AMP-S). Under these conditions, the glucose moiety of alpha-D-[14C]glucose 1-diphosphate is transferred to limit dextrin forming alpha(1----4) glycosidic bonds and simultaneously releasing pyrophosphate as shown by 31P NMR spectroscopy. Thus, specific structural requirements invoked to explain the reactions of pyridoxal(5')diphospho(1)-alpha-D-glucose need not to be assumed in the case of the reactions of alpha-D-glucose 1-diphosphate. Dianions isomorphous to phosphate activate pyridoxal phosphorylase regardless of their pK values while the same anions, when bound covalently to pyridoxal, are inactive. Thus, anions bound noncovalently to pyridoxal phosphorylase act differently than anions linked covalently to pyridoxal, such as the 5'-phosphate group of pyridoxal 5'-phosphate, which is postulated to be part of a proton donor-acceptor pathway. The reaction of 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol (heptenitol) with phosphorylase yields, in the presence of orthophosphate as a glycosyl acceptor, 1-deoxy-D-gluco-heptulose 2-phosphate (heptulose-2-P). This sugar phosphate is unreactive but a potent competitive inhibitor for rabbit muscle phosphorylase b and potato phosphorylase with respect to alpha-D-glucose 1-phosphate: Ki = 14 X 10(-6) M and 1.9 X 10(-6) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible prenatal diagnosis of type III glycogenosis.

This paper reports the first prenatal diagnosis in a pregnancy at risk for type III glycogenosis using a reliable test for amylo-1,6-glucosidase in cultured amniotic fluid cells. This test, based upon the different rates of glycogen and phosphorylase limit dextrin hydrolysis, was found to be more reliable in detecting a deficiency of amylo-1,6-glucosidase activity than either phosphorylase limit dextrin hydrolysis alone or [14C]glucose incorporation using leukocytes and cultured cells.