The cluster structure of barley amylopectins of different genetic backgrounds

The unit chains of amylopectin are organized into clusters. In this study, the cluster structure was analysed in detail in four different genotypes of barley, of which two possessed the amo1 genetic background. Amylose content of the barley starches differed from 0 to 32.6%. Isolated amylopectin was hydrolysed with α-amylase from Bacillus subtilis into domains, defined as groups of clusters, which were size-fractionated by methanol. The domain fractions were further treated with α-amylase to release single clusters. Amylopectin, domains and clusters were subsequently treated with phosphorylase and β-amylase to produce φ,β-limit dextrins and the detailed internal structures of these different structure levels were investigated. Analysis was performed with gel-permeation and anion-exchange chromatography. Equal amount of A-chains were detected in all barleys, but the distribution of B-chains differed. At least two types of domain structures were identified in all four barley varieties. Large domains were built up by large clusters and small domains by small clusters. In all four barley samples the number of long chains was small suggesting that shorter chains with a degree of polymerization of 25-35 also are involved in the interconnection of clusters. The cluster structure of the amylopectin correlated with the genetic background. The two barley samples with amo1 genetic background possessed a more dense structure. Internal chain lengths in these two barleys were shorter resulting in larger domains built up by larger clusters.     

Structures of clusters in sweetpotato amylopectin

Sweetpotato amylopectin was subjected to partial hydrolysis by α-amylase from Bacillus amyloliquefaciens to release the clusters. Clusters were then fractionated and precipitated by methanol and structurally characterized by gel-permeation chromatography and high-performance anion-exchange chromatography. An initial stage of α-amylolysis on the amylopectin isolated mostly domains but also clusters. A second stage of α-amylolysis on the domains and clusters further isolated their respective clusters and sub-clusters. All the domains, clusters and sub-clusters were sequentially subjected to phosphorolysis and β-amylolysis to obtain their internal part. The degree of polymerization of the clusters in the form of φ,β-limit dextrins were from 58 to 86. Each domain contained 2–8 clusters. Two types of clusters were structurally identified. Type A clusters were larger and contained about 12 chains per cluster with higher degree of branching (DB), whereas those of type B were smaller and contained about eight chains per cluster with lower DB.     

Synthesis of phenyl 2-acetamido-2-deoxy-4-O-α-l-fucopyranosyl-β-d-glucopyranoside and p-nitrophenyl 2-acetamido-2-deoxy-6-O-α-l-fucopyranosyl-β-d-glucopyranoside

Maize and potato amylopectin (57 and 64%, respectively) were recovered as non-cyclic products from 4-h digests of the starches with cyclodextrin glycosyltransferase {(1→4)-α-d-glucan:[(1→4)-α-d-glucopyranosyl]transferase (cyclising), EC 2.4.1.19} from Klebsiella pneumoniae M 5 al. Besides smaller saccharides, highly branched fragments of different sizes (average d.p. 40–140) were obtained by fractionation. The extents of beta-amylolysis varied between 24 and 37%, indicating that the clusters were not equally susceptible to attack by cyclodextrin glycosyltransferase. The fragments of potato amylopectin still contained larger amounts of material of high molecular weight. Accordingly, part of the longer B-chains of the basic structure were protected from the enzymic attack, presumably because of interchain branches. By debranching with pullulanase, it was evident that the beta-limit dextrins of the fragments of potato amylopectin were composed of longer B-chains (average chainlength 17.8) than those of maize amylopectin (average chain-length 14.1). The A/B-chain ratios, which were calculated from h.p.l.c. data for the debranched beta-limit dextrins, were 1.22 (maize) and 1.06 (potato). Some structural differences between potato and maize amylopectin are discussed.     

Substituent distribution in highly branched dextrins from methylated starches

Granular potato starch and amylopectin potato starch were methylated to molar substitutions (MS) up to 0.29. Extensive alpha-amylase digestion gave mixtures of partially methylated oligomers. Precipitation of larger fragments by methanol yielded mainly alpha-limit dextrins (84-99%). Methanol precipitates were extensively digested with beta-amylase yielding alpha,beta-limit dextrins. The average substitution level of branched glucose residues in the dextrins thus obtained was determined after per deuteriomethylation by using FAB mass spectrometry, and compared with that of the linearly linked glucose residues. The present work demonstrates that methylation does not show any preference for substitution at either branched or linearly linked glucose residues, taking into account the inherently lower amount of substitution sites at branched residues. The results corroborate earlier studies wherein it was found that substituents in branched regions are distributed almost randomly. In addition, the data enable the determination of the average degree of branching of partially methylated dextrins.     

Structural heterogeneity in waxy-rice starch

Alpha-amylase of B. amyloliquefaciens was used for the structural characterization of the amylopectin from waxy-rice starch. Fractions of -dextrins with a degree of polymerization (d.p.) <5000 were isolated from amylopectin hydrolysates after 1 and 3 h. φ,β-Limit dextrins were prepared by successive phosphorolysis and beta-amylolysis of the fractions and these were analysed by a second alpha-amylolysis. Based on the hydrolysis pattern, the limit dextrins were divided into two major groups, A and B, which possessed units of clusters of d.p. 100–200 and 90–130, respectively. An extensive alpha-amylolysis resulted in characteristic distributions of dextrins with d.p. <80 which represented branched building blocks. Type A dextrins possessed more larger building blocks with d.p. 40, but less intermediate and small blocks, than type B. The φ,β-limit dextrin of the original amylopectin had a distinct distribution enriched in small building blocks. A model is proposed in which the two types of dextrins originate from regular and less regular structural domains of the amylopectin fraction within the starch granules.     

Effects of substrate branching characteristics on kinetics of enzymatic depolymerizaion of mixed linear and branched polysaccharides: I. Amylose/amylopectin alpha-amylolysis

Hydrolysis reactions of homopolysaccharides, which differ in their degree of branching, and mixtures of linear and branched polymers were carried out with alpha-amylase. The branching structures of both the original amylopectin substrate and the cluster domains of amylopectin, obtained by ethanol precipitation of the products of the action of alpha-amylase, were characterized via enzymatic digestion with debranching enzyme (i.e., isoamylase), followed by the fractions of the resulting products using gel filtration chromatography. The structural properties (i.e., molecular weight, molecular weight distribution, and branching characteristics) of the resulting products during depolymerization of amylose, amylopectin and their mixtures via alpha-amylase were characterized by size exclusion chromatography coupled with a low angle laser right scattering (SEC/LALLS) technique. It was determined that substrate branching characteristics strongly influence both the observed enzymatic activity as well as the enzyme's action pattern. A simplified kinetic model that represents the hydrolysis reactions of amylose and amylopectin mixtures via endo-acting alpha-amylase is proposed. We found that that reaction kinetics (i.e., enzyme affinity) was also governed by the substrate's conformation in solution. The relationships between the mass fraction of branched polymers and the kinetic parameters during alpha-amylolysis were compared with those predicted by the kinetic model. Excellent agreement was found between the model predictions and the experimental observations. The results reported here imply and interrelationship between enzyme action and polymeric substrate structural properties. (c) 1994 John Wiley & Sons, Inc.     

Growth Inhibition of Lactobacillus casei Phage J1 by Dehydroacetate

The fine structures of amylopectin and intermediate material characteristic of amylomaize starch were investigated by chemical and enzymatic means. In comparison with waxy-maize amylopectin, that of amylomaize starch was found to possess a img/ approximately 10 glucose units longer. Unit-chain profiles of waxy and amylomaize amylopectins revealed that the clear difference lay simply in the relative amounts of two unit-chain fractions. By fractionations of debranched β-limit dextrins, it was demonstrated that the img/ of the internal chains in amylomaize amylopectin was 9 glucose units longer than that in waxy-maize amylopectin. In addition, the proportion of maltose and maltotriose fractions in the debranched dextrin for amylomaize amylopectin was noticeably smaller than found for waxy-maize amylopectin. These data suggest a lesser branching frequency of outer branches in amylomaize amylopectin, confirming the previous proposal that this amylopectin has longer inner and outer branches than those of normal amylopectin.

As for amylomaize intermediate material, the average degree of polymerization was estimated to be 250 to 300 glucose units per molecule. It was also indicated that there were 5 or 6 glucose residues corresponding to the non-reducing end in the molecule. The unit-chain profile of the intermediate material implied that this molecule was mainly composed of branches with img/ around 50. Moreover, the presence of only small amounts of maltose and maltotriose fractions was demonstrated by the unit-chain distribution of this β-limit dextrin. These findings indicate that amylomaize intermediate material is totally consistent with a branched glucan having a low molecular weight, proposing that this anomalous glucan has such a fine structure that four or five branches with img/ around 50 are linked to a main linear chain of 100 to 150 glucose units.     

Determination of depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate/flow-injection analysis system

This study reports on the determination of the depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using flow-injection analysis with fluorescence detection and 2-p-toluidinylnaphthalene-6-sulfonate as the fluorescent probe. The experimental data points, corresponding to the evolution of the concentration of "detectable" substrate with depolymerization time, were fit to a single exponential decay curve in the case of amylose and to a double exponential decay curve in the cases of amylopectin and soluble starch. For all the assayed substrates, the determined depolymerization rates at time zero correlated well with the initial enzyme and substrate concentrations through the usual Michaelis-Menten hyperbola. Therefore, this methodology allows the determination of alpha-amylase activity using any of these substrates. For amylopectin and soluble starch, the value of the total depolymerization rate at any depolymerization time was the result of the additive contribution of two partial depolymerization rates. In contrast, the total depolymerization rate for amylose was always a single value. These results, in conjunction with the relative time evolution of the two partial depolymerization rates (for amylopectin and soluble starch), are in good agreement with a linear molecular structure for amylose, a "grape-like" cluster molecular structure for amylopectin, and an extensively degraded grape-like cluster structure for soluble starch.     

Structures of building blocks in clusters of sweetpotato amylopectin

φ,β-Limit dextrins of domains and clusters of sweetpotato amylopectin were subjected to extensive hydrolysis by Bacillus amyloliquefaciens α-amylase to release building blocks and reveal the internal structures of clusters. The composition of building blocks was analyzed by size-fractionation, gel permeation chromatography, and high performance anion exchange chromatography. Different domains and clusters had structurally similar building blocks with around three chains per building block and internal chain length around 2.9. Singly branched and doubly branched building blocks were the largest and second largest groups in the clusters. Type A clusters had more large building blocks and contained 5–6 blocks per cluster with an inter-block chain length (IB-CL) of 7.0, whereas type B clusters had less large building blocks and contained 3–4 blocks per cluster with IB-CL 7.9. Models on how the building blocks could be organized into type A and type B clusters are discussed.     

Heterogeneity in branching of amylopectin

The angular dependence of scattered light from amylopectin and its β-limit dextrin, the mean square radius of gyration and the molecular weights M_w and M_n have been calculated on the basis of the cascade branching theory for the homogeneously branched model by Meyer &; Bernfeld (1940) (Model I) and for the two heterogeneously branched structures suggested by French (1972) (Model II) and by Robin et al. (1974, 1975) (Model III). The calculations take into account the particularities of topology in branched molecules and the experimentally determined ratio of the number of A- and B-chains, A/B = 1. Furthermore, an average branching density of 4% and an interconnecting chain length of ovbar|n_i2 = 22, found by gel permeation chromatography (GPC) after debranching, were used. The constraints lead to the conclusion that amylopectin is heterogeneously branched. Densely branched clusters containing 3·22 branching units are interconnected by longer chains of 22 units in length. Comparison of the calculated angular dependence of light scattering with measurements from a maize amylopectin β-limit dextrin in 1 n NaOH solution gives strong evidence for a modified Robin-Mercier model. The modification consists of the conclusion that the interconnecting chains are preferentially B-chains, such that these chains carry on the average 1·4 clusters, while Robin and Mercier assume exactly 2 clusters. Our result is in agreement with the distribution of chain length found after debranching the amylopectin β-limit dextrin.     

Effects of amylopectin structure and molecular weight on microstructural and rheological properties of mixed beta-lactoglobulin gels

Nongelling amylopectin fractions from potato and barley have been used to form mixed beta-lactoglobulin gels. The amylopectin fractions were produced by varying the time of alpha-amylase hydrolysis followed by sequential ethanol precipitation. The molecular weights, radius of gyration, chain length distribution, and viscosity of the fractions were established. The mixed gels were analyzed rheologically with dynamic mechanical analysis in shear and microstructurally with light microscopy, transmission electron microscopy, and nuclear magnetic resonance spectroscopy. The result of the gel studies clearly showed that small differences in the molecular weight of amylopectins have a significant influence on the kinetics of protein aggregation and thereby on the gel microstructure and the rheological behavior of the gel. Both an increase in the molecular weight and a higher concentration of amylopectins resulted in a more open protein network structure, with thicker strands of larger and more close-packed beta-lactoglobulin clusters, which showed a larger storage modulus. The transmission electron micrographs revealed that degraded amylopectins were enclosed inside the protein clusters in the mixed gels, whereas nondegraded amylopectin was only found outside the protein clusters. The volume-weighted mean value of the molecular weight of the amylopectins was found to vary between 3.2 x 10(4) and 5.0 x 10(7) Da and the ratio of gyration between 14 and 61 nm. The maximum in chain length distribution was generally somewhat distributed toward longer chain lengths for potato compared to barley, but the differences in chain length distribution were minor compared to those seen in the molecular weight and ratio of gyration between the fractions.     

The distribution of covalently bound phosphate in the starch granule in relation to starch crystallinity

Five selected starches with a 60-fold span in their content of monoesterified starch phosphate were investigated with respect to distribution of glucose 6-phosphate and glucose 3-phosphate residues, amylopectin chain length distributions and gelatinisation properties. The distribution of starch phosphate in the starch granules was determined by preparation of N?geli dextrins followed by quantitative 31P-nuclear magnetic resonance spectroscopy. Total starch phosphate content was positively correlated to the unit chain lengths of the amylopectin as well as to the chain lengths of the corresponding N?geli dextrins. The major part (68-92%) of the total starch phosphate content was partitioned to the hydrolysed (amorphous) parts. Starch-bound glucose 6-phosphate per milligram of starch was 2-fold enriched in the amorphous parts, whereas phosphate groups bound at the 3-position were more evenly distributed. The gelatinisation temperatures of the native starches as determined by differential scanning calorimetry were positively correlated (R(2)=0.75) to starch phosphate content, while crystallinity (gelatinisation enthalpy) and crystal heterogeneity (endotherm peak width) showed no correlations to starch phosphate content. The relations between starch molecular structure, architecture and functional properties are discussed.     

The structure of high molecular weight dextrins obtained from potato starch by treatment with Bacillus macerans enzyme

Bacillus macerans enzyme (BME)-derived high molecular weight dextrins, which are by-products in the course of the industrial production of cylodextrins, were isolated and their chemical structures were characterized.Dextrin I was obtained in a yield of about 24% from BME-hydrolyzate (a mixture of dextrin and cylodextrins, 50% each) of potato starch by fractionation with an ultrafiltrator having a membrane of cut-off molecular weight 2.0 × 10⁴. Dextrin II was obtained in a yield of about 15% from BME-hydrolyzate (a mixture of dextrins and cyclodextrins, 70 : 30) of Dextrin I by the same method.Dextrin I and II consisted of dextrin having molecular weights over 20 × 10⁶ and dextrins having molecular weights 4 × 10³−1 × 10⁵ in the ratio of 80 : 12 and 66: 15, respectively.The results of hydrolysis by β-amylase and methylation analysis indicated that the average, exterior and interior chain lenghts of the dextrins having molecular weights over 20 × 10⁶ and 4 × 10³−1 × 10⁵ from Dextrin I were 16.5, 8.2 and 7.3, and 11.5, 6.9 and 3.6, respectively, than those from Dextrin II were 13.6, 4.7 and 9.9, and 10.4, 5.1 and 4.3, respectively.     

Action of Pseudomonas isoamylase on various branched oligo- and poly-saccharides

Pseudomonas isoamylase (EC 3.2.1.68) hydrolyzes (1 → 6)-α-D-glucosidic linkages of amylopectin, glycogen, and various branched dextrins and oligosaccharides. The detailed structural requirements for the substrate are examined qualitatively and quantitatively in this paper, in comparison with the pullulanase of Klebsiella aerogenes. As with pullulanase. Ps. isoamylase is unable to cleave D-glucosyl stubs from branched saccharides. Ps. isoamylase differs from pullulanase in the following characteristics: (1) The favored substrates for Ps. isoamylase are higher-molecular-weight polysaccharides. Most of the branched oligosaccharides examined were hydrolyzed at a lower rate, 10% or less of the rate of hydrolysis of amylopectin. (2) Maltosyl branches are hydrolyzed off by Ps. isoamylase very slowly in comparison with maltotriosyl branches. (3)Pr. isoamylase requires a minimum of three D-glucose residues in the B- or C-chain.     

Exoenzymic activity of alpha-amylase immobilized on a phenol-formaldehyde resin

Amylose and amylopectin from two starch sources were partially degraded by alpha-amylase immobilized on a phenol-formaldehyde resin. The degradation products were fractionated by gel-permeation chromatography and high-pressure, liquid chromatography. Two distinct fractions were obtained from tapioca amylose. One is a fragment having a molecular weight exceeding 200,000, and the other consists of oligosaccharides of low molecular weight with a degree of polymerization of 1–8. In contrast, treatment of tapioca amylose with soluble alpha-amylase produces a single fraction, nearly all of which has a molecular weight of <35,000, with only traces of small oligosaccharides detectable by high-pressure, liquid chromatography. Even wider differences were observed in degradation products from tapioca amylopectin. Similar activity-patterns were obtained with immobilized and soluble enzymes, using corn amylose and corn amylopectin as substrates. Immobilization of alpha-amylase on the resin apparently restricts the activity of the enzyme to the ends of the starch molecules, making it appear to be limited to exoenzymic activity.     

Cyclodextrin glycosyltransferase from Bacillus circulans DF 9R: Activity and kinetic studies

Activity characteristics and kinetic aspects of a cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans DF 9R were studied. A mixture of α-, β- and γ-cyclodextrins (CDs), glucose, maltose and negligible amounts of longer linear dextrins were produced from gelatinized amylose, amylopectin and starch from different sources. In the coupling reaction, CDs were the substrates in the presence of acceptors such as maltose and/or longer oligosaccharides. From oligosaccharides formed by three or more glucose units, this enzyme produced linear chains of several lengths which were then cyclized. CGTase catalytic efficiency was compared employing an analytical grade starch and cassava starch for food use. Since the results obtained were similar for both starches, the use of an economic starch is an advantage. CGTase was inhibited by the substrate and its own products. Starch concentrations over 20 mg/mL inhibited the cyclizing activity. CDs behaved as competitive inhibitors and maltose as an uncompetitive inhibitor while maltotriose showed a mixed inhibition pattern. Limit dextrins showed a scarce inhibitory effect on enzyme activity. CD production could be improved with an ultrafiltration membrane reactor for continuous removal of the products; the starch concentration should be maintained below an inhibitory concentration and limit dextrins would remain in the reactor without affecting enzyme activity.     

The structural analysis and enzymic synthesis of a pentasaccharide alpha-limit dextrin formed from amylopectin by Bacillus subtilis alpha-amylase

Crystalline Bacillus subtilis alpha-amylase hydrolyses arnylopectin to a mixture Of D-gIUCOSe, maltose, and branched oligosaccharides (alpha-limit dextrins). The smallest such dextrin formed under the conditions of the experiment is a pentasaccharide. A combination of methylation analysis, periodate oxidation, and frag- mentation analysis with acid narrowed the pentasaccharide structure to two possibilities, but failed to distinguish between them. A rigid proof that the dextrin has the structure 6'-a-maltosylmaltotriose was obtained by application of enzymic degradation. Finally, the structure of the pentasaccharide was confirmed by enzymic synthesis. It is shown that the structural analysis of such oligosaccharides, derived from amylopectin, can be made by the use of enzymes alone, without resort to the more time-consuming, less-specific, and less-sensitive methods of chemical analysis. Conclusions are drawn regarding the action pattern of the B. subtilis arnylase.     

The composition of peptidochitodextrins from sarcophagid puparial cases

1. N-Bromosuccinimide cleaved proteins and pigments from fly puparia, increasing the chitin:protein ratio from 0.5 to 1.5. The product afforded subfractions (ratio 5:1) of molecular weights of 1200 and 1600 devoid of aromatic residues and N-terminal beta-alanine, direct aryl links between polysaccharide chains being discounted. 2. The chitin-protein complex decreased in molecular weight when treated with Pronase, which suggested polypeptide bridges within the native chitin micelle. The limit dextrins generated by chitinase were mixtures of unsubstituted dextrins and peptidylated oligosaccharides, with the former predominating. 3. Peptidochitodextrins of similar molecular weight but markedly different solubility were prepared, which were indistinguishable with respect to amino acid, glucosamine, acetyl, X-ray or infrared characteristics. It is suggested that physical interactions contribute to the stability of the integument in addition to the covalent bonds that form during sclerotization.     

Building block organisation of clusters in amylopectin from different structural types

Clusters of chains consisting of tightly branched units of building blocks were isolated from 10 amylopectin samples possessing the 4 types of amylopectin with different internal unit chain profiles previously described. It was shown that clusters in types 1 and 2 amylopectins are larger than in types 3 and 4, but the average cluster size did not correspond to the ratio of short to long chains of the amylopectins. The size-distribution of the building blocks, having one or several branches, possessed generally only small differences between samples. However, the length of the interblock segments followed the type of amylopectin structure, so that type 1 amylopectins had shortest and type 4 the longest segments. The chains in the clusters were divided into characteristic groups probably being involved in the interconnection of two, three, and four - or more - building blocks. Long chains were typically found in high amounts in clusters from type 4 amylopectins, however, all cluster samples contained long chains. The results are discussed in terms of the building block structure of amylopectin, in which the blocks together with the interblock segments participate in a branched backbone building up the amorphous lamellae inside growth rings of the starch granules. In such a model, amylopectins with proportionally less long chains (types 1 and 2) possess a more extensively branched backbone compared to those with more long chains (types 3 and 4).     

Purification and characterization of the extracellular alpha-amylase from Clostridium acetobutylicum ATCC 824.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography (mono Q) and gel filtration (Superose 12). The enzyme had an isoelectric point of 4.7 and a molecular weight of 84,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a monomeric protein, the 19-amino-acid N terminus of which displayed 42% homology with the Bacillus subtilis saccharifying alpha-amylase. The amino acid composition of the enzyme showed a high number of acidic and hydrophobic residues and only one cysteine residue per mole. The activity of the alpha-amylase was not stimulated by calcium ions (or other metal ions) or inhibited by EDTA, although the enzyme contained seven calcium atoms per molecule. alpha-Amylase activity on soluble starch was optimal at pH 5.6 and 45 degrees C. The alpha-amylase was stable at an acidic pH but very sensitive to thermal inactivation. It hydrolyzed soluble starch, with a Km of 3.6 g . liter-1 and a Kcat of 122 mol of reducing sugars . s-1 . mol-1. The alpha-amylase showed greater activity with high-molecular-weight substrates than with low-molecular-weight maltooligosaccharides, hydrolyzed glycogen and pullulan slowly, but did not hydrolyze dextran or cyclodextrins. The major end products of maltohexaose degradation were glucose, maltose, and maltotriose; maltotetraose and maltopentaose were formed as intermediate products. Twenty seven percent of the glucoamylase activity generally detected in the culture supernatant of C. acetobutylicum can be attributed to the alpha-amylase.