The Mechanism for the Synthesis of Starch and Its Relationship to the Newly Proposed Structural Model for DNA. Part I.

The physical and chemical properties of starch and glycogen can be explained on the basis that glycogen is converted into amylose and amylopectin by a debranching enzyme (isoamylase) which removes the external α-1,6-linked branches of the glycogen. This precursor glycogen exists in all corn endosperm examined: ae high amylose, normal dent, waxy, and sweet corn. Analyses of amylopectins show that they behave as statistical condensation polymers (the model-V of Erlander and French) which have been debranched. Further support for the mechanism is given by the fact that the chain length and degree of branching of the interior part of all amylopectins is essentially the same as that of its precursor glycogen. Any mechanism for starch synthesis must, however, consider the complexing of enzymes because both amylopectin and glycogen behave as if they were strongly complexed to proteins. Thus proteases can destroy these complexes and viscosity differences of various barley geneotype starches can be correlated to protein content. Acid hydrolysis studies on corn amylopectins suggest that the type of protein complexed with amylopectin changes with both endosperm maturity and corn variety. Amylose synthesis from debranched chains can be correlated with the ability of chromosomes to regulate when and in what sequence an enzyme is synthesized. The proposed model for desoxyribonucleic acid (DNA) – which is a hollow cylinder with the bases on the outside – can explain how these regulators (genetic repressors and inducers) can function. „Proofs”︁ for the Watson-Crick model such as X-ray analyses, hypochromism, and molecular dimensions are shown to be erroneous.     

Starch Biosynthesis. 3: The Glycogen Precursor Mechanism Using Phosphorylase in the Production of the Precursor Glycogen

Based on the previously proposed glycogen precursor mechanism (S. R. Erlander, Enzymologia 19 (1958), 273–283), it is now proposed that phosphorylase is the primary enzyme for the production of linear chains in the precursor glycogen. The mechanism involves the translocation of ADPglucose (ADPGlu), and is suppressed by ATP because of a reverse of the ADPglucose pyrophosphorylase (ADPG pp) mechanism. Soluble starch synthase II (SSS II) is a back-up system, involves the translocation of glucose-6-phosphate (Glu-6-P), and is activated, not suppressed, by ATP, and is used if phosphorylase or the ADPGlu translocator is destroyed. Each system is independent and produces products which suppress the other. Hence, only one system works at a time. Both mechanisms produce a glycogen precursor and both are dependent upon ADPGlu pp. The initial higher radioactivity of amylose and the constant yield of amylose can be explained by a three or four day biosynthesis of this glycogen, followed by the removal of the glycogen's exterior branches by debranching enzymes to produce amylose and amylopectin. These removed branches are first degraded (using SSS I and II) to ADPGlu, which is the only source of ADPGlu for amylose synthesis. The retention of the polymodal behavior of debranched amylopectin in going from Bomi to shx barley amylopectins is most likely due to a change from phosphorylase to SSS II since both debranched amylopectins produce Poisson distributions.     

Rice Starch Molecular Size and its Relationship with Amylose Content

The composition and starch molecular structure of eight rice varieties were studied. Waxy and non‐waxy (long‐, medium‐, and short‐grain) rice varieties from California and Texas were used. The amylose contents were measured using the Concanavalin A method and were found to be related to the type of rice: waxy ≈ 1.0%, short and medium grain 8.7–15.4%, and long grain 17.1–19.9%. The weight‐average molar masses (M_w) of the starches varied from 0.52 to 1.96×10⁸ g/mol. As would be expected, a higher M_w of rice starch correlated to lower amylose content. The range of M_w of amylopectin was 0.82 to 2.50 ×10⁸ g/mol, and there was also a negative correlation of amylopectin M_w with amylose content. Amylose M_w ranged from 2.20 to 8.31×10⁵ g/mol. After debranching the amylopectin with isoamylase, the weight‐average degree of polymerization (DP_w) for the long‐chain fraction correlated positively with a higher amylose content. California and Texas varieties were significantly different in their amylose content, starch M_w (short‐ and medium‐grain only), and amylopectin M_w (p < 0.05).     

Untersuchungen an enzymatisch modifizierten verzweigten Polysacchariden. II. Sternpolymere aus Glykogen und Amylopektin als Strukturmodelle für Stärke

Studies on Enzymatically Modified Branched Polysaccharides. II. Star-shaped Polymers with Glycogen and Amylopectin as Structure Models for Starch . The number and length of outer chains of amylopectin and glycogen were varied (a) by partial debranching of amylopectin with pullulanase followed by synthesis with potato phosphorylase or, (b) using muscle phosphorylase without previous debranching. The resulting star molecules constructed of amylose chains grafted onto amylopectin or glycogen may be regarded as models for starch and conceivable intermediate products of starch components.     

Purification and Characterization of Extracellular Isoamylase from Flavobacterium sp

An extracellular isoamylase from Flavobacterium sp., was purified by fractionation with ammonium sulfate, DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose column chromatography. Single band of the debranching activity of the purified enzyme was detected by polyacrylamide gel electrophoresis. The enzyme efficiently hydrolyzed α-1,6-glucosidic linkage of glycogen and amylopectin and formed amylose chains, but did not hydrolyze pullulan. The enzyme released maltotriose from ß-limit dextrin of waxy maize amylopectin and glycogen, but no detectable maltose and glucose. Action of the isoamylase is similar to other microbial isoamylases but its physical properties are different.     

Varietal Differences in Properties Among High Amylose Rice Starches

Among three high-amylose starches differing in gel consistency, the hard gel starch (IR42), corresponding to harder cooked rice, had higher amylograph consistency and setback, higher gel viscosity in 0.2 N KOH and higher alkali viscograph peak than starch with soft (IR32) or medium (IR36) gel consistency. IR42 starch had less extractable starch and amylose in boiling water than IR32 and IR36 starches. The three starches had similar amyloses; the differences in gel consistency were due to the amylopectin fraction. IR42 amylopectin had higher iodine affinity, more long-chain linear fractions on isoamylase debranching and gel filtration or 1-butanol precipitation, and less DP_n 16–17 fraction than IR32 and IR36 amylopectins.     

Studies on the Origin of Amylose and Amylopectin in Starch Granules. II. Changes in Amylose Content and Enzyme Activities in Cultures of Polytoma uvella

Changes in amylose percentage and the activities of starch-synthesizing enzymes were followed in cultures of Polytoma uvella over a 6-day period. Phosphorylase, ADPG-glucosyltransferase, amylose content, ADPG and UDPG concentrations reached a peak in 96 hrs. old cultures. At this stage the cells of shaken cultures contained much higher enzyme activities and much more starch than those of stagnant cultures. After 96 hrs. phosphorylase and transferase activities declined sharply, amylose percentage decreased from 13.5 % to 6 %, but starch production continued, be it at a lower rate. Since Q-enzyme activity increased during this period, it is suggested that the decrease in amylose percentage was due to preponderant production of amylopectin. A high correlation existed between amylose percentage and starchbound transferase. Experiments were initiated to bring about differential inactivation of phosphorylase and ADPG-glucosyl-transferase by growing cultures at higher temperatures.     

Retrogradation of Sweetpotato Starch

Hardness and the percentage of leaked water of sweetpotato starch gels after storage were investigated as indexes of starch retrogradation. Starches of some varieties of sweetpotato were retrograded rapidly, but those of others were not retrograded during storage for one week. After one month of storage, starches of all varieties were highly retrograded, but the varietal order of hardness and the percentage of leaked water were almost the same as that after storage for one week. The study of chain length distribution by gel permeation chromatography after debranching by isoamylase showed that the hardness and the percentage of leaked water from the gel were positively correlated with amylose content and the proportion of Fr 1, the fraction of the highest molecular weight, containing amylose and extremely long chains of amylopectin, and negatively correlated with the proportion of Fr 3, the fraction with the lowest molecular weight, containing shorter chains of amylopectin. In addition, it was demonstrated by high performance anion exchange chromatography that the proportion of extra‐short chains (around DP 10) of amylopectin after isoamylase treatment was negatively correlated with the retrogradation index of starch. These results suggest that retrogradation of sweetpotato starch was promoted by amylose and extremely long chains of amylopectin and was inhibited by extra‐short chains (around DP 10) of amylopectin.     

Production and Application of a Maltogenic Amylase by a Strain of Thermomonospora viridis TF-35

An extracellular and thermostable maltogenic amylase-producing moderate thermophile (Thermomonospora viridis TF-35), which grew well at 28–60°C, with optima at 45°C and pH 7, was isolated from soil. Maximal enzyme production was attained after aerobical cultivation for 32 h at 42°C with a medium (pH 7.3) composed of 2% (w/v) soluble starch, 2% gelatin hydrolyzate, 0.1% K₂HPO₄ and 0.02% MgSO₄ · 7H₂O. The partially purified enzyme, which was most active at 60°C and pH 6.0 and stabilized with Ca²⁺, converted about 65, 80, 75, 75, 65 and 60% of maltotriose, maltotetraose, maltopentaose, amylose, amylopectin and glycogen into maltose as a major product under the conditions used, respectively. Glucose and small amounts of maltooligosaccharides were also formed concomitantly as by-products. The molar ratio of maltose to glucose from maltotriose were larger than 1 during all stages of the hydrolysis. About 70 and 76% of 25% (w/v) potato starch liquefites having a 3.5 DE value were converted into maltose by the enzyme in the absence and presence of pullulanase during the saccharification, respectively. About 90 and 94% of the starch liquefites were also converted into maltose with relatively low contents of maltooligosaccharides by the cooperative 2 step reaction with the enzyme after obtaining starch hydrolyzates containing about 85 and 90% maltose by the simultaneous actions of soybean ß-amylase and debranching enzymes.     

银杏支链淀粉分子结构的研究

应用酶法研究了银杏支链淀粉的精细分子结构。结果显示银杏支链淀粉的平均链长、外链和内链长分别为２５、１７和７：用异淀粉酶和普鲁兰酶作用于银杏支链淀粉β－极限糊精,得到银杏支链淀粉的Ａ、Ｂ链比值为１．６８：１,分支化度为２．６８。此结果也证实支链淀粉在生物体内的形成并非来源于糖原的前体物质。     

Lintnerization of Two Amylose‐free Starches of A‐ and B‐Crystalline Types,Respectively

Waxy maize starch (WMS) and potato amylopectin starch (PAPS), representing amylose‐free A‐ and B‐crystalline granules, respectively, were subjected to hydrolysis in diluted hydrochloric acid (lintnerization). The solubilization rate of the granules was dependent on the temperature, but there were only small differences between the samples. The compositions of the lintners obtained at 29°C were also similar, though the sensitivity to enzymatic debranching was different. The degree of debranching increased significantly if pullulanase and isoamylase were used successively. Because some branches remained resistant to enzymatic attack, the degree of scattered branching was difficult to estimate. The amount and composition of β‐limit dextrins suggested, however, that both starches contained scattered branches. Long chains and the shortest chains in the amylopectin molecules were sensitive to lintnerization and therefore probably located outside the crystallites. The composition of the PAPS lintners was dependent on the temperature of the lintnerization, whereas that of WMS lintners was not. The composition of a lintner from normal potato starch was compared with PAPS, and it is concluded that the presence of amylose has a higher effect than the type of crystallinity on the lintnerization results.     

Comparison Between Granular Starch Hydrolyzing Enzyme and Conventional Enzymes for Ethanol Production from Maize Starch with Different Amylose: Amylopectin Ratios

The effect of enzyme treatments (granular starch hydrolyzing and conventional enzymes) and different amylose: amylopectin ratios of maize starch on ethanol production was evaluated. For starch treatments, amylose: amylopectin ratios were prepared by mixing commercially available Hylon VII (70% amylose and 30% amylopectin) and Amioca (˜100% amylopectin) starches. For maize treatments, waxy, high amylose and regular dent hybrids were used to represent varying amylose: amylopectin ratios. All hydrolyses followed by fermentations were conducted at 15% solids content. Differences were observed in ethanol yields among granular starch hydrolyzing and conventional enzymes. Differences also were observed in ethanol yields between different amylose: amylopectin ratios for pure starch and maize samples. For starch samples, final ethanol concentrations varied from 2.2 to 9.1% (v/v) for fermentation with granular starch hydrolyzing enzyme and from 6.7 to 9.3% (v/v) for conventional enzymes. Higher ethanol concentrations were observed for Amioca starch for both enzymes. For maize samples, final ethanol concentrations were highest for waxy maize for both granular starch hydrolyzing (8.2%, v/v) and conventional (8.2%, v/v) enzymes. Lowest ethanol concentrations were observed for high‐amylose maize samples for granular starch hydrolyzing (6.3%, v/v) and conventional (5.2%, v/v) enzymes.     

Structural characterizations and digestibility of debranched high-amylose maize starch complexed with lauric acid

Structural characterizations and digestibility of debranched high-amylose maize starch complexed with lauric acid (LA) were studied. The cooked starch was debranched by using pullulanase and then complexed. Light microscopy showed that the lipids complexed starches had irregularly-shaped particles with strong birefringence. Gel-permeation chromatograms revealed that amylopectin degraded to smaller molecules during increasing debranching time, and the debranch reaction was completed at 12 h. Debranching pretreatment and prolonged debranching time (from 2 h to 24 h) could improve the formation of starch lipids complex. X-ray diffraction pattern of the amylose–lipid complexes changed from V-type to a mixture of B- and V-type polymorphs and relative crystallinity increased as the debranching time increased from 0 to 24 h. In DSC thermograms, complexes from debranched starch displayed three separated endotherms: the melting of the free lauric acid, starch–lipid complexes and retrograded amylose, respectively. The melting temperature and enthalpy changes of starch–lipid complex were gradually enhanced with the increasing of debranching time. However, no significant enthalpy changes were observed from retrograded amylose during the starch–lipid complex formation. Rapidly digestible starch (RDS) content decreased and resistant starch (RS) content increased with the increasing of debranching time, while the highest slowly digestible starch (SDS) content was founded at less debranching time of 2 h. The crystalline structures with dense aggregation of helices from amylose-LA complex and retrograded amylose could be RS, while SDS mostly consisted of imperfect packing of helices between amylopectin residue and amylose or LA.     

Amylose Content and Chain Profile of Amylopectin from Normal,High Amylose and Waxy Barleys

The amylose content and the chain profile of amylopectin from normal, waxy and high amylose barley starches were determined after enzymatic debranching and gel permeation chromatography and the degree of branching of the amylopectin was analysed by 1H-n.m.r. spectroscopy. The normal barley starch contained around 30%, the high amylose around 40% and the waxy starch 9% amylose. The amylopectin of the high amylose starches had longer chains than those of the normal or waxy starches, especially in the molecular weight interval 5,400-8,000, but less of those below 2,400 in molecular weight. The chain length of amylopectin from high amylose barley was on average 5 units longer than those of normal or waxy barleys.     

Starch Biosynthesis. I. The Size Distributions of Amylose and Amylopectin and Their Relationships to the Biosynthesis of Starch

Equations for the size distributions of both linear and branched polymers were applied to debranched amylopectin, linear amylose, and branched amylose polymers. The experimental size distribution of linear amylose corresponds to the broad size distribution of an A–B condensation polymer, whereas that of debranched amylopectin linear chains corresponds to the much narrower Poisson size distribution. These dramatic differences illustrate that different types of enzymes synthesize the linear chains of amylose as compared to those of amylopectin. These results support the previously proposed mechanism. The polymodal behavior of debranched amylopectin is due to the existance of individual Poisson-type polymers created by tier structures in a statistically formed precursor glycogen. Equations were developed which enable the calculation of the percentages of these individual Poisson polymers. When applied to the differences between shx and Bomi barley amylopectins, it is concluded that both studies agree that two different short, inner tier, A-chains exist, where the longer chain is located in the more external third tier in the amylopectins. In amylose, three different polymers exist: A linear amylose A–B type condensation polymer, a branched amylose which behaves as a statistical A–R–B₂ type polymer, and an intermediate, non-statistically branched amylose polymer.     

Pisum sativum and Vicia faba Carbohydrates: Structural Studies of Starches

The fine structure of laboratory purified broadbean and smooth pea starches, with an amylose content of 32-34%, has been studied by pullulanase debranching, before or after beta-amylolysis, and by the properties of the chemically fractionated amylose and amylopectin. The enzymatic study has shown the presence of the three chain populations (DP > 60, 45 and 15) observed with other starches. The linear DP 15 and 45 chains occur in a ratio of 8.5 for broadbean and 9.75 for pea, which indicates an amylopectin similar to cereal starches. The λ_max, beta-amylolysis limit and intrinsic viscosity of the two amylopectins confirm the cereal-like nature. The two amylose components are not completely linear according to their beta-amylolysis limit of 81.5% which corroborates the in complete debranching of the total starch. The physical structure, studied by X-ray diffractometry, is of C-type. By submitting legume starch granules to mild acid hydrolysis (lintnerization), a residue has been obtained from both starches, which has an increase in the crystalline fraction, with a tendency towards the A-type pattern for broadbean and the B-type for pea. The crystallites are mainly formed of linear chains (CL 15) with some singly branched material (DP 25). Gelatinization of starch granules occurs at 44-65-86°C for broadbean with a heat of gelatinization of 3.8 cal g^?1 and at 48-61-80°C for pea with a heat of gelatinization of 3.2 cal g^?1.     

Amylose and β‐Glucan Content of New Waxy Barleys

Starch was isolated from four new waxy barleys and compared with normal and high‐amylose barley starch. The waxy barley samples were selected lines from crosses of Swedish hulled and naked barley cultivars with the cultivar Azhul as donor of the waxy gene. The starches from the waxy barley samples were found to contain 0.7–2.6% amylose when determined iodimetrically by amperometric titration and 0.0–0.9% when determined by size exclusion chromatography after debranching. However, Sepharose CL‐2B elution profiles of the starches detected by iodine staining showed that all four waxy samples were free from detectable amounts of amylose. The amylopectin starches were found to contain a small polysaccharide fraction with molecular size smaller than amylopectin, with an iodine staining λ_max range of 550–600 nm. The water extractable and acid extractable β‐glucan contents in the waxy barley cultivars were generally found to be higher than those in normal barley.     

芋头淀粉的研究

本文研究了芋头淀粉的性质,实验采用电子显微镜拍摄了芋头淀粉颗粒的形态;运用X射线衍射仪测定了X-光衍射图样及结晶结构;通过重结晶法制得纯度较高的芋头直链淀粉和支链淀粉,利用高效液相色谱仪测定了芋头淀粉及其直、支链淀粉淀粉的重均聚合度、数均聚合度、分子量分布及分散度等,并与大米淀粉进行了比较.研究了芋头淀粉糊在不同质量分数、pH值以及不同蔗糖添加量的条件下,Micro Visco-Amylo-Graph黏度曲线的变化情况.本研究为进一步了解芋头淀粉的特性及应用开发提供了一定的理论依据.     

The Fine Structure of Corn Starches of Various Amylose-Percentage: Waxy,Normal and Amylomaize

The fine structure of high-amylose corn starches has been studied after dispersion of the starch and fractionation into their components amylose and amylopectin. The resulting amylopectin fraction reported in the literature possesses anomalous properties with regard to the waxy and normal amylopectin. However, the experimental results obtained by different authors for determining the structure lead to controversal explanations. Therefore, using an enzymic method, which permits the direct examination of the constitutive chains of the starch, the fine structure of the amylopectin of an amylomaize starch (64% amylose) has been investigated and compared with those of waxy and normal starches. The pullulanase – debranched chains are fractionated by gel permeation and their linearity are checked under the action of β-amylase. The inner chains of the amylopectin fraction are studied after debranching of the β-limit dextrins. The results show the identity between amylopectins from waxy and normal starches. The amylopectin fraction of amylomaize has its own structure with longer inner chains than those of waxy-maize amylopectin.     

Bestimmung der Molekulargewichtsverteilung in nativen Stärken durch Gelchromatographie

Determination of the Distribution of Molecular Weights in Native Starches by Gel Chromatography . The distribution of the molecular weights of polysaccharides in corn, potatoe and rice starch were investigated by fractionation of starch polysaccharides in a gel chromatographic system. The amylopectin component of the dispersed starch was excluded from the gel. Therefore the conclusion is likely that the molecular weight of amylopectin is higher than 20 × 10⁶. The gel columns were calibrated by chromatography of dextran fractions of known molecular weight. By comparison of the elution volumes of dextran fractions of know molecular weight with the elution volumes of amylose fractions it was possible to determine their molecular weights. From curves of molecular distribution the amount of amylose as percentage of total amylose can be determined. The average molecular weight of potatoe amylose (M̄_w 50%) is about 400 000 whereas it amounts to 100 000–200 000 in the case of rice and corn starch. The amylose of a “High Amylose Starch” (about 75% amylose) has a molecular weight of 50 000.