Carbon isotope ratios of amylose,amylopectin and mutant starches

Carbon isotope ratios (expressed as δ¹³C values) were determined for various sources of starch and the starch fractions amylose and amylopectin. The δ¹³C values of amylose were consistently less negative, 0.4–2.3 ‰, than those of amylopectin in kernal starch from maize (Zea mays) and barley (Hordeum vulgare) and in tuber starch from potato (Solanum tuberosum). Kernel starch isolated from the maize mutants wx1 and ae1, with known genetic lesions in the starch biosynthetic pathway, also showed significant differences in δ¹³C values. Collectively, these results suggest that variation in carbon isotope ratios in the amylose and amylopectin components of starch may be attributed to isotopic discrimination by the enzymes involved in starch biosynthesis.     

Distribution of methyl substituents in amylose and amylopectin from methylated potato starches

Granular potato starches were methylated in aqueous suspension with dimethyl sulfate to molar substitution (MS) values up to 0.29. Fractions containing mainly amylose or amylopectin were obtained after aqueous leaching of the derivatised starch granules. Amylopectin in these fractions was precipitated with Concanavalin A to separate it from amylose. Amylose remained in solution and was enzymatically converted into D-glucose for quantification, thereby taking into account the decreased digestibility due to the presence of methyl substituents. It was found that the MS of amylose was 1.6-1.9 times higher than that of amylopectin in methylated starch granules. The distributions of methyl substituents in trimers and tetramers, prepared from amylose- or amylopectin-enriched fractions, were determined by FAB mass spectrometry and compared with the outcome of a statistically random distribution. It turned out that substituents in amylopectin were distributed heterogeneously, whereas substitution of amylose was almost random. The results are rationalised on the basis of an organised framework that is built up from amylopectin side chains. The crystalline lamellae are less accessible for substitution than amorphous branching points and amylose.     

Structural and thermodynamic properties of rice starches with different genetic background Part 2. Defectiveness of different supramolecular structures in starch granules

High-sensitivity differential scanning microcalorimetry (HSDSC), small-angle X-ray scattering (SAXS), light (LM) and scanning electronic (SEM) microscopy techniques were used to study the defectiveness of different supramolecular structures in starches extracted from 11 Thai cultivars of rice differing in level of amylose and amylopectin defects in starch crystalline lamellae. Despite differences in chain-length distribution of amylopectin macromolecules and amylose level in starches, the invariance in the sizes of crystalline lamellae, amylopectin clusters and granules was established. The combined analysis of DSC, SAXS, LM and SEM data for native starches, as well as the comparison of the thermodynamic data for native and annealed starches, allowed to determine the structure of defects and the localization of amylose chains in crystalline and amorphous lamellae, defectiveness of lamellae, clusters and granules. It was shown that amylose “tie chains”, amylose–lipid complexes located in crystalline lamellae, defective ends of double helical chains dangling from crystallites inside amorphous lamellae (“dangling” chains), as well as amylopectin chains with DP 6–12 and 25–36 could be considered as defects. Their accumulation can lead to a formation of remnant granules. The changes observed in the structure of amylopectin chains and amylose content in starches are reflected in the interconnected alterations of structural organization on the lamellar, cluster and granule levels.     

Structural and thermodynamic properties of rice starches with different genetic background Part 1. Differentiation of amylopectin and amylose defects

A combined DSC - HPAEC-PAD approach, gel permeation chromatography and mild long-term acidic hydrolysis were employed to study the effects of amylopectin chain-length distributional and amylose defects on the assembly structures of amylopectin (crystalline lamellae, amylopectin clusters) in A-type polymorphic starches extracted from 11 Thai cultivars of rice with different amylose level. Joint analysis of the data allowed determining the contributions of different populations of amylopectin chains to the thermodynamic melting parameters of crystalline lamellae. It was shown that amylopectin chains with DP 6-12 and 25or=37 could be related to chains stabilizing these structures. The total effect of amylose and amylopectin defects can be described by means of Thomson-Gibbs' equation. The increase of defects in the assembly structures is accompanied by rise of the rates of acidic hydrolysis of both amorphous and crystalline parts in starches.     

Structural parameters of amylopectin clusters and semi-crystalline growth rings in wheat starches with different amylose content

Small-angle X-ray scattering (SAXS) and scanning electron microscopy (SEM) were used to investigate the internal structure of wheat starch granules with different amylose content. Different approaches were used for treatment (interpretation) of SAXS data to assess the values of structural parameters of amylopectin clusters and the size of crystalline and amorphous lamella in different wheat starches. The average values of the semi-crystalline growth rings thickness in starches have been determined and the relationship between structural characteristics and thermodynamic melting parameters is discussed.     

Modulation of substrate preference of thermus maltogenic amylase by mutation of the residues at the interface of a dimer

To elucidate the relationship between the substrate size and geometric shape of the catalytic site of Thermus maltogenic amylase, Gly50, Asp109, and Val431, located at the interface of the dimer, were replaced with bulky amino acids. The k(cat)/K(m) value of the mutant for amylose increased significantly, whereas that for amylopectin decreased as compared to that of the wild-type enzyme. Thus, the substituted bulky amino acid residues modified the shape of the catalytic site, such that the ability of the enzyme to distinguish between small and large molecules like amylose and amylopectin was enhanced.     

Biochemical characterization of alpha-amylase from the yeast Cryptococcus flavus

During our screening of amylolytic microorganisms from Brazilian fruits, we isolated a yeast strain classified as Cryptococcus flavus. When grown on starch-containing medium this strain exhibited the highest amylase production after 24 h of cultivation. The extracellular amylase from C. flavus was purified from the culture broth by a single step using chromatography on a Sephacryl S-100 column. The enzyme was purified 16.14-fold with a yield of 50.21% of the total activity. The purified enzyme was a glycoprotein with an apparent molecular mass of 75 and 84.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The enzyme lost approximately 50% of the molecular mass after treatment with glycosidases. The major end products of starch, amylose, amylopectin, pullulan and glycogen were maltose and maltotriose. The K(m) value for the pure enzyme was 0.056 mg ml(-1) with soluble starch as the substrate. Enzyme activity was optimal at pH 5.5 and 50 degrees C. The enzyme retained 90% of the activity after incubation at 50 degrees C for 60 min and was inhibited by Cu(2+), Fe(2+) and Hg(2+).     

Locations of hypochlorite oxidation in corn starches varying in amylose content

The general oxidation mechanism by hypochlorite on starch has been well studied, but the information on the distribution of the oxidation sites within starch granules is limited. This study investigated the locations where the oxidation occurred within corn starch granules varying in amylose content, including waxy corn starch (WC), common corn starch (CC), and 50% and 70% high-amylose corn starch (AMC). Oxidized corn starches were surface gelatinized by 13 M LiCl at room temperature to different extents (approximately 10%, 20%, 30%, and 40%). The surface-gelatinized remaining granules were separated and studied for structural characteristics including carboxyl content, amylose content, amylopectin chain-length distribution, thermal properties, and swelling properties. Oxidation occurred mostly at the amorphous lamellae. More carboxyl groups were found at the periphery than at the core of starch granules, which was more pronounced in oxidized 70% AMC. More amylose depolymerization from oxidation occurred at the periphery of CC. For WC and CC, amylopectin long chains (>DP 36) were more prone to depolymerization by oxidation. The gelatinization properties as measured by differential scanning calorimetry also supported the changes in amylopectin fine structure from oxidation. Oxidized starches swelled to a greater extent than their unmodified counterparts at all levels of surface removal. This study demonstrates that the locations of oxidation and physicochemical properties of oxidized starches are affected by the molecular arrangement within starch granules.     

Physicochemical properties of endosperm and pericarp starches during maize development

Endosperm starch and pericarp starch were isolated from maize (B73) kernels at different developmental stages. Starch granules, with small size (2–4 μm diameter), were first observed in the endosperm on 5 days after pollination (DAP). The size of endosperm-starch granules remained similar until 12DAP, but the number increased extensively. A substantial increase in granule size was observed from 14DAP (diameter 4–7 μm) to 30DAP (diameter10–23 μm). The size of starch granules on 30DAP is similar to that of the mature and dried endosperm-starch granules harvested on 45DAP. The starch content of the endosperm was little before 12DAP (less than 2%) and increased rapidly from 10.7% on 14DAP to 88.9% on 30DAP. The amylose content of the endosperm starch increased from 9.2% on 14DAP to 24.2% on 30DAP and 24.4% on 45DAP (mature and dried). The average amylopectin branch chain-length of the endosperm amylopectin increased from DP23.6 on 10DAP to DP26.9 on14DAP and then decreased to DP25.4 on 30DAP and DP24.9 on 45DAP. The onset gelatinization temperature of the endosperm starch increased from 61.3 °C on 8DAP to 69.0 °C on 14DAP and then decreased to 62.8 °C on 45DAP. The results indicated that the structure of endosperm starch was not synthesized consistently through the maturation of kernel. The pericarp starch, however, showed similar granule size, starch content, amylose content, amylopectin structure and thermal properties at different developmental stages of the kernel.     

Characterization of barley starches of waxy, normal, and high amylose varieties

Four varieties of barley starches, W.B. Merlin, glacier, high amylose glacier, and high amylose hull-less glacier, were isolated from barley seeds. Apparent and absolute amylose contents, molecular size distributions of amylose and amylopectin, amylopectin branch-chain-length distributions, and Naegeli dextrin structures of the starches were analyzed. W.B. Merlin amylopectin had the longest detectable chain length of DP 67, whereas glacier, high amylose glacier and high amylose hull-less glacier amylopectins had the longest detectable chain length of DP 82, 79, and 78, respectively. All the four starches displayed a substantially reduced proportion of chains at DP 18–21. Amylopectins of high amylose varieties did not show significantly larger proportions of long chains than that of normal and waxy barley starch. Onset gelatinization temperatures of all four barley starches ranged from 55.0 to 56.5°C. Absolute amylose contents of W.B. Merlin, glacier, high amylose glacier, and high amylose hull-less glacier were 9.1, 29.5, 44.7, and 43.4%, respectively; phospholipid contents were 0.36, 0.78, 0.79, and 0.97%, respectively.     

Structural characteristics and physicochemical properties of oxidized corn starches varying in amylose content

The effects of amylose content on the extent of oxidation and the distribution of carboxyl groups in hypochlorite-oxidized corn starches were investigated. Corn starches including waxy corn starch (WC), common corn starch (CC), and 50% and 70% high-amylose corn starches (AMC) were oxidized with NaOCl at three concentrations (0.8%, 2%, and 5%). Carboxyl and carbonyl content of oxidized starches increased with increasing NaOCl concentration. High-AMC (70%) had slightly higher carboxyl and carbonyl contents at 0.8% NaOCl, whereas WC had significantly higher carboxyl and carbonyl contents at 2% and 5% NaOCl levels. Carbohydrate profiles by high-performance size-exclusion chromatography indicate that amylose was more susceptible to depolymerization than amylopectin. Degradation of amylopectin long chains (DP >24) was more pronounced in WC and CC than in AMCs. The crystalline lamellae of WC started to degrade at 2% NaOCl, but those of the other corn starches remained intact even at 5% NaOCl level according to X-ray crystallinity. By using anion-exchange chromatography for separation and size-exclusion chromatography for characterization, carboxyl groups were found to be more concentrated on amylopectin than on amylose, particularly in AMCs. Oxidation decreased gelatinization temperature and enthalpy with WC showing the most decrease and 70% AMC showing the least. The gelatinization enthalpy of 50% AMC decreased significantly faster than those of CC and 70% AMC after 0.8% oxidation. Retrogradation of amylopectin slightly increased after oxidation with increasing oxidation level. The peak viscosities of oxidized WC and CC were higher than those of their native counterparts at 0.8% NaOCl, but this increase was not observed in AMCs. The setback viscosities of 2% NaOCl-oxidized 50% and 70% AMCs were much higher than those of the unmodified counterparts. The extent of oxidation and physicochemical properties of oxidized starches varied greatly with the amylase:amylopectin ratio of corn starches. Amylose was suggested to play an important role in controlling the oxidation efficiency.     

Retrograded starches as potential anodes in lithium-ion rechargeable batteries

Retrograded starch is a crystal formed by starch molecules with hydrogen bonds. Many literatures have reported its physicochemical character, but its crystal structure is so far unclear. As we isolate amylose and amylopectin from retrograded maize, sweet potato and potato starches in 4.0M KOH solutions and make them retrograde alone in neutral solution (adjusted by HCl) to form crystal, a new phenomenon appears, crystals of KCl do not appear in retrograded potato amylose, potato amylopectin, and maize amylose, indicating that those crystals may absorb K(+) and (or) Cl(-), and those ions probably act with aldehyde of starch or hydroxy of fatty acid attached in starch, such characteristic may make retrograded starches replace graphite as anode with high-capacity in lithium-ion rechargeable batteries.     

Internal structure and physicochemical properties of corn starches as revealed by chemical surface gelatinization

The organization of amylose and amylopectin within starch granules is still not well elucidated. This study investigates the radial distribution of amylose and amylopectin in different corn starches varying in amylose content (waxy corn starch (WC), common corn starch (CC), and 50% and 70% amylose corn starches (AMC)). Corn starches were surface gelatinized by 13 M LiCl at room temperature to different extents (approximately 10%, 20%, 30%, and 40%). The gelatinized surface starch and remaining granules were characterized for amylose content, amylopectin chain-length distribution, thermal properties, swelling power (SP), and water solubility index (WSI). Except for the outmost 10% layer, the amylose content in CC increased slightly with increasing surface removal. In contrast, amylose was more concentrated at the periphery than at the core for 50% and 70% AMC. The proportion of amylopectin A chains generally decreased while that of B1 chains generally increased with increasing surface removal for all corn starches. The gelatinization enthalpy usually decreased, except for 70% AMC, whereas the retrogradation enthalpy relatively remained unchanged for CC but increased for WC, 50% and 70% AMC with increasing surface removal. The SP and WSI increased with increasing surface removal for all corn starches, with WC showing a significant increase in SP after the removal of the outmost 10% layer. The results of this study indicated that there were similarities and differences in the distribution of amylose and amylopectin chains along the radial location of corn starch granules with varying amylose contents. More amylose-lipid complex and amylopectin long chains were present at the periphery than at the core for amylose-containing corn starches.     

Structural and thermodynamic properties of starches extracted from GBSS and GWD suppressed potato lines

A combined DSC–SAXS approach was employed to study the effects of amylose and phosphate esters on the assembly structures of amylopectin in B-type polymorphic potato tuber starches. Amylose and phosphate levels in the starches were specifically engineered by antisense suppression of the granule bound starch synthase (GBSS) and the glucan water dikinase (GWD), respectively. Joint analysis of the SAXS and DSC data for the engineered starches revealed that the sizes of amylopectin clusters, thickness of crystalline lamellae and the polymorphous structure type remained unchanged. However, differences were found in the structural organization of amylopectin clusters reflected in localization of amylose within these supramolecular structures. Additionally, data for annealed starches shows that investigated potato starches possess different types of amylopectin defects. The relationship between structure of investigated potato starches and their thermodynamic properties was recognized.     

Structure function relationships of transgenic starches with engineered phosphate substitution and starch branching

Potato tuber starch was genetically engineered in the plant by the simultaneous antisense suppression of the starch branching enzyme (SBE) I and II isoforms. Starch prepared from 12 independent lines and three control lines were characterised with respect to structural and physical properties. The lengths of the amylopectin unit chains, the concentrations of amylose and monoesterified phosphate were significantly increased in the transgenically engineered starches. Size exclusion chromatography with refractive index detection (SEC-RI) indicated a minor decrease in apparent molecular size of the amylose and the less branched amylopectin fractions. Differential scanning calorimetry (DSC) revealed significantly higher peak temperatures for gelatinisation and retrogradation of the genetically engineered starches whereas the enthalpies of gelatinisation were lower. Aqueous gels prepared from the transgenic starches showed increased gel elasticity and viscosity. Principle component analysis (PCA) of the data set discriminated the control lines from the transgenic lines and revealed a high correlation between phosphate concentration and amylopectin unit chain length. The PCA also indicated that the rheological characteristics were primarily influenced by the amylose concentration. The phosphate and the amylopectin unit chain lengths had influenced primarily the pasting and rheological properties of the starch gels.     

Structures of starches from rice mutants deficient in the starch synthase isozyme SSI or SSIIIa

Amylose and amylopectin of rice mutants deficient in a starch synthase (SS) isozyme in the endosperm, either SSI (ΔSSI) or SSIIIa (ΔSSIIIa), were structurally altered from those of their parent (cv. Nipponbare, Np). The amylose content was higher in the mutants (Np, 15.5%; ΔSSI, 18.2%; ΔSSIIIa, 23.6%), and the molar ratio of branched amylose and its side chains was increased. The chain-length distribution of the β-amylase limit dextrins of amylopectin showed regularity, which appeared consistent with the generally accepted cluster structure, and the degrees of polymerization found at the intersections were taken as the boundaries of the B-chain fractions. The mole % of the B(1)-B(3) fractions was changed slightly in ΔSSI, which is consistent with the proposed role of SSI in elongating the external part of clusters. In ΔSSIIIa, a significant increase in the B(1) fraction and a decrease in the B(2) and B(3) fractions were observed. The internal chain length of the B(2) and B(3) fractions appeared to be slightly altered, suggesting that the deficiency in SS affected the actions of branching enzyme(s).     

Preparation and characterization of soluble starches having different molecular sizes and composition,by acid hydrolysis in different alcohols

Potato and waxy-maize starches were separately modified for 1 h at 65° with 0.36% hydrochloric acid in methanol, ethanol, 2-propanol, and 1-butanol. All of the modified starches were readily soluble in hot water, to give crystal-clear solutions up to a concentration of at least 20% (w/v). The modified granules were studied by light-microscopy and iodine-iodide staining. All of the modified starches retained their granule appearance, although with various degrees of damage that progressively increased from methanol to 1-butanol. Both hydrolysis and alcoholysis occurred, but to different extents in the different alcohols. The highest proportion of alcoholysis occurred in methanol where 50% of the resulting molecules were glycosides, the lowest in 1-butanol where 6% were glycosides. The number-average molecular weights of the modified starches also progressively decreased from 126,670 for the methanol-modified waxy-maize starch to 4,750 for the 1-butanol-modified potato starch. The methanol- and ethanol-modified potato starches were fractionated into amylose and amylopectin components. The 2-propanol- and 1-butanol-modified potato starches gave only an amylopectin component. The amylose components were characterized by gel-permeation chromatography on Bio-Gel A-5m, and the amylopectin components, on Bio-Gels A-150m and A-0.5m. The molecular sizes of the amylose and amylopectin components progressively decreased from methanol- to 1-butanol-modified starches. Furthermore, the polymodal composition of the amylopectin component was decreased to give a more homogeneous product. Waxy-maize starch was modified in methanol and 2-propanol and gave products that were of lower molecular size and more homogeneous than the polymodal native starch. It is shown that the differential effect of the different alcohols on the modification of the starch granules is produced by effecting different concentrations of acid inside the granule, where hydrolysis occurs in the 10–12% of water contained in the granule. It is postulated that 2-propanol and 1-butanol dissolve the double-helical, crystalline regions in the starch granule to give different types of products under otherwise identical conditions of modification.     

Multiple substitutions of methionine 129 in human prion protein reveal its importance in the amyloid fibrillation pathway

The role of the polymorphism Met or Val in position 129 in the human prion protein is well documented regarding disease susceptibility and clinical manifestations. However, little is known about the molecular background to this phenomenon. We investigated herein the conformational stability, amyloid fibrillation kinetics, and seeding propensity of different 129 mutants, located in β-strand 1 of PrP (Met(129) (WT), M129A, M129V, M129L, M129W, M129P, M129E, M129K, and M129C) in HuPrP(90-231). The mutations M129V, M129L, M129K, and M129C did not affect stability (midpoints of thermal denaturation, T(m) = 65-66 °C), whereas the mutants M129A and M129E and the largest side chain M129W were destabilized by 3-4 °C. The most destabilizing substitution was M129P, which lowered the T(m) by 7.2 °C. All mutants, except for M129C, formed amyloid-like fibrils within hours during fibril formation under near physiological conditions. Fibril-forming mutants showed a sigmoidal kinetic profile and showed shorter lag times during seeding with preformed amyloid fibrils implicating a nucleated polymerization reaction. In the spontaneous reactions, the lag time of fibril formation was rather uniform for the mutants M129A, M129V, and M129L resembling the wild type. When the substituted amino acid had a distinct feature discriminating it from the wild type, such as size (M129W), charge (M129E, M129K), or rotational constraint (M129P), the fibrillation was impeded. M129C did not form ThT/Congo red-positive fibrils, and non-reducing SDS-PAGE of M129C during fibrillation conditions at different time points revealed covalent dimer formation already 15 min after fibrillation reaction initiation. Position 129 appears to be a key site for dictating PrP receptiveness toward recruitment into the amyloid state.     

Contribution of the region Glu181 to Val200 of the extracellular loop of the human P2X1 receptor to agonist binding and gating revealed using cysteine scanning mutagenesis1

At the majority of mutants in the region Glu181-Val200 incorporating a conserved AsnPheThrΦΦxLys motif cysteine substitution had no effect on sensitivity to ATP, partial agonists, or methanethiosulfonate (MTS) compounds. For the F185C mutant the efficacy of partial agonists was reduced by ∼ 90% but there was no effect on ATP potency or the actions of MTS reagents. At T186C, F188C and K190C mutants ATP potency and partial agonists responses were reduced. The ATP sensitivity of the K190C mutant was rescued towards WT levels by positively charged (2-aminoethyl)methanethiosulfonate hydrobromide and reduced by negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate. Both MTS reagents decreased ATP potency at the T186C mutant, and abolished responses at the F195C mutant. ³²P-2-azido ATP binding to the mutants T186C and K190C was sensitive to MTS reagents consistent with an effect on binding, however binding at F195C was unaffected indicating an effect on gating. The accessibility of the introduced cysteines was probed with (2-aminoethyl)methanethiosulfonate hydrobromide-biotin, this showed that the region Thr186-Ser192 is likely to form a beta sheet and that accessibility is blocked by ATP. Taken together these results suggest that Thr186, Phe188 and Lys190 are involved in ATP binding to the receptor and Phe185 and Phe195 contribute to agonist evoked conformational changes.