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In this paper, a detailed characterization of the mechanisms at the origin of the shape-memory effect in amorphous potato starch is presented. Using different treatments (annealing) and preparation methods (hot casting and extrusion), the local structures responsible for the shape-memory were disrupted, as evidenced in the first part of the article detailing the macroscopic properties: mechanical, calorimetric and shape-memory. In the second part the macromolecular scale is investigated using X-rays diffraction and CP-MAS NMR, and thus allows making the link between the structural differences and the macroscopic properties. Finally we discuss the origin of shape-memory in amorphous starch., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The many studies about amylolysis have collected considerable information regarding the contribution of the starch physico-chemical properties. But the inherent elaborate and variable structure of granular starch and, consequently, the multifactorial condition of the system hinders the interpretation of the experimental results. The immediate benefit of multivariate statistical analysis approaches with that regard is twofold: considering the factors, possibly interrelated, all together and not independently, providing a first estimation of the magnitude and confidence level of the relations between factors and amylolysis kinetic parameters. Based on data of amylolysis of 13 starch samples from wild type, single and double mutants of maize by porcine pancreatic α-amylase (PPA), a multivariate analysis is proposed. Amylolysis progress-curves were fitted by a Weibull function, as proposed in a previous work, to extract three kinetic parameters: the reaction rate coefficient during the first time-unit, k, the reaction rate retardation over time, h, and the final hydrolysis extent, X∞. Multivariate models relate the macromolecular composition and the fractions of crystalline polymorphic types to the kinetic parameters. h and X∞ are found to be highly related to the measured properties. Thus the amylose content appears to be significantly correlated to the hydrolysis rate retardation, which sheds some light on the probable contribution of the amylose molecules contained in the granules. The multivariate models give correct prediction performances except for k whose a part of variability remains unexplained. A further analysis points out the extent of the characterisation effort of the granule structure needed to extend the fraction of explained variability., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Two empirical models, a conventional first-order kinetics and a fractal-like first-order kinetic model were tested for analysing the hydrolysis of 13 wild type, single and double mutants of maize starch by porcine pancreatic α-amylase (PPA). The major difference between the two models is an additional parameter, the fractal kinetics exponent h, which enables to characterise a decrease of the reaction rate coefficient over time. The fractal-like kinetic model should be preferred to characterise the amylolysis for 10 mutants out of 13 as sugary-2 and amylose-extender curves exhibit clear reaction rate retardation, unlike normal maize and waxy maize. Analysis of the model parameter values reveals two groups of kinetics for the maize mutants: amylose-extender, normal and waxy on one hand, sugary-2 on the other hand. Possible relations between the parameters of the model and granule composition and structure are discussed., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

1-10% starch/clay bionanocomposites with shape memory properties were obtained by melt processing. X-ray diffraction (XRD) and TEM evidenced the presence of a major fraction of clay tactoids, consisting of 4-5 stacked crystalline layers, with a thickness of 6.8 nm. A significant orientation of the nanoparticles induced by extrusion was also observed. Tensile tests performed above the glass transition of the materials showed that the presence of clay nanoparticles leads to higher elastic modulus and maximum stress, without significant loss in elongation at break which typically reached 100%. Samples submitted to a 50% elongation and cooled below the glass transition showed shape memory behavior. Like unreinforced starch, the bionanocomposites showed complete shape recovery in unconstrained conditions. In mechanically constrained conditions, the maximum recovered stress was significantly improved for the bionanocomposites compared to unreinforced starch, opening promising perspectives for the design of sensors and actuators., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Brachypodium distachyon is a non-domesticated cereal. Nonetheless, Brachypodium was recently introduced as a model plant for temperate cereals. This study compares grain starch metabolism in Brachypodium and barley (Hordeum vulgare). In Brachypodium, we identified and annotated 28 genes involved in starch metabolism and identified important motifs including transit peptides and putative carbohydrate-binding modules (CBMs) of the families CBM20, CBM45, CBM48, and CBM53. Starch content was markedly lower in Brachypodium grains (12%) compared to barley grains (47%). Brachypodium starch granules were doughnut shaped and bimodally distributed into distinct small B-type (2.5-10 µm) and very small C-type (0.5-2.5 µm) granules. Large A-type granules, typical of cereals, were absent. Starch-bound phosphate, important for starch degradation, was 2-fold lower in Brachypodium compared with barley indicating different requirements for starch mobilization. The amylopectin branch profiles were similar and the amylose content was only slightly higher compared with barley cv. Golden Promise. The crystallinity of Brachypodium starch granules was low (10%) compared to barley (20%) as determined by wide-angle X-ray scattering (WAXS) and molecular disorder was confirmed by differential scanning calorimetry (DSC). The expression profiles in grain for most genes were distinctly different for Brachypodium compared to barley, typically showing earlier decline during the course of development, which can explain the low starch content and differences in starch molecular structure and granule characteristics. High transitory starch levels were observed in leaves of Brachypodium (2.8% after 14h of light) compared to barley (1.9% after 14h of light). The data suggest important pre-domesticated features of cereals., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Background: Native starch accumulates as granules containing two glucose polymers: amylose and amylopectin. Phosphate (0.2-0.5%) and proteins (0.1-0.7%) are also present in some starches. Phosphate groups play a major role in starch metabolism while granule-bound starch synthase 1 (GBSS1) which represents up to 95% of the proteins bound to the granule is responsible for amylose biosynthesis., Methods: Synchrotron micro-X-ray fluorescence (μXRF) was used for the first time for high-resolution mapping of GBSS1 and phosphate groups based on the XRF signal of sulfur (S) and phosphorus (P), respectively. Wild-type starches were studied as well as their related mutants lacking GBSS1 or starch-phosphorylating enzyme., Results: Wild-type potato and maize starch exhibited high level of phosphorylation and high content of sulfur respectively when compared to mutant potato starch lacking glucan water dikinase (GWD) and mutant maize starch lacking GBSS1. Phosphate groups are mostly present at the periphery of wild-type potato starch granules, and spread all over the granule in the amylose-free mutant. P and S XRF were also measured within single small starch granules from Arabidopsis or Chlamydomonas not exceeding 3-5μm in diameter., Conclusions: Imaging GBSS1 (by S mapping) in potato starch sections showed that the antisense technique suppresses the expression of GBSS1 during biosynthesis. P mapping confirmed that amylose is mostly present in the center of the granule, which had been suggested before., General Significance: μXRF is a potentially powerful technique to analyze the minor constituents of starch and understand starch structure/properties or biosynthesis by the use of selected genetic backgrounds., (© 2013.)

Lowering of the CO₂ concentration in the environment induces development of a pyrenoidal starch sheath, as well as that of pyrenoid and CO₂-concentrating mechanisms, in many microalgae. In the green algae Chlamydomonas and Chlorella, activity of granule-bound starch synthase (GBSS) concomitantly increases under these conditions. In this study, effects of the GBSS-defective mutation (sta2) on the development of pyrenoidal starch were investigated in Chlamydomonas. Stroma starch- and pyrenoid starch-enriched samples were obtained from log-phase cells grown with air containing 5% CO₂ (high-CO₂ conditions favouring stromal starch synthesis) and from those transferred to low-CO₂ conditions (air level, 0.04% CO₂, favouring pyrenoidal starch synthesis) for 6h, respectively. In the wild type, total starch content per culture volume did not increase during the low-CO₂ conditions, in spite of the development of pyrenoidal starch, suggesting that degradation of some part of stroma starch and synthesis of pyrenoid starch simultaneously occur under these conditions. Even in the GBSS-deficient mutants, pyrenoid and pyrenoid starch enlarged after lowering of the CO₂ concentration. However, the morphology of the pyrenoid starch was thinner and more fragile than the wild type, suggesting that GBSS does affect the morphology of pyrenoidal starch. Surprisingly normal GBSS activity is shown to be required to obtain the high A-type crystallinity levels that we now report for pyrenoidal starch. A model is presented explaining how GBSS-induced starch granule fusion may facilitate the formation of the pyrenoidal starch sheath., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Synchrotron UV fluorescence microscopy was used for the first time to visualize the adsorption and diffusion of an enzyme while degrading a solid substrate. The degradation pathway of single starch granules by two amylases, optimized for biofuel production and industrial starch hydrolysis, was followed by tryptophan fluorescence (excitation at 280 nm, emission filter at 300-400 nm) and visible light imaging. Thus, both the adsorption of enzyme onto starch granules at 283 nm resolution and the resulting morphological changes were recorded at different stages of hydrolysis. It is the first time that amylases were localized on starch without staining or adding a fluorescent probe at such high resolution. This technique presents a very high potential for imaging proteins in complex systems. Its sensitivity was demonstrated by the detection of GBSS (the granular bound starch synthase) at high recording times, GBSS being present at very low levels in maize starch granules.

A new α-amylase from Rhizomucor sp. (RA) was studied in detail due to its very efficient hydrolysis of raw starch granules at low temperature (32 °C). RA contains a starch binding domain (SBD) connected to the core amylase catalytic domain by a O-glycosylated linker. The mode of degradation of native maize starch granules and, in particular, the changes in the starch structure during the hydrolysis, was monitored for hydrolysis of raw starch at concentrations varying between 0.1 and 31%. RA was compared to porcine pancreatic α-amylase (PPA), which has been widely studied either on resistant starch or as a model enzyme in solid starch hydrolysis studies. RA is particularly efficient on native maize starch and release glucose only. The hydrolysis rate reaches 75% for a 31% starch solution and is complete at 0.1% starch concentration. The final hydrolysis rate was dependent on both starch concentration and enzyme amount applied. RA is also very efficient in hydrolyzing the crystalline domains in the maize starch granule. The major A-type crystalline structure is more rapidly degraded than amorphous domains in the first stages of hydrolysis. This is in agreement with the observed preferential hydrolysis of amylopectin, the starch constituent that forms the backbone of the crystalline part of the granule. Amylose-lipid complexes present in most cereal starches are degraded in a second stage, yielding amylose fragments that then reassociate into B-type crystalline structures, forming the final resistant fraction.

The nature of the cytoplasmic pathway of starch biosynthesis was investigated in the model heterotrophic dinoflagellate Crypthecodinium cohnii. The storage polysaccharide granules were shown to be composed of both amylose and amylopectin fractions with a chain length distribution and crystalline organization very similar to those of green algae and land plant starch. Preliminary characterization of the starch pathway demonstrated that C. cohnii contains multiple forms of soluble starch synthases and one major 110-kDa granule-bound starch synthase. All purified enzymes displayed a marked substrate preference for UDP-glucose. At variance with most other microorganisms, the accumulation of starch in the dinoflagellate occurs during early and mid-log phase, with little or no synthesis witnessed when approaching stationary phase. In order to establish a genetic system allowing the study of cytoplasmic starch metabolism in eukaryotes, we describe the isolation of marker mutations and the successful selection of random recombinant populations after homothallic crosses.

Eukaryotic cells are composed of a variety of membrane-bound organelles that are thought to derive from symbiotic associations involving bacteria, archaea, or other eukaryotes. In addition to acquiring the plastid, all Archaeplastida and some of their endosymbiotic derivatives can be distinguished from other organisms by the fact that they accumulate starch, a semicrystalline-storage polysaccharide distantly related to glycogen and never found elsewhere. We now provide the first evidence for the existence of starch in a particular species of single-cell diazotrophic cyanobacterium. We provide evidence for the existence in the eukaryotic host cell at the time of primary endosymbiosis of an uridine diphosphoglucose (UDP-glucose)-based pathway similar to that characterized in amoebas. Because of the monophyletic origin of plants, we can define the genetic makeup of the Archaeplastida ancestor with respect to storage polysaccharide metabolism. The most likely enzyme-partitioning scenario between the plastid's ancestor and its eukaryotic host immediately suggests the precise nature of the ancient metabolic symbiotic relationship. The latter consisted in the export of adenosine diphosphoglucose (ADP-glucose) from the cyanobiont in exchange for the import of reduced nitrogen from the host. We further speculate that the monophyletic origin of plastids may lie in an organism with close relatedness to present-day group V cyanobacteria.

The nature of the cytoplasmic pathway of starch biosynthesis was investigated in the model glaucophyte Cyanophora paradoxa. The storage polysaccharide granules are shown to be composed of both amylose and amylopectin fractions, with a chain length distribution and crystalline organization similar to those of green algae and land plant starch. A preliminary characterization of the starch pathway demonstrates that Cyanophora paradoxa contains several UDP-glucose-utilizing soluble starch synthase activities related to those of the Rhodophyceae. In addition, Cyanophora paradoxa synthesizes amylose with a granule-bound starch synthase displaying a preference for UDP-glucose. A debranching enzyme of isoamylase specificity and multiple starch phosphorylases also are evidenced in the model glaucophyte. The picture emerging from our biochemical and molecular characterizations consists of the presence of a UDP-glucose-based pathway similar to that recently proposed for the red algae, the cryptophytes, and the alveolates. The correlative presence of isoamylase and starch among photosynthetic eukaryotes is discussed.

Films of acid-hydrolyzed hydroxypropylated pea starch with average molecular weight M w ranging from 3.3 x 10 (4) g/mol to 1.6 x 10 (6) g/mol were prepared from 25% (w/w) solution by casting. The structure of the films was investigated by means X-ray diffraction and calorimetry, evidencing a B-type crystalline structure. In similar drying conditions, 25 degrees C and 40% of relative humidity, the crystallinity varied from 24% for the low molecular weight (A5) to almost none for the highest molecular weight (A160). The influence of the drying temperature was also investigated. A reduction of the crystallinity from 16% to almost none was found when increasing temperature from 25 to 65 degrees C. The glass transition temperature ( T g) at different water contents was determined. The difference of T g between the first and the second scan was interpreted by changes in the water distribution between phases into the B-type crystalline structure. Mechanical properties of the films determined by tensile tests and by DMTA in the glassy state showed no effect of the average molecular weight or of crystallinity. In contrast, thermomechanical experiments by DMTA showed that the average molecular weight of the sample influenced the mechanical relaxation and the moduli in the rubbery state.

The nature of the periplastidial pathway of starch biosynthesis was investigated with the model cryptophyte Guillardia theta. The storage polysaccharide granules were shown to be composed of both amylose and amylopectin fractions with a chain length distribution and crystalline organization very similar to those of starch from green algae and land plants. Most starch granules displayed a shape consistent with biosynthesis occurring around the pyrenoid through the rhodoplast membranes. A protein with significant similarity to the amylose-synthesizing granule-bound starch synthase 1 from green plants was found as the major polypeptide bound to the polysaccharide matrix. N-terminal sequencing of the mature protein proved that the precursor protein carries a nonfunctional transit peptide in its bipartite topogenic signal sequence which is cleaved without yielding transport of the enzyme across the two inner plastid membranes. The enzyme was shown to display similar affinities for ADP and UDP-glucose, while the V(max) measured with UDP-glucose was twofold higher. The granule-bound starch synthase from Guillardia theta was demonstrated to be responsible for the synthesis of long glucan chains and therefore to be the functional equivalent of the amylose-synthesizing enzyme of green plants. Preliminary characterization of the starch pathway suggests that Guillardia theta utilizes a UDP-glucose-based pathway to synthesize starch.

Whereas Glc is stored in small-sized hydrosoluble glycogen particles in archaea, eubacteria, fungi, and animal cells, photosynthetic eukaryotes have resorted to building starch, which is composed of several distinct polysaccharide fractions packed into a highly organized semicrystalline granule. In plants, both the initiation of polysaccharide synthesis and the nucleation mechanism leading to formation of new starch granules are currently not understood. Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, defines the tiniest eukaryote with one of the smallest genomes. We show that it accumulates a single starch granule at the chloroplast center by using the same pathway as higher plants. At the time of plastid division, we observe elongation of the starch and division into two daughter structures that are partitioned in each newly formed chloroplast. These observations suggest that in this system the information required to initiate crystalline polysaccharide growth of a new granule is contained within the preexisting polysaccharide structure and the design of the plastid division machinery.

This study investigates whether it is possible to produce an amylose-free potato starch by displacing the amylose enzyme, granule-bound starch synthase I (GBSSI), from the starch granule by engineered, high-affinity, multiple-repeat family 20 starch-binding domains (SBD2, SBD3, SBD4, and SBD5). The constructs were introduced in the amylose-containing potato cultivar (cv. Kardal), and the starches of the resulting transformants were compared with those of SBD2-expressing amylose-free ( amf) potato clones. It is shown that a correctly sized protein accumulated in the starch granules of the various transformants. The amount of SBD accumulated in starch increased progressively from SBD to SBD3; however, it seemed as if less SBD4 and SBD5 was accumulated. A reduction in amylose content was not achieved in any of the transformants. However, it is shown that SBDn expression can affect physical processes underlying granule assembly, in both genetic potato backgrounds, without altering the primary structure of the constituent starch polymers and the granule melting temperature. Granule size distribution of the starches obtained from transgenic Kardal plants were similar to those from untransformed controls, irrespective of the amount of SBDn accumulated. In the amf background, granule size is severely affected. In both the Kardal and amf background, apparently normal oval-shaped starch granules were composed of multiple smaller ones, as evidenced from the many “Maltese crosses” within these granules. The results are discussed in terms of different binding modes of SBD. [ABSTRACT FROM AUTHOR]

Starch defines a semicrystalline polymer made of two different polysaccharide fractions. The A- and B-type crystalline lattices define the distinct structures reported in cereal and tuber starches, respectively. Amylopectin, the major fraction of starch, is thought to be chiefly responsible for this semicrystalline organization while amylose is generally considered as an amorphous polymer with little or no impact on the overall crystalline organization. STA2 represents a Chlamydomonas reinhardtii gene required for both amylose biosynthesis and the presence of significant granule-bound starch synthase I (GBSSI) activity. We show that this locus encodes a 69 kDa starch synthase and report the organization of the corresponding STA2 locus. This enzyme displays a specific activity an order of magnitude higher than those reported for most vascular plants. This property enables us to report a detailed characterization of amylose synthesis both in vivo and invitro . We show that GBSSI is capable of synthesizing a significant number of crystalline structures within starch. Quantifications of amount and type of crystals synthesized under these conditions show that GBSSI induces the formation of B-type crystals either in close association with pre-existing amorphous amylopectin or by crystallization of entirely de novo synthesized material. [ABSTRACT FROM AUTHOR]

Starch granules derived from certain tuber or root crops exhibit a B-type polymorphic pattern and amylopectin with a high content of long B-chains and comparatively long segments between the building blocks. Here, four B-crystalline starches are selected to study their morphology and molecular composition, including lintnerization conducted at two different temperatures. The structure of the granules suggests that the B-type crystalline starches can be divided into two distinct groups, with potato and edible canna forming one group having large granules with typical "growth rings" and the molecular structure of the lintners being dependent on the temperature of lintnerization. The other group consists of shoti and lesser yam starches possessing granules with alternating "granular slices" instead of rings or shells and the molecular composition of the lintners is not dependent on the temperature. It is found that the former group of starch possess lower gelatinization temperatures and swell at lower temperatures than the latter group, suggesting a more labile granular structure of potato and canna starches compared to shoti and lesser yam. As the properties are not related to the amylose or phosphate content of the granules, the result suggests that B-crystalline starch granules are found as at least two distinct structural types with unique architectures. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this work was to characterize the amylopectin of low amylose content cassava starches obtained from transgenesis comparatively with a natural waxy cassava starch (WXN) discovered recently in CIAT (International Center for Tropical Agriculture). Macromolecular features, starch granule morphology, crystallinity and thermal properties of these starches were determined. of amylopectin from the transgenic varieties are lower than WXN. Branched and debranched chain distributions analyses revealed slight differences in the branching degree and structure of these amylopectins, principally on DP 6–9 and DP>37. For the first time, a deep structural characterization of a series of transgenic lines of waxy cassava was carried out and the link between structural features and the mutated gene expression approached. The transgenesis allows to silenced partially or totally the GBSSI, without changing deeply the starch granule ultrastructure and allows to produce clones with similar amylopectin as parental cassava clone. [Copyright &y& Elsevier]

Abstract: Two new mutant cassava starches with extreme amylose contents (0 and 30–31%) have been recently reported. These mutants are drastically different from normal cassava starch whose amylose content typically ranges between 15 and 25%. The new mutants were compared with five normal cassava starches (ranging from 16.8 to 21.5% amylose) and commercial versions of amylose-free or normal potato and maize starch. Macromolecular features, crystallinity, granule sizes, and thermal properties of these starches were compared. The structure of cassava amylopectin was not modified by the waxy mutation and waxy cassava starch exhibited properties similar to the ones of waxy maize starch. Waxy cassava and maize show similar and of amylopectin (between 408×106 gmol−1 and 520×106 gmol−1; 277–285nm, respectively), whereas waxy potato amylopectin has lower and . On the contrary, the higher-amylose mutations induced by gamma rays radiation in cassava, modified deeply the branching pattern of amylopectin as well as the starch characteristics and properties: and decreased, while branching degree increased. These modifications resulted in changes in starch granule ultrastructure (e.g. decreased starch crystallinity), a weak organized structure, and increased susceptibility to mild acid hydrolysis. The distinctive properties of the new cassava starches demonstrated in this article suggest new opportunities and commercial applications for tropical sources of starch. [Copyright &y& Elsevier]

Abstract: Background: Glycogen and starch branching enzymes catalyze the formation of α(1→6) linkages in storage polysaccharides by rearrangement of preexisting α-glucans. This reaction occurs through the cleavage of α(1→4) linkage and transfer in α(1→6) of the fragment in non-reducing position. These enzymes define major elements that control the structure of both glycogen and starch. Methods: The kinetic parameters of the branching enzyme of Rhodothermus obamensis (RoBE) were established after in vitro incubation with different branched or unbranched α-glucans of controlled structure. Results: A minimal chain length of ten glucosyl units was required for the donor substrate to be recognized by RoBE that essentially produces branches of DP 3–8. We show that RoBE preferentially creates new branches by intermolecular mechanism. Branched glucans define better substrates for the enzyme leading to the formation of hyper-branched particles of 30–70nm in diameter (dextrins). Interestingly, RoBE catalyzes an additional α-4-glucanotransferase activity not described so far for a member of the GH13 family. Conclusions: RoBE is able to transfer α(1→4)-linked-glucan in C4 position (instead of C6 position for the branching activity) of a glucan to create new α(1→4) linkages yielding to the elongation of linear chains subsequently used for further branching. This result is a novel case for the thin border that exists between enzymes of the GH13 family. General significance: This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis. It defines new approach to produce highly branched α-glucan particles in vitro. [Copyright &y& Elsevier]

Abstract: A new α-amylase from Anoxybacillus flavothermus (AFA) was found to be effective in hydrolyzing raw starch in production of glucose syrup at temperatures below the starch gelatinization temperature. AFA is very efficient, leading to 77% hydrolysis of a 31% raw starch suspension. The final hydrolysis degree is reached in 2–3h at starch concentrations lower than 15% and 8–24h at higher concentrations. AFA is also very efficient in hydrolyzing the crystalline domains in the starch granule. The major A-type crystalline structure is more rapidly degraded than amorphous domains in agreement with the observed preferential hydrolysis of amylopectin. Amylose–lipid complexes are degraded in a second step, yielding amylose fragments which then re-associate into B-type crystalline structures forming the final α-amylase resistant fraction. The mode of action of AFA and the factors limiting complete hydrolysis are discussed in details. [Copyright &y& Elsevier]