On the cluster structure of amylopectin.

KEY MESSAGE: Two opposing models for the amylopectin structure are historically and comprehensively reviewed, which leads us to a better understanding of the specific fine structure of amylopectin. Amylopectin is a highly branched glucan which accounts for approximately 65-85 of starch in most plant tissues. However, its fine structure is still not fully understood due to the limitations of current methodologies. Since the 1940 s, many scientists have attempted to elucidate the distinct structure of amylopectin. One of the most accepted concepts is that amylopectin has a structural element known as "cluster", in which neighboring side chains with a degree of polymerization of ≥ 10 in the region of their non-branched segments form double helices. The double helical structures are arranged in inter- and intra-clusters and are the origin of the distinct physicochemical and crystalline properties of starch granules. Several models of the cluster structure have been proposed by starch scientists worldwide during the progress of analytical methods, whereas no direct evidence so far has been provided. Recently, Bertoft and colleagues proposed a new model designated as "the building block and backbone (BB) model". The BB model sharply contrasts with the cluster model in that the structural element for the BB model is the building block, and that long chains are separately synthesized and positioned from short chains constituting the building block. In the present paper, we conduct the historical review of the cluster concept detailing how and when the concept was established based on experimental results by many scientists. Then, differences between the two opposing concepts are explained and both models are critically discussed, particularly from the point of view of the biochemical regulation of amylopectin biosynthesis.

Insights into waxy maize starch degradation by sulfuric acid: Impact on starch structure, pasting, and rheological property.

This study investigated the effects of acid degradation of amylopectin on the structure, pasting, and rheological properties of waxy maize starch. It is found that: 1) the amount of amylopectin short-chains with degree of polymerization (DP) ~ 15-50 increased while that of amylopectin long-chains with DP ~ 50-200 decreased by acid hydrolysis; 2) acid hydrolysis produced smaller amylopectin molecules with a narrower size distribution; 3) acid hydrolysis had a minor effect on the crystalline and granular structures of native starch; 4) the pasting viscosity of acid hydrolyzed starch during heating and the consistency coefficient, K, of starch gels increased, whereas the flow behavior index, n, decreased. Correlation analysis was used to clarify the molecular causes for the variations of pasting and rheological properties of acid hydrolyzed starch.

Influence of amylose content and oxidation level of potato starch on acetylation, granule structure and radicals' formation.

This study was aimed at determining the effect of the amylose content of starch and oxidation level of potato starch on the structure of starch granules, and susceptibility to chemical modification (acetylation) and subsequent generation of radicals. Potato starch and waxy potato starch were oxidised with sodium hypochlorite applied in doses corresponding to 10, 20, and 30gCl/kg starch, and then acetylated with acetic acid anhydride. The carboxyl, carbonyl, acetyl groups were determined in modified starches. Structural properties of starch granules were evaluated based on gelatinisation, crystallinity, specific surface, intrinsic viscosity, and microphotographs by SEM microscope. The electron paramagnetic resonance (EPR) measurements were carried out to establish starch susceptibility to radical creation upon chemical modification and UV radiation. The amount of formed radicals was treated as a measure of the starch structure stability. The higher amount of amylose and the highest level of oxidation led to strong starch structure destruction and consequently facilitated radical generation. Study results showed also that amylose content as well as the degree of starch oxidation modified consecutive acetylation process. The different effectiveness of the acetylation processes influenced the morphology and structure of starch granules.

Amylopectin-g-poly(methylacrylate-co-sodium acrylate): An efficient Cd(II) binder.

Synthesis, characterization and Cd(II) adsorption studies of a novel biodegradable graft copolymer based on partially hydrolysed polymethylacrylate (PMA) grafted amylopectin was reported, which was prepared by first grafting of PMA chains onto the amylopectin backbone followed by partial alkaline hydrolysis. The hydrolysed graft copolymer (PHAP) was characterized by measuring saponification equivalent (SE), FTIR, (1)H NMR and (13)C NMR spectroscopy and thermal analysis (TG/DTG). The graft copolymer was biodegradable. Various operating variables affecting the metal sorption such as, amount of adsorbent, solution pH, contact time, temperature and Cd(II) solution concentration were studied which showed that the maximum adsorption of Cd(II) was found at pH 5.5, temperature 90°C, time 120min, polymer dose, 0.02g/L and initial Cd(II) concentration, 50mg/L. The adsorption data were well described by the pseudo-second-order and Langmuir isotherm model. Metal complexation studies were carried out experimentally using UV-visible, FTIR spectroscopy and theoretically using Density Functional Theory by Gaussian 09 and Gauss view 5.0 programmes which confirms a square planer geometry involving Cd(II) and COO(-) groups. Calculation of the various thermodynamic parameters was also done. The negative value of free energy change indicates the spontaneous nature of the adsorption.

Role of molecular entanglements in starch fiber formation by electrospinning.

We have demonstrated a method of fabricating pure starch fibers with an average diameter in the order of micrometers. In the present study, correlation between the rheological properties of starch dispersions and the electrospinnability was attempted via the extrapolation of the critical entanglement concentration, which is the boundary between the semidilute unentangled regime and the semidilute entangled regime. Dispersions of high amylose starch containing nominally 80% amylose (Gelose 80) required 1.2-2.7 times the entanglement concentration for effective electrospinning. Besides starch concentration, molecular conformation, and shear viscosity were also of importance in determining the electrospinnability. The rheological properties and electrospinnability of different starches were studied. Hylon VII and Hylon V starches, containing nominally 70 and 50% amylose, respectively, required concentrations of 1.9 and 3.7 times their entanglement concentrations for electrospinning. Only poor fibers were obtained from mung bean starch, which contains about 35% amylose, while starches with even lower amylose contents could not be electrospun.

Integrated functions among multiple starch synthases determine both amylopectin chain length and branch linkage location in Arabidopsis leaf starch.

This study assessed the impact on starch metabolism in Arabidopsis leaves of simultaneously eliminating multiple soluble starch synthases (SS) from among SS1, SS2, and SS3. Double mutant ss1- ss2- or ss1- ss3- lines were generated using confirmed null mutations. These were compared to the wild type, each single mutant, and ss1- ss2- ss3- triple mutant lines grown in standardized environments. Double mutant plants developed similarly to the wild type, although they accumulated less leaf starch in both short-day and long-day diurnal cycles. Despite the reduced levels in the double mutants, lines containing only SS2 and SS4, or SS3 and SS4, are able to produce substantial amounts of starch granules. In both double mutants the residual starch was structurally modified including higher ratios of amylose:amylopectin, altered glucan chain length distribution within amylopectin, abnormal granule morphology, and altered placement of α(1→6) branch linkages relative to the reducing end of each linear chain. The data demonstrate that SS activity affects not only chain elongation but also the net result of branch placement accomplished by the balanced activities of starch branching enzymes and starch debranching enzymes. SS3 was shown partially to overlap in function with SS1 for the generation of short glucan chains within amylopectin. Compensatory functions that, in some instances, allow continued residual starch production in the absence of specific SS classes were identified, probaby accomplished by the granule bound starch synthase GBSS1.

Characterization of the surface morphology of durum wheat starch granules using atomic force microscopy.

Knowledge of the structure and properties of microscopic surfaces of durum wheat starch granules is essential for understanding the functional and physico-chemical properties. The nanoscale surface undulations on the starch granules inside durum wheat macroscopically influence the milling properties. The objective of this study was to visualize the surface morphology and the size of starch grains of vitreous and nonvitreous durum wheat kernels using atomic force microscopy. The distribution of starch granules in the vitreous and nonvitreous durum wheat starch samples were examined and compared. The results of our study confirm the 'blocklet' model of the ultrastructure of the starch granule surface. Image contrast enhancement using UV/ozone treatment of microtomed starch samples improved the imaging of growth rings on the starch samples. The observation of growth rings in the nonvitreous starch granule surfaces indicates that amylopectin is more common than amylose in nonvitreous starch when compared with vitreous starch.

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.

Antisense inhibition of isoamylase alters the structure of amylopectin and the physicochemical properties of starch in rice endosperm.

This is the first report on regulation of the isoamylase1 gene to modify the structure of amylopectin and properties of starch by using antisense technology in plants. The reduction of isoamylase1 protein by about 94% in rice endosperm changed amylopectin into a water-insoluble modified amylopectin and a water-soluble polyglucan (WSP). As compared with wild-type amylopectin, the modified amylopectin had more short chains with a degree of polymerization of 5-12, while their molecular sizes were similar. The WSP, which structurally resembled the phytoglycogen in isoamylase-deficient sugary-1 mutants, accounted for about 16% of the total alpha-polyglucans in antisense endosperm, and it was distributed throughout the whole endosperm unlike in sugary-1 mutant. The reduction of isoamylase activity markedly lowered the gelatinization temperature from 54 to 43 degrees C and the viscosity, and modified X-ray diffraction pattern and the granule morphology of the starch. The activity of pullulanase, the other type of starch debranching enzyme, in the antisense endosperm was similar to that in wild-type, whereas it is deficient in sugary-1 mutants. These results indicate that the isoamylase1 is essential for amylopectin biosynthesis in rice endosperm, and that alteration of the isoamylase activity is an effective means to modify the physicochemical properties and granular structure of starch.

Surface composition and morphology of starch, amylose, and amylopectin films.

The surfaces of solution-cast films of starch, amylose, and amylopectin were examined with scanning electron microscopy (SEM), atomic force microscopy (AFM), electron spectroscopy for chemical analysis (ESCA), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The surface topography visualized by SEM showed that amylopectin films were very smooth whereas amylose and starch films were rougher. It appears that crystallinity or phase separation in the bulk of the film affects the surface topography. AFM showed that the outmost surfaces of all films were covered with small protrusions, 15-35 nm wide and 1-4 nm high. Studies with ESCA revealed the presence of 3-8% nitrogen on the surfaces. ToF-SIMS indicated that the nitrogen originates from protein because ionic fragments from amino acids and the peptide backbone were found. Extracts from the top surface layer of the starch film showed protein bands in gel electrophoresis (SDS-PAGE) around 60 kDa, which is in the same molecular weight range as the biosynthesizing enzyme GBSS I present in starch granules. The proteins apparently phase separated during film formation and migrated to the surface, resulting in an extensive enrichment of proteins in the film surface, where about 8% of the protein is present in the top 0.01% of the film. We believe that the protrusions observed with AFM could be one or a few proteins aggregated side by side.

An alternative technique to reveal polysaccharides in Toxoplasma gondii tissue cysts.

Ultrathin sections of tissue cysts isolated from the brain of Toxoplasma gondii infected mice were submitted to two different methodologies derived from the periodic acid - Schiff's reagent (PAS) technique. The use of osmium tetroxide vapor as a developing agent of the aldehyde oxidation to reveal polysaccharides with periodic acid resulted in positive reaction in amylopectin granules in bradyzoites, as well as in the wall and matrix of the cysts, with excellent increment of the ultrastructural morphology. This technique can be used for study of T. gondii-host cell intracellular cycle, the differentiation tachyzoite-bradyzoite, and also for the formation of cysts into the host cells.

Two loci control phytoglycogen production in the monocellular green alga Chlamydomonas reinhardtii.

The STA8 locus of Chlamydomonas reinhardtii was identified in a genetic screen as a factor that controls starch biosynthesis. Mutations of STA8 cause a significant reduction in the amount of granular starch produced during nutrient limitation and accumulate phytoglycogen. The granules remaining in sta8 mutants are misshapen, and the abundance of amylose and long chains in amylopectin is altered. Mutations of the STA7 locus, which completely lack isoamylase activity, also cause accumulation of phytoglycogen, although sta8 and sta7 mutants differ in that there is a complete loss of granular starch in the latter. This is the first instance in which mutations of two different genetic elements in one plant species have been shown to cause phytoglycogen accumulation. An analytical procedure that allows assay of isoamylase in total extracts was developed and used to show that sta8 mutations cause a 65% reduction in the level of this activity. All other enzymes known to be involved in starch biosynthesis were shown to be unaffected in sta8 mutants. The same amount of total isoamylase activity (approximately) as that present in sta8 mutants was observed in heterozygous triploids containing two sta7 mutant alleles and one wild-type allele. This strain, however, accumulates normal levels of starch granules and lacks phytoglycogen. The total level of isoamylase activity, therefore, is not the major determinant of whether granule production is reduced and phytoglycogen accumulates. Instead, a qualitative property of the isoamylase that is affected by the sta8 mutation is likely to be the critical factor in phytoglycogen production.

Internal structure of the starch granule revealed by AFM.

Atomic force microscopy images of sectioned native corn starch granules show evidence of the well-known radial organisation of the starch macromolecules, with the less-ordered hilum region near to the centre. Native granules show blocks 400-500 nm in size that span the growth rings. Lintnerised starch granules, where a mild acid hydrolysis has been used to remove the amorphous and less crystalline parts of the granule, clearly show smaller 'blocklets' within the rings approximately 10-30 nm in size. This level of organisation within the growth rings corresponds to the blocklet or superhelix structures that have been proposed in the literature for the association or clustering of amylopectin helices. Mechanical property imaging techniques have provided enhanced contrast to view this morphology, and shown the deformability of the starch structure under contact mode imaging conditions.