Molecular and supra-molecular structure of waxy starches developed from cassava (Manihot esculenta Crantz).

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. M¯(w) 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.

Hydrolysis of concentrated raw starch: A new very efficient α-amylase from Anoxybacillus flavothermus.

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.

Molecular size and mass distributions of native starches using complementary separation methods: asymmetrical flow field flow fractionation (A4F) and hydrodynamic and size exclusion chromatography (HDC-SEC).

Starch consists of a mixture of two α-glucans built mainly upon α-(1,4) linkages: amylose, an essentially linear polymer, and amylopectin, a branched polymer containing 5-6% of α-(1,6) linkages. The aim of the present work was to analyze the structural properties of native starches displaying different amylose-to-amylopectin ratios and arising from different botanical sources, using asymmetrical flow field flow fractionation (A4F) and a combination of hydrodynamic and size-exclusion chromatography (HDC-SEC) coupled with multiangle laser light scattering, online quasi-elastic light scattering, and differential refractive index techniques. The procedure, based upon dimethyl sulfoxide pretreatment and then solubilization in water, generates a representative injected sample without altering the initial degree of polymerization. The amylopectin weight-average molar masses and radii of gyration were around 1.0 × 10(8)-4.8 × 10(8) g mol(-1) and 110-267 nm, respectively. For each starch sample, the hydrodynamic radius (R(H)) distributions and the molar mass distributions obtained from the two fractionation systems coupled with light scattering techniques were analyzed. The size determination scales were extended by means of R(H) calibration curves. HDC-SEC and A4F data could be matched. However, A4F enabled a better separation of amylopectins and therefore an enhanced structural characterization of the starches. The two advantages of this experimental approach are (1) it can directly obtain distributions as a function of both molar mass and size, while taking account of sample heterogeneity, and (2) it is possible to compare the results obtained using the different techniques through the direct application of R(H) distributions.

In depth study of a new highly efficient raw starch hydrolyzing α-amylase from Rhizomucor sp.

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.

In situ tracking of enzymatic breakdown of starch granules by synchrotron UV fluorescence microscopy.

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.

Online determination of structural properties and observation of deviations from power law behavior.

Synthetic amyloses, pullulans, phytoglycogen, rabbit liver glycogen, oyster glycogen, and dextrans were studied using high-performance size-exclusion chromatography combined with multiangle laser light scattering (MALLS) and online quasi-elastic light scattering (QELS), which provided the RH distributions up to 65 nm. Different structural parameters were extracted from entire molar mass distributions, including the slope of the log-log plot of R H(i) versus M(i)and the rho(i )= R(Gi)/R(Hi)ratio. This approach enabled to observe deviations from the De Gennes scaling law concept. Evidences that the power laws do not obey the general universality were furnished by the observation of strong deviations in the relation between radii and molar masses for the branched polysaccharides, a decrease of rho-parameter with molar mass toward values much lower than theoretically expected, and the fact that relation between rho-parameter and apparent segment density did not show the expected power law decrease with an exponent of 1/3. The universality of scaling behavior seems no longer to be realized if structural heterogeneity governs the system.

Branching features of amylopectins and glycogen determined by asymmetrical flow field flow fractionation coupled with multiangle laser light scattering.

The aim of this work was to characterize starch polysaccharides using asymmetrical flow field flow fractionation coupled with multiangle laser light scattering. Amylopectins from eight different botanical sources and rabbit liver glycogen were studied. Amylopectins and glycogen were completely solubilized and analyzed, and high mass recoveries were achieved (81.7-100.0%). Amylopectin Mw, RG, and the hydrodynamic coefficient nuG (the slope of the log-log plot of RGi vs Mi) were within the ranges 1.05-3.18 x 10(8) g mol(-1), 163-229 nm, 0.37-0.49, respectively. The data were also considered in terms of structural parameters. The results were analyzed by comparison with the theory of hyperbranched polymers (Flory, P. J. Principles of Polymer Chemistry; Cornell University Press: Ithaca, NY, 1953; Burchard, W. Macromolecules, 1977, 10, 919-927). This theory, based upon the ABC model, has been shown to underestimate the branching degrees of amylopectins. However, quantitative agreement with the data in the literature was found for amylopectins when using the ABC model modified by the introduction of a multiplying factor, determined from previously described amylopectin structures in terms of the number of branching point calculations.

Soluble starch synthase I: a major determinant for the synthesis of amylopectin in Arabidopsis thaliana leaves.

A minimum of four soluble starch synthase families have been documented in all starch-storing green plants. These activities are involved in amylopectin synthesis and are extremely well conserved throughout the plant kingdom. Mutants or transgenic plants defective for SSII and SSIII isoforms have been previously shown to have a large and specific impact on the synthesis of amylopectin while the function of the SSI type of enzymes has remained elusive. We report here that Arabidopsis mutants, lacking a plastidial starch synthase isoform belonging to the SSI family, display a major and novel type of structural alteration within their amylopectin. Comparative analysis of beta-limit dextrins for both wild type and mutant amylopectins suggests a specific and crucial function of SSI during the synthesis of transient starch in Arabidopsis leaves. Considering our own characterization of SSI activity and the previously described kinetic properties of maize SSI, our results suggest that the function of SSI is mainly involved in the synthesis of small outer chains during amylopectin cluster synthesis.

Mutants of Arabidopsis lacking a chloroplastic isoamylase accumulate phytoglycogen and an abnormal form of amylopectin.

Mutant lines defective for each of the four starch debranching enzyme (DBE) genes (AtISA1, AtISA2, AtISA3, and AtPU1) detected in the nuclear genome of Arabidopsis (Arabidopsis thaliana) were produced and analyzed. Our results indicate that both AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of DBE named Iso1, the major isoamylase activity found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content in both lines and to the accumulation of water-soluble polysaccharides whose structure is similar to glycogen. In addition, the residual amylopectin structure in the corresponding mutant lines displays a strong modification when compared to the wild type, suggesting a direct, rather than an indirect, function of Iso1 during the synthesis of amylopectin. Mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure. This result suggests that this isoamylase-type of DBE plays a major role during starch mobilization. The analysis of the Atpu1 single-mutant lines did not lead to a distinctive phenotype. However, Atisa2/Atpu1 double-mutant lines display a 92% decrease in starch content. This suggests that the function of pullulanase partly overlaps that of Iso1, although its implication remains negligible when Iso1 is present within the cell.

Origin of the limited alpha-amylolysis of debranched maltodextrins crystallized in the A form: a TEM study on model substrates.

The detailed ultrastructure of a new type of resistant starch and the way that it is modified during hydrolysis by alpha-amylases were studied by transmission electron microscopy (TEM) on model starch crystals. The selected substrates were waxy maize starch lintners and A-type crystals prepared from low degree of polymerization (DP) amylose. A model describing the stacking of double helices is proposed for A-type low DP amylose crystals. The enzymatic hydrolysis of both lintners and low DP crystals has been shown to occur by the side of double helices and not their ends. The results were transposed to a new type of resistant starch (RS) produced by debranching maltodextrins in concentrated solutions. This product presents A-type crystallinity contrary to all other known classified RS. Moreover it consists of low DP chains similar to the model crystals studied and yields similar electron diffraction patterns to those of A-type low DP crystals. The similarities in the morphology of these substrates with that of the studied RS led us to attribute its resistance to its particularly dense and compact morphology, resulting from the epitaxial growth of elementary crystalline A-type platelets. In the resulting structure, the accessibility of double helices to alpha-amylase is strongly reduced by aggregation.

Volumetric behaviour of maltose-water, maltose-glycerol and starch-sorbitol-water systems mixtures in relation to structural relaxation.

The densities of amorphous maltose-water, maltose-glycerol and starch-sorbitol-water mixtures were measured using a vibrating-tube density meter and pycnometry. The volumetric change on mixing was investigated through the calculation of the quantity DeltaV/V, the difference between (experimental) volume of the mixture and the linear composition weighted pure constituent volumes (ideal mixing). For all of the systems studied the quantity DeltaV/V was negative and approached a minimum of -0.03 and -0.015 at mass fractions of maltose in the region of 0.75 and 0.85 for the maltose-water and maltose-glycerol mixtures, respectively. Results are discussed in the context of volume change due to structural relaxation of vitreous materials and related to the phenomenon of antiplasticisation.

Structural relaxation and physical ageing of starchy materials.

The structural relaxation during the ageing of an amorphous maltose and a starch-sorbitol mixture was examined using a range of physical techniques. Heat capacity, measured by differential scanning calorimetry, showed an overshoot in the glass-transition region, the size of which was temperature and time dependent. Volume relaxation measurements were made at different ageing temperatures in the range T(g) -15 to -30 K. The volume decreased with increasing ageing time, in an essentially linear fashion with log time. The mechanical behaviour of the materials showed a progressive embrittlement on ageing. For both materials, the mechanical relaxation time increased with ageing, and the material became stiffer. Investigation of the effect of physical ageing on transport properties was also performed using conductivity measurements on a maltose-water-KC1 mixture. A decay in conductivity, which was almost linear with log time, was observed. The structural relaxation was modelled using the Tool-Narayanaswamy approach to describe the calorimetric data.