3D imaging of enzymes working in situ.

Today, development of slowly digestible food with positive health impact and production of biofuels is a matter of intense research. The latter is achieved via enzymatic hydrolysis of starch or biomass such as lignocellulose. Free label imaging, using UV autofluorescence, provides a great tool to follow one single enzyme when acting on a non-UV-fluorescent substrate. In this article, we report synchrotron DUV fluorescence in 3-dimensional imaging to visualize in situ the diffusion of enzymes on solid substrate. The degradation pathway of single starch granules by two amylases optimized for biofuel production and industrial starch hydrolysis was followed by tryptophan autofluorescence (excitation at 280 nm, emission filter at 350 nm). The new setup has been specially designed and developed for a 3D representation of the enzyme-substrate interaction during hydrolysis. Thus, this tool is particularly effective for improving knowledge and understanding of enzymatic hydrolysis of solid substrates such as starch and lignocellulosic biomass. It could open up the way to new routes in the field of green chemistry and sustainable development, that is, in biotechnology, biorefining, or biofuels.

SAXS conformational tracking of amylose synthesized by amylosucrases.

Amylose, a linear polymer of α(1,4)-linked glucosyl units and a major constituent of starch granules, can also be enzymatically synthesized in vitro from sucrose by bacterial amylosucrases. Depending on the initial sucrose concentration and the enzyme used, amylose oligomers (or polymers) are formed and self-associate during synthesis into various semicrystalline morphologies. This work describes for the first time a synchrotron SAXS study of the structure in solution of two amylosucrases, namely, NpAS and the thermostable DgAS, under conditions of polymer synthesis and, simultaneously, the amylose conformation. The structure in solution of both amylosucrases during the reaction was shown to be similar to the known crystallographic structures. The conformation of amylose produced at an early stage consists of a mixture of wormlike chains and double helical cylindrical structures. In the case of NpAS, in a second stage, individual double helices pack into clusters before crystallizing and precipitating. Amylose produced by DgAS never self-associates in such clusters due to the higher temperature used for amylose synthesis. All the dimensions determined for wormlike chains and cylindrical conformations at different times of NpAS synthesis are in very good agreement with structural features usually observed on gels of amylose extracted from starch. This provides new insights in understanding the mechanisms of amylose gelation.

Carboxymethylcellulose/single walled carbon nanotube complexes.

In this work, Carboxymethylcellulose/single walled carbon nanotube hybrids are prepared. Our goal is to use the non-covalent association of an anionic polysaccharide and single walled carbon nanotubes to prepare a biocompatible complex which preserves the intrinsic properties of the nanotubes. The hybrids are characterized by Raman scattering, Emission spectroscopy and Atomic Force Microscopy. A comparison is made with sodium dodecyl benzene sulfonate dispersed nanotubes. We show that carboxymethylcellulose forms an apparently non-helicoidal superstructure with carbon nanotubes which leads to their individualization. Emission and Raman spectroscopies suggest that the interaction occurs preferentially with semiconducting nanotubes. Further sugar chemistry may open different functionalization opportunities that will contribute to the design of biosensors and open this field of applications.

Structural investigation of amylose complexes with small ligands: helical conformation, crystalline structure and thermostability.

Crystalline amylose complexes were prepared with decanal, 1-butanol, menthone and alpha-naphtol. Their crystalline structure and the related helical conformation, determined by wide angle X-ray diffraction (WAXD) and 13C CPMAS solid state NMR, were assigned to V6I, V6II, V6III and V8 types, respectively. It was possible to propose some hypotheses on the possible nature of interactions and especially intra-/inter-helical inclusion. Some shifts in the NMR C1 carbon signals were attributed to the presence of ligand in specific sites inside the structure for a same type of V6 helical conformation. Moreover, the crystallinity and polymorphic changes induced by desorption/rehydration were studied. A general increase of the carbon resonances sharpness upon rehydration has been observed, but also a V6II-V6I transition when decreasing the water content. Differential scanning calorimetry (DSC) experiments were also performed to approach the thermostability of the four types of complex and also the way they form again after melting/cooling sequences.

Structural investigation of amylose complexes with small ligands: inter- or intra-helical associations?

Highly crystalline amylose complexes with menthone (1) and linalool (2) were analysed by wide-angle X-ray diffraction and solid-state nuclear magnetic resonance (NMR). The complexes, after partial water desorption in a controlled atmosphere (aw=0.75), displayed a typical V-isopropanol structure, showing the presence of ligand inside or between the helices in the crystalline domains. Sequential washing of the powdered complexes with ethanol before and after desorption permitted probing the intra- and inter-helical inclusions. High resolution magic angle spinning (HRMAS) recordings were used to compare the chemical shifts of free and bound aroma which allowed a proposal that some hydrogen bonding is involved in the amylose complexing. Moreover, it showed that free aroma was completely removed by ethanol washing. Using cross polarization magic angle spinning (CPMAS) and X-ray scattering experiments, it was demonstrated that the V-isopropanol type was retained for linalool whatever the treatment used. On the contrary, the measurement shifts toward the V-6I amylose hydrate (V-h) type for menthone after ethanol washing before the desorption step, reflecting the disappearance of inter-helical associations between menthone and amylose. The stability of the complex prepared with linalool shows that this ligand is more strongly associated to amylose helices. The discrepancies observed in the chemical shifts attributed to carbons C1 and C4 in CPMAS spectra of V-isopropanol and V-h forms could be attributed either to a deformation of the single helix (with possible inclusion of the ligand inside) or to the presence of the ligand between helices (only water molecules are present in the V-h form).

Split crystallization during debranching of maltodextrins at high concentration by isoamylase.

Debranching and crystallization occurring during the enzymatic treatment of 25% (w/v) aqueous solutions of maltodextrins by isoamylase at 52 degrees C were studied. The morphology as well as the crystal and molecular structures of the precipitates formed at different stages of the reaction were characterized. Two types of resulting products, differing in terms of structure and morphology, were evidenced. A loose B-type network, containing linear and branched chains of highest molecular weight, was mainly formed during the first 12 h of reaction, whereas aggregates of A-type lamellar crystals, made of short linear chains, were predominantly obtained between 12 and 48 h. The aggregation behavior as a function of temperature and molecular weight distribution of such substrates was discussed and compared to that of related starch products.

NMR local range investigations in amorphous starchy substrates I. Structural heterogeneity probed by (13)C CP-MAS NMR.

The (13)C CP-MAS (Cross Polarization and Magic Angle Spinning) NMR signatures of a series of amorphous and semi-crystalline samples prepared from various starchy substrates (native potato starch, amylopectin, amylose) following different techniques of preparation (casting, freeze drying, solvent exchange) are compared. Decompositions of the C1 resonance spectra reveal the existence of four or five main types of alpha(1-4) linkages, which can be quantified. The influence of the intrinsic primary structure (linear or branched) and of the preparation procedure on conformational changes and resulting crystallinity are interpreted in terms of distributions of average glycosidic linkages dihedral angles (Phi, Psi). The role of hydration is also considered. An improved understanding at different structural levels is obtained in relation to local and intermediate range orders. Such information may be useful for the understanding of the structural evolution of a large variety of starchy substrates before or after treatments widely used in industrial processes.

NMR local range investigations in amorphous starchy substrates: II-Dynamical heterogeneity probed by (1)H/(13)C magnetization transfer and 2D WISE solid state NMR.

In the preceding paper, we have investigated the structural heterogeneous character of a series of amorphous samples prepared from various starchy substrates (native potato starch, amylopectin and amylose) following different techniques of preparation (casting, freeze drying and solvent exchange). Spectral decompositions of the C1 resonances of the (13)C CP-MAS (Cross Polarization and Magic Angle Spinning) spectra under (1)H decoupling have shown the existence of five main types of alpha(1-4) linkages. In this part, 2D solid state NMR WISE experiments and the (13)C/(1)H magnetization transfer in CP as a local probe for both structures and dynamics were used. The (13)C CP magnetization curves versus contact time of each C1 component in each recorded spectrum were fitted with an analytic function taking into account two (1)H reservoirs. Interpretation of the characteristic times derived from fitting yields some improvements on the knowledge of the heterogeneity of the samples and on the water molecules distribution.

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.

Photodegradation of cassava and corn starches.

The baking expansion properties of sour cassava starch (Polvilho azedo) are attributable to photochemical starch degradation induced by heterolactic fermentation after sun-drying. This study investigated the effects of UV irradiation on the different structural levels of cassava starch as compared to those of corn starch and dextrins. Photosensitive compounds excited at 360 and 290 nm in cassava starch were photodegraded when starch was exposed to sunlight or 360 nm irradiation. UV irradiation depolymerized cassava and corn starches, inducing modifications due, at least in part, to a mechanism involving free radicals. Lactic acid was also photodegraded. Photodegradation induced by UV absorption could have been due to fluorescent chromophores found in starches and nonfluorescent chromophores present in glucosidic units.

The substitution pattern in cationised and oxidised potato starch granules.

The distribution pattern of substituents within the granules and the components of two cationised and two oxidised potato starches was studied. The level of crystallinity in wet-cationised (WC) and hypochlorite oxidised (HO) starch granules was similar to that of native starch granules but lower in dry-cationised (DC) and peroxide oxidised (PO) granules. However, the melting temperature of DC granules remained similar to native granules but was decreased in the other samples. With all modified starches, the initial rate of acid hydrolysis (lintnerisation) was increased compared to native granules. The degree of substitution decreased only slightly in WC granules after the lintnerisation, whereas virtually all the substituted glucosyl units in DC starch were hydrolysed already at initial stages. The decrease of substituents in the HO and PO starches was intermediate. The starches were partly resistant to the action of isoamylase and the successive beta-amylolysis, suggesting that substituents were found both close to the branches and near the nonreducing ends in the amylopectin component. It is suggested that the DC starch was preferentially cationised at the surface of the granules, whereas WC and oxidised starches were modified throughout the granules.

Physicochemical behaviors of sugars, lipids, and gluten in short dough and biscuit.

The structure of short dough and biscuit has been characterized at a macroscopic level (dimensions, bulk structure) and a microscopic level (starch damage, protein aggregates, microstructure) by physical and biochemical methods. The baking process of short dough induces a large decrease of the product bulk density from 1.26 to 0. 42 (+/-0.01) g.cm(-)(3) for final biscuit, leading to a cellular solid with a thin colored surface and a porous inner structure. Proteins appear aggregated in biscuit when compared to short dough, whereas starch granules remain almost intact in biscuits. The components which are involved in the cohesiveness of short dough and biscuit final structure have been identified. They suggest that short dough is a suspension of solid particles in a liquid phase being an emulsion of lipids in a concentrated sugar solution. The role of sugars in biscuit structure suggest that biscuit structure is a composite matrix of protein aggregates, lipids and sugars, embedding starch granules.

Amylose chain behavior in an interacting context. III. Complete occupancy of the AMY2 barley alpha-amylase cleft and comparison with biochemical data.

In the first two papers of this series, the tools necessary to evaluate substrate ring deformations were developed, and then the modeling of short amylose fragments (maltotriose and maltopentaose) inside the catalytic site of barley alpha-amylase was performed. In this third paper, this docking has been extended to the whole catalytic cleft. A systematic approach to extend the substrate was used on the reducing side from the previous enzyme/pentasaccharide complex. However, due to the lack of an obvious subsite at the nonreducing side, an alternate protocol has been chosen that incorporates biochemical information on the enzyme and features on the substrate shape as well. As a net result, ten subsites have been located consistent with the distribution of Ajandouz et al. (E. H. Ajandouz, J. Abe, B. Svensson, and G. Marchis-Mouren, Biochimica Biophysica Acta, 1992, Vol. 1159, pp. 193-202) and corresponding binding energies were estimated. Among them, two extreme subsites (-6) and (+4), with stacking residues Y104 and Y211, respectively, have strong affinities with glucose rings added to the substrate. No other deformation has been found for the new glucose rings added to the substrate; therefore, only ring A of the DP 10 fragment has a flexible form when interacting with the inner stacking residues Y51. Global conservation of the helical shape of the substrate can be postulated in spite of its significant distortion at subsite (-1).

Ultrastructural aspects of phytoglycogen from cryo-transmission electron microscopy and quasi-elastic light scattering data.

Phytoglycogen particles extracted from the sugary maize mutant su 1 and dispersed in water were studied using transmission electron microscopy (TEM) and light scattering. Dried specimens were either negatively stained with uranyl acetate or shadowed with W/Ta. Frozen-hydrated unstained particles embedded in a thin film of vitreous ice were also observed using cryo-TEM. The particles exhibited a spheroidal shape, with a diameter ranging from 30 to 100 nm. Some of them presented a multilobular morphology and appeared to be formed by smaller subunits, 20-30 nm in diameter, resembling the described beta-particles for animal glycogen. The diameter of stained and ice-embedded particles was measured from electron micrographs. The corresponding size distribution histograms showed that the average weight diameter of ice-embedded particles was higher than that of stained ones. In the latter case, a shrinkage of the particle was believed to occur during the drying process. Light scattering experiments confirmed the diameter of ice-embedded particles and indicated that they could be considered as uniformly dense spheroidal objects.

The influence of alterations in ADP-glucose pyrophosphorylase activities on starch structure and composition in potato tubers.

In order to examine whether alterations in the supply of precursor molecules into the starch biosynthetic pathway affected various characteristics of the starch, starch was isolated from potato (Solanum tuberosum L.) tubers containing reduced amounts of the enzyme ADP-glucose pyrophosphorylase (AGPase). It was found that although the type of crystalline polymorph in the starch was not altered, the amylose content was severely reduced. In addition, amylopectin from the transgenic plants accumulated more relatively short chains than that from control plants and the sizes of starch granules were reduced. The starch granules from the transgenic plants contained a greater amount of granule-bound starch synthase enzyme, which led to an increase in the maximum activity of the enzyme per unit starch tested. The K(m) for ADP-glucose was, at most, only slightly altered in the transgenic lines. Potato plants containing reduced AGPase activity were also transformed with a bacterial gene coding for AGPase to test whether this enzyme can incorporate phosphate monoesters into amylopectin. A slight increase in phosphate contents in the starch in comparison with the untransformed control was found, but not in comparison with starch from the line with reduced AGPase activity into which the bacterial gene was transformed.Key words: ADP-glucose pyrophosphorylase. Amylopectin structure. Amylose. Solanum (starch. tuber). Starch granule size. Starch phosphorylation

Starch granules: structure and biosynthesis.

The emphasis of this review is on starch structure and its biosynthesis. Improvements in understanding have been brought about during the last decade through the development of new physicochemical and biological techniques, leading to real scientific progress. All this literature needs to be kept inside the general literature about biopolymers, despite some confusing results or discrepancies arising from the biological variability of starch. However, a coherent picture of starch over all the different structural levels can be presented, in order to obtain some generalizations about its structure. In this review we will focus first on our present understanding of the structures of amylose and amylopectin and their organization within the granule, and we will then give insights on the biosynthetic mechanisms explaining the biogenesis of starch in plants.

Amylose is synthesized in vitro by extension of and cleavage from amylopectin.

Amylose synthesis was obtained in vitro from purified Chlamydomonas reinhardtii starch granules. Labeling experiments clearly indicate that initially the major granule-bound starch synthase extends glucans available on amylopectin. Amylose synthesis occurs thereafter at rates approaching or exceeding those of net polysaccharide synthesis. Although these results suggested that amylose originates from cleavage of a pre-existing external amylopectin chain, such transfer of chains from amylopectin to amylose was directly evidenced from pulse-chase experiments. The structure of the in vitro synthesized amylose could not be distinguished from in vivo synthesized amylose by a variety of methods. Moreover high molecular mass branched amylose synthesis preceded that of the low molecular mass, suggesting that chain termination occurs consequently to glucan cleavage. Short pulses of synthesis followed by incubation in buffer with or without ADP-Glc prove that transfer requires the presence of the glucosyl-nucleotide. Taken together, these observations make a compelling case for amylopectin acting as the in vivo primer for amylose synthesis. They further prove that extension is followed by cleavage. A model is presented that can explain the major features of amylose synthesis in plants. The consequences of intensive amylose synthesis on the crystal organization of amylopectin are reported through wide angle x-ray analysis of the in vitro synthesized polysaccharides.

Digestion of carbohydrate from white beans (Phaseolus vulgaris L.) in healthy humans.

Resistant starch (RS) is thought to be present in large amounts in legume seeds; however, it has never been quantified in healthy humans. RS from cooked (atmospheric pressure) white beans was quantified in humans and pigs, and characterized to explain its low digestibility. Six human volunteers were intubated to collect ileal digesta after an experimental meal composed of orange juice, butter and 167 g beans (dry matter basis). The reliability of the intubation method was examined in a pig study in which it was compared with another collection method, ileal cannulation. Chemical analyses, microscopy and size exclusion chromatography were performed on human and pig digesta. The pig study showed that the intubation method may underestimate the quantity of RS. However, no chemical/physical difference was observed between the RS collected by the two techniques. In the human study, 16.5 +/- 1.3% (11.3 g) of the ingested starch was recovered as RS. The microscopy of the digesta showed that part of the RS was enclosed in the cell walls. Although the RS was composed mainly of alpha-glucan molecules with a degree of polymerization (DP) 40 to 60, oligosaccharides and large molecules of DP > 400 were also present. Retrogradation was not found to be the main factor responsible for starch malabsorption. We conclude that white beans may contain a large amount of RS formed mainly by partially degraded molecules protected by the cell walls during their transit through the gut.

Starches from A to C. Chlamydomonas reinhardtii as a model microbial system to investigate the biosynthesis of the plant amylopectin crystal.

Wide-angle powder x-ray diffraction analysis was carried out on starch extracted from wild-type and mutant Chlamydomonas reinhardtii cells. Strains containing no defective starch synthases as well as mutants carrying a disrupted granule-bound starch synthase structural gene displayed the A type of diffraction pattern with a high degree of crystallinity. Mutants carrying a defect for the major soluble starch synthase (SSS), SSS II, were characterized by a switch to the B type of diffraction pattern with very low crystallinity. Mutant strains carrying SSS I as the only glucan elongation enzyme regained some of their crystallinity but switched to the C type of diffraction pattern. Differential scanning calorimetry analysis correlated tightly with the x-ray diffraction results. Together with the electron microscopy analyses, these results establish C. reinhardtii as a microbial model system displaying all aspects of cereal starch synthesis and structure. We further show that SSS II is the major enzyme involved in the synthesis of crystalline structures in starch and demonstrate that SSS I alone builds a new type of amylopectin structure.