Structure studies of amylose-V complexes and retrograded amylose by action of alpha amylases, and a new method for preparing amylodextrins

Human-salivary, porcine-pancreatic, and Bacillus subtilis alpha amylases were used to study the structure of amylose-V complexes with butyl alcohol, tert-butyl alcohol, 1,1,2,2-tetrachloroethane, and 1-naphthol, and of retrograded amylose. Alpha amylase hydrolyzes the amorphous, folding areas on the surfaces of the lamella of packed helices, with the formation of resistant, amylodextrin fragments. Their degree of polymerization (d.p.) corresponds to the diameter of the helices and the folding length of the chain. The resistant fragments were fractionated on a column of Bio-Gel A-0.5m. Gel filtration of human-salivary and porcine-pancreatic alpha amylase hydrolyzates gave resistant fragments whose peak fractions, i.e., the three pooled fractions from the gel-filtration column with the highest amount of carbohydrate, had a d.p. of 75 +/- 4 for the amylose complex with butyl alcohol, 90 +/- 3 for those with tert-butyl alcohol and tetrachloroethane, and 123 +/- 2 for that with 1-naphthol. These d.p. values correspond to helices of six residues per turn with a folding length of 10 nm, seven residues per turn with a folding length of 10 nm, and eight residues per turn with a folding length of 12 nm (or nine residues per turn with a folding length of 10 nm), respectively. Acid hydrolysis of retrograded amylose gave a resistant fragment having an average d.p. of 32, human-salivary and porcine-pancreatic alpha amylases gave a resistant fragment of d.p. 43, and Bacillus subtilis alpha amylase gave a resistant fragment of d.p. 50. A structure for retrograded amylose is proposed in which there are crystalline, double-helical regions that are 10 nm long, interspersed with amorphous regions. The amorphous regions are hydrolyzed by acid and by alpha amylases, leaving the crystalline regions intact. The differences in the sizes of the resistant amylodextrins depend on the differences in the specificities of the hydrolyzing agents: acid hydrolyzes right up to the edge of the crystalline region, whereas the alpha amylases hydrolyze up to some point several D-glucosyl residues away from the crystalline region, leaving "stubs" on the ends of the amylodextrins whose sizes are dependent on the sizes of the binding sites of the individual alpha amylases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of tailor made short chain amylose–lipid complexes using varying reaction conditions

When amylose was synthesized using potato phosphorylase in the presence of amylose complexing lipids, monodisperse populations of amylose–lipid complexes were formed. Enzyme dosage and glucose-1-phosphate (glc-1-P)/primer ratio influenced the reaction rate of the enzymic synthesis, presumably by changing the balance between amylose synthesis and amylose–lipid complexation and precipitation, and impacted the molecular weight of the complexes. Lipid characteristics affected the dissociation properties and amylose chain lengths of the amylose–lipid complexes presumably by determining the minimal amylose chain length necessary for complexation and precipitation. Tailor made short chain amylose–lipid complexes can hence be produced by choosing the appropriate reaction conditions. We propose a synthesis mechanism in which the primer is elongated until an amylose chain is obtained which is of sufficient length to complex a first lipid. Further chain extension then occurs, together with subsequent complexation until the complex becomes insoluble and precipitates.     

Thermal dissociation of amylose-fatty acid complexes

Complexes saturated with fatty acids of increasing chain length (10:0 to 20:0), prepared by neutralisation of an alkaline solution of amylose, were shown by differential scanning calorimetry not to contain uncomplexed fatty acids. Insoluble complexes, prepared by dilution with water of a solution of amylose in methyl sulphoxide, contained uncomplexed fatty acids. The dissociation temperature of the complexes increased with increasing chain length of the fatty acids, but the dissociation enthalpy was practically independent of the chain length. Dissociation of the complexes (originating from alkaline solution) at 135°, followed by annealing for 16 h at 90° and rapid cooling, increased the dissociation temperature. The results are discussed in the light of a stoichiometric relationship between fatty acids and amylose.     

Influence of chain length of short monodisperse amyloses on the formation of A- and B-type X-ray diffraction patterns

Amyloses of uniform length were obtained by phosphorlytic synthesis (DP 20–35) and by preparative g.p.c. fractionation by an α-amylolytic digest of amylose on Bio-Gel P-4 (DP 3–20). Crystalline precipitates formed from pure aqueous solution on standing at ambient or lower temperatures gave the A-type X-ray pattern for malto-oligomers of DP 10–12 and the B-type pattern for DP 13 and all longer chains. With these carefully purified samples a mixture of the A- and B-amylose pattern was not observed. Malto-oligomers shorter than DP 10 did not crystallize. The findings support recent studies, indicating that the average chain length of amylopectin and the X-ray type of various starches are closely related. The reason for an influence of chain length on crystalline packing remains to be resolved.     

Amylose–lipid complexation: a new fractionation method

Amylose fractions of different peak Degree of Polymerisation (DP) (DP20, DP60, DP400, DP950) were complexed with docosanoic acid (C22) and glyceryl monostearate (GMS) at 60 and 90 °C. Complexation yields, relative crystallinities, dissociation temperatures and enthalpies increased with amylose chain lengths (DP20–DP60–DP400). Relative crystallinities and thermal stabilities of the DP950-complexes were slightly lower than those of the other amylose fractions, probably due to increased conformational disorders, resulting in crystal defaults. Molecular weight distributions of the complexes revealed that, irrespective to the complexation temperature, the critical DP for complex formation and precipitation was 35 and 40 for complexes with GMS and C22, respectively, corresponding to the length needed to accommodate two GMS- or C22-molecules within an amylose helix. Complexation of dextrins with a well-chosen lipid, allows to separate starch derived dextrins with a predictable critical chain length as border. Dextrins, of sufficient DP will complex and precipitate, while the shorter dextrins will remain in solution.     

Monte Carlo simulation of multiple attack mechanism of α-amylase

Porcine pancreatic α-amylase (EC 3.2.1.1) produces short maltooligosaccharides from a single enzyme-substrate complex without dissociation by multiple or repetitive attack. Multiple attack is caused by relative sliding of the enzyme along the product chain of the enzyme-product complex without dissociation to form another productive complex. The Monte Carlo method was applied to the multiple attack mechanism to predict product distribution from amylose and amylopectin molecules of arbitrary sizes. The position of the initial attack to make the enzyme-substrate complex and branched reaction paths from the enzyme-product complex were selected by random numbers and probabilities. A simulated product distribution from 100,000 samples of amylose of chain length greater than 80 agreed completely with experimental data at the early stage of hydrolysis of amylose of mean chain length 90. On the other hand, the simulated product distribution from amylopectin agreed with experimental data of potato amylopectin when the effective chain length of the A chain was 9. Since the mean chain length of the A chain of potato amylopectin is 15, it is possible that amylopectin is partially compact in solution, so that the enzyme can recognize and act only on the outer side of the A chain at the early stage of digestion. © 1996 John Wiley & Sons, Inc.     

Inclusion/exclusion of fatty acids in amylose complexes as a function of the fatty acid chain length

Structural models are proposed for amylose-fatty acid complexes depending on the respective chain lengths of their constituents. The three studied fatty acids induce the Vh amylose crystalline type. However, in contrast to lauric and palmitic acids, caprylic acid is not present in crystals. On the basis of the relative amounts of amylose and fatty acid determined in complexes and previous results of molecular modelling, inclusion of lauric and palmitic acids inside the amylose helices is proposed; the acyl chains are included in crystalline areas and the car☐ylic groups in amorphous areas. The absence of caprylic acid in crystals could be due to the solubility of this compound in the crystallization medium.     

Monitoring the crystallization of amylose–lipid complexes during maize starch melting by synchrotron x‐ray diffraction

Most starch granules exhibit a natural crystallinity, with different diffraction patterns according to their botanical origin: A‐type from cereals and B‐type from tubers. The V polymorph results essentially from the complexing of amylose with compounds such as iodine, alcohols, or lipids. The intensity and nature of phase transitions (annealing, melting, polymorphic transitions, recrystallization, etc.) induced by hydrothermal treatments in crystalline structures are related to temperature and water content. Despite its small concentration, the lipid phase present mainly in cereal starches has a large influence on starch properties, particularly in complexing amylose. The formation of Vh crystalline structures was observed by synchrotron x‐ray diffraction in native maize starch heated at intermediate and high moisture contents (between 19 and 80%). For the first time, the crystallization of amylose–lipid complexes was evidenced in situ by x‐ray diffraction without any preliminary cooling, at heating rates corresponding to the usual conditions for differential scanning calorimetry experiments. For higher water contents, the crystallization of Vh complexes clearly occurred at 110–115°C. For intermediate water contents, mixed A + Vh (or B + Vh for high amylose starch) diffraction diagrams were recorded. Two mechanisms can be involved in amylose complexing: the first relating to crystallization of the amylose and lipid released during starch gelatinization, and the second to crystalline packing of separate complexed amylose chains (amorphous complexes) present in native cereal starches. © 1999 John Wiley & Sons, Inc. Biopoly 50: 99–110, 1999     

A possible structure of retrograded maize starch speculated by UV and IR spectra of it and its components

“Retrogradation” has been used to describe the changes that occur in starch after gelatinization, from an initially amorphous state to a more ordered or crystalline state, which has a significant impact on starch application in food, textiles and materials fields. But mechanism of starch retrogradation is still unclear until now and there is no breakthrough in this area. Here we are speculating a possible structure of retrograded maize starch by UV (binding with iodine) and IR spectra of it and its compositions. We speculate that nucleation of retrograded starch origins from combination of reducing end of amylopectin and non-reducing end of amylose, and retrogradation terminates at combining of non-reducing end of amylopectin and reducing end of amylose. The chain length of resistant digestion retrograded starch should be nearly same. The hydroxyl associated with sixth carbon atoms of glucan must form hydrogen bond with other hydroxyl of starch.     

Amylopectin molecular structure reflected in macromolecular organization of granular starch

For lintners with negligible amylose retrogradation, crystallinity related inversely to starch amylose content and, irrespective of starch source, incomplete removal of amorphous material was shown. The latter was more pronounced for B-type than for A-type starches. The two predominant lintner populations, with modal degrees of polymerization (DP) of 13-15 and 23-27, were best resolved for amylose-deficient and A-type starches. Results indicate a more specific hydrolysis of amorphous lamellae in such starches. Small-angle X-ray scattering showed a more intense 9-nm scattering peak for native amylose-deficient A-type starches than for their regular or B-type analogues. The experimental evidence indicates a lower contrasting density within the "crystalline" shells of the latter starches. A higher density in the amorphous lamellae, envisaged by the lamellar helical model, explains the relative acid resistance of linear amylopectin chains with DP > 20, observed in lintners of B-type starches. Because amylopectin chain length distributions were similar for regular and amylose-deficient starches of the same crystal type, we deduce that the more dense (and ordered) packing of double helices into lamellar structures in amylose-deficient starches is due to a different amylopectin branching pattern.     

Potato phosphorylase catalyzed synthesis of amylose-lipid complexes

On-line size-exclusion chromatography monitoring of potato phosphorylase catalyzed amylose synthesis--starting from alpha-D-glucose-1-P and maltohexaose--revealed rather monodisperse amylose populations. In the presence of lipids, amylose-lipid complexes spontaneously formed and precipitated. They were recovered by centrifugation, freeze-dried, and characterized by wide-angle X-ray diffraction and differential scanning calorimetry. The presence of lipids during amylose synthesis led to lower amylose degrees of polymerization (DP). Lipid chain length defined amylose DP, which increased in the order myristic acid (C14), glyceryl monostearate (GMS), stearic acid (C18), and docosanoic acid (C22). The thermal stability of the complexes increased in the same manner, with the C22 complexes having the highest dissociation temperature. In addition, we hypothesized that these results provide additional evidence for the fringed micellar organization of (semi-enzymically synthesized) amylose-lipid complexes.     

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.     

Synthesis and characterization of 2,3-di-O-alkylated amyloses: hydrophobic substitution destabilizes helical conformation

Amylose was selectively alkylated in the 2,3-O position of each anhydroglucose unit after trityl protection of the 6-OH groups. Alkyl iodides of varying chain length (C(2), C(5), C(8)) were coupled to amylose, and degrees of substitution (DSs) were varied between 0.3 and 1.8, as assessed by NMR analysis. Increasing amounts of methyl groups per anhydroglucose unit increased solubility in nonaqueous media, while at the same time reducing the ability of modified amylose to form a complex with iodine. The tendency to form inclusion complexes with the surfactant N-dodecyl pyridinium bromide decreased in the order beta-cyclodextrin > amylose approximately solubilized starch, indicating that the frozen macrocycle of beta-cyclodextrin was the most efficient inclusion host. Introduction of the bulky trityl group abolished the helical amylose conformation, which is not readily reassumed in the presence of hydrophobic substitution of the C2 and C3 positions. These results indicated that a polar outer surface is necessary but not sufficient for the formation of a stable amylose helix.     

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 2-3 h at starch concentrations lower than 15% and 8-24 h 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.     

Formation and crystallization of amylose–monoglyceride complex in a starch matrix

The formation of amylose–lipid complexes in a gelatinized potato starch matrix was investigated using potato starch and glycerol monopalmitin. These complexes exist in two forms, with the amounts of each of the forms being dependent on the temperatures and durations of the pre-treatments.

Differential scanning calorimetry (DSC) was used to analyze transition temperatures and melting enthalpies, and thereby determine the amount of the complexes in the samples. X-ray diffraction analysis was used to investigate their crystallinity.

In measurements with DSC, form I started to melt at 88.5°C, and form II at 112.9°C. When complex form II was preheated at 100 or 110°C, its melting point rose to 116.3 and 119.7°C, respectively, because of an annealing effect. The same phenomenon occurred with complex form I: when preheated at 90°C, its melting point rose to 96.8°C. The crystal formation of form II appeared to be slower when treated at 110°C than at 100°C. Their maximum melting enthalpies were reached after about 24 h and 4 h of preheating, respectively. In X-ray diffraction analyses, form II showed a V-pattern, but form I did not. This indicates that form II is more crystalline than form I. It was possible to transform form I into form II when it was heat treated, because form I was then partially or totally melted.

As a comparison, the charged substance cetyltrimethylammonium bromide created complex form I with amylose in the starch matrix, but not form II.     

Effect of amylose deposition on potato tuber starch granule architecture and dynamics as studied by lintnerization

The effect of amylose deposition on the amylopectin crystalline lamellar organization in potato starch granules was studied by mild acid, so‐called lintnerization, of potato tuber starch transgenically engineered to deposit different levels of amylose. The starch granules were subjected to lintnerization at different temperatures (25, 35, and 45°C) and to two levels of solubilization, ～ 45 and 80%. The rate of the lintnerization increased with temperature but was suppressed by amylose. The molecular size of the lintner dextrins increased with temperature, but this effect was suppressed by the presence of amylose. At high temperatures and low‐amylose content, the degree of branches was high with the concomitant increase in size in the dextrins. A portion of the branches was resistant to debranching enzymes possibly due to specific structural formations. The effects of temperature suggested a unique granular architecture of potato starch, and a model showing the dependence of temperature on the dynamic arrangement of amylopectin and amylose in the crystalline and amorphous lamellae for the potato starch is suggested. © 2012 Wiley Periodicals, Inc.     

On the Structure of a New C-Glycosyl Flavone,Embinin, Isolated from the Petals of Iris germanica Linnaeous*1

Complexes of amylose with several kinds of n-aliphatic ketones having different chain lengths, different positions and numbers of carbonyl groups in the molecules were prepared. The unit cell dimensions of the complexes were calculated in both the wet and dried states by means of X-ray diffraction analysis. Both the 6₁- and 7₁-helix amyloses were presented in these complexes. It was found that the helix packing diameter of the amylose-ketone complex changes depending upon the linear chain length of the ketone molecule complexed.     

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 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.     

Amylose–dye complexes in cationic micelles: an optical spectroscopy study

The formation of amylose complexes with rose bengal (RB), erythrosine B (ER), and phenolphthalein (PP) in the presence of the cationic detergent tetradecyltrimethylammonium bromide (TTABr) was studied using optical spectroscopy methods. Absorption spectroscopy, steady-state fluorescence spectroscopy and picosecond time-resolved fluorescence spectroscopy were used to derive association constants k_s of the dyes, critical micelle concentration (CMC) values and structural information on the complexes formed. It seems that PP fits very well into amylose sites, where it forms an efficient inclusion complex with k_s=44,500 M⁻¹. The molecular diameter of RB is too big to fit the amylose cavity. Only part of the xanthene unit may be adopted in the helical cavity of amylose, whereas most of the interaction occurs through electrostatic and/or dipole–dipole interactions with the amylose chain. The ER molecule is an intermediate case, because it may fit the amylose cavity or adsorb on the amylose surface to form a complex. The presence of a surfactant in the amylose–ligand system increases the association constant for all dyes. In the presence of amylose, a decrease of the detergent CMC value of about one order of magnitude is observed. It is probable that the increased number of micelles incorporate more dyes into the amylose vicinity, which finally changes the structure of the amylose chain. On a macro scale, it was noted that the samples with dyes and detergent have a lower tendency to precipitate and the gelation process is delayed compared to that in water.