Structure function relationships of transgenic starches with engineered phosphate substitution and starch branching

Potato tuber starch was genetically engineered in the plant by the simultaneous antisense suppression of the starch branching enzyme (SBE) I and II isoforms. Starch prepared from 12 independent lines and three control lines were characterised with respect to structural and physical properties. The lengths of the amylopectin unit chains, the concentrations of amylose and monoesterified phosphate were significantly increased in the transgenically engineered starches. Size exclusion chromatography with refractive index detection (SEC-RI) indicated a minor decrease in apparent molecular size of the amylose and the less branched amylopectin fractions. Differential scanning calorimetry (DSC) revealed significantly higher peak temperatures for gelatinisation and retrogradation of the genetically engineered starches whereas the enthalpies of gelatinisation were lower. Aqueous gels prepared from the transgenic starches showed increased gel elasticity and viscosity. Principle component analysis (PCA) of the data set discriminated the control lines from the transgenic lines and revealed a high correlation between phosphate concentration and amylopectin unit chain length. The PCA also indicated that the rheological characteristics were primarily influenced by the amylose concentration. The phosphate and the amylopectin unit chain lengths had influenced primarily the pasting and rheological properties of the starch gels.     

Structural and physicochemical characterisation of rye starch

The gelatinisation, pasting and retrogradation properties of three rye starches isolated using a proteinase-based procedure were investigated and compared to those of wheat starch isolated in a comparable way. On an average, the rye starch granules were larger than those of wheat starch. The former had very comparable gelatinisation temperatures and enthalpies, but slightly lower gelatinisation temperatures than wheat starch. Under standardised conditions, they retrograded to a lesser extent than wheat starch. The lower gelatinisation temperatures and tendencies of the rye starches to retrograde originated probably from their higher levels of short amylopectin (AP) chains [degree of polymerisation (DP) 6–12] and their lower levels of longer chains (DP 13–24) than observed for wheat starch. The rapid visco analysis differences in peak and end viscosities between the rye starches as well as between rye and wheat starches were at least partly attributable to differences in the levels of AP short chains and in average amylose molecular weight. The AP average chain lengths and exterior chain lengths were slightly lower for rye starches, while the interior chain lengths were slightly higher than those for wheat starch.     

Molecular structure of amylopectin from Amaranth starch and its effect on physicochemical properties

The molecular structure of amylopectin and its varphi,beta-limit dextrins from starch of 13 amaranth cultivars was determined by HPAEC-PAD after debranching. Chain length profiles of amylopectins showed bimodal distributions. The molar-based ratios of the average chain lengths of amylopectins (CLap) ranged from 17.41 to 18.22. The molar-based average chain lengths (CLld) and average B-chain lengths (BCLld) of varphi,beta-limit dextrins varied from 7.68 to 8.05, and from 14.10 to 14.73, respectively. Correlation analysis indicated that most structural parameters were positively correlated with thermal properties with few exceptions, whereas the content of fraction fa' ("'" stands for molar-based chain length ratio) was negatively correlated with the thermal properties. Pasting properties of cold paste viscosity (CPV) and setback were also correlated with amylopectin structural parameters.     

The effect of mutant genes at the r, rb, rug3, rug4, rug5 and lam loci on the granular structure and physico-chemical properties of pea seed starch

The granular structure and gelatinisation properties of starches from a range of pea seed mutants were studied. Genes which affect the supply of substrate during starch synthesis (rb, rug3, rug4) affected the total crystallinity and possibly increased the content of A polymorphs in the starch. Conversely, genes directly affecting the synthesis of starch polymers (r, rug5, lam) increased the content of B polymorphs, but had a minimal effect on total crystallinity. During gelatinisation, starches from the rb, rug3, rug4 and lam mutants had narrow endothermic peaks which were similar to starch from the wild-type, although all the starches had different peak temperatures and enthalpy changes. Starches from r and rug5 mutants were very different to all other starches, having a very wide transition during gelatinisation. In addition, the amylopectin in starch from these mutants had altered chain lengths for those parts of the polymer which form the ordered structures in the granule.     

Phosphate esters in amylopectin clusters of potato tuber starch

Starch phosphate is important in starch metabolism and in order to deduce its location and structural effects in clusters and building blocks of amylopectin, these were isolated from a normal potato (WT) and two starches with antisense suppressed glucan water dikinase (asGWD) activity and starch branching enzyme (asSBE) activity possessing suppressed and increased phosphate contents, respectively. Neutral N-chains and phosphorylated P-chains of the amylopectin macromolecules were similar in WT and asGWD, whereas asSBE possessed considerably longer P-chains. Cluster β-limit dextrins were isolated by α-amylase treatment and successive β-amylolysis. Cluster sizes were generally smaller in asSBE. The building block composition of neutral N-clusters were very similar in WT and asGWD, while asSBE was different, containing less blocks with degree of polymerization (DP)>14. Phosphate content of the P-clusters of WT and asGWD was rather similar, while asSBE contained highly phosphorylated P-clusters with proportionally more P-chains and a low degree of branching. The average chain lengths of the P-clusters were, however, similar in all samples. Our data demonstrate only minor effect on the cluster structure in relation to phosphate deposition suggesting conserved reaction patterns of starch phosphorylation. Models are suggested to account for the principle structural and functional effects of starch phosphate esters.     

Physico-chemical and degradative properties of in-planta re-structured potato starch

Starch re-structured directly in potato tubers by antisense suppression of starch branching enzyme (SBE), granule bound starch synthase (GBSS) or glucan water dikinase (GWD) genes was studied with the aim at disclosing the effects on resulting physico-chemical and enzyme degradative properties. The starches were selected to provide a combined system with specific and extensive alterations in amylose and covalently esterified glucose-6-phosphate (G6P) contents. As an effect of the altered chemical composition of the starches their hydrothermal characteristics varied significantly. Despite of the extreme alterations in phosphate content, the amylose content had a major affect on swelling power, enthalpy for starch gelatinization and pasting parameters as assessed by Rapid Visco Analysis (RVA). However, a combined influence of the starch phosphate and long glucan chains as represented by high amylose or long amylopectin chain length was indicated by their positive correlation to the final viscosity and set back (RVA) demonstrating the formation of a highly hydrated and gel-forming system during re-structuring of the starch pastes. Clear inverse correlations between glucoamylase-catalyzed digestibility and amylopectin chain length and starch phosphate and lack of such correlation with amylose content indicates a combined structuring role of the phosphate groups and amylopectin chains on the starch glucan matrix.     

Chemical and physical properties of ginkgo (Ginkgo biloba) starch

Starch isolated from mature Ginkgo biloba seeds and commercial normal maize starches were subjected to α-amylolysis and acid hydrolysis. Ginkgo starch was more resistant to pancreatic α-amylase hydrolysis than the normal maize starch. The chain length distribution of debranched amylopectin of the starches was analyzed by using high performance anion-exchange chromatography equipped with an amyloglucosidase reactor and a pulsed amperometric detector. The chain length distribution of ginkgo amylopectin showed higher amounts of both short and long chains compared to maize starch. Naegeli dextrins of the starches prepared by extensive acid hydrolysis over 12 days demonstrated that ginkgo starch was more susceptible than normal maize to acid hydrolysis. Ginkgo dextrins also demonstrate a lower concentration of singly branched chains than maize dextrins, and unlike maize dextrin, debranched ginkgo shows no multiple branched chains. The ginkgo starch displayed a C-type X-ray diffraction pattern, compared to an A-type pattern for maize. Ginkgo starch and maize starch contained 24.0 and 17.6% absolute amylose contents, respectively.     

Consequences of antisense RNA inhibition of starch branching enzyme activity on properties of potato starch

Antisense constructs containing cDNAs for potato starch branching enzyme (SBE) were introduced into potato (Solanum tuberosum L.). A population of transgenic plants were generated in which tuber SBE activity was reduced by between 5 and 98% of control values. No significant differences in amylose content or amylopectin branch length profiles of transgenic tuber starches were observed as a function of tuber SBE activity. Starches obtained from low SBE activity plants showed elevated phosphorous content. ³¹P n.m.r. analysis showed that this was due to proportionate increases in both 3- and 6-linked starch phosphates. A consistent alteration in starch gelatinisation properties was only observed when the level of SBE activity was reduced to below ˜5% of that of control values. Starches from these low SBE activity plants showed increases of up to 5 °C in d.s.c. peak temperature and viscosity onset temperature. Studies on melting of crystallites obtained from linear (1 → 4)-- -glucan oligomers suggest that an average difference of double helix length of about one glucose residue might be sufficient to account for the observed differences in gelatinisation properties. We speculate that the modification of gelatinisation properties at low SBE activities is due to a subtle alteration in amylopectin branch patterns resulting in small changes in double helix lengths within granules.     

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.     

A combined reduction in activity of starch synthases II and III of potato has novel effects on the starch of tubers

A chimeric antisense construct has been used to generate transgenic potatoes ( Solanum tuberosum L.) in which activities of both of the main starch synthases responsible for amylopectin synthesis in the tuber (SSII and SSIII) are reduced. The properties of starch from tubers of these plants have been compared with those of starches from transgenic plants in which activity of either SSII or SSIII has been reduced. Starches from the three types of transgenic plant are qualitatively different from each other and from the starch of control plants with unaltered starch synthase activities, with respect to granule morphology, the branch lengths of amylopectin, and the gelatinisation behaviour analysed by viscometry. The effects of reducing SSII and SSIII together cannot be predicted from consideration of the effects of reducing these two isoforms individually. These results indicate that different isoforms of starch synthase make distinct contributions to the synthesis of amylopectin, and that they act in a synergistic manner, rather than independently, during amylopectin synthesis.     

Importance of isoforms of starch-branching enzyme in determining the structure of starch in pea leaves

The impact of a mutation at the r locus of peas ( Pisum sativum L.) on the structure of starch in the leaf has been studied. The mutation specifically eliminates the A class of isoform of starch-branching enzyme (SBE A) from the leaf, causing a 10-fold reduction in the total activity of the enzyme. Gel-permeation chromatography and thymol precipitation show that wild-type leaf starch consists of polymers with the general characteristics of amylose and amylopectin, although amylose is only a very minor component of the starch. High-performance anion exchange chromatography (HPAEC) of debranched amylopectin reveals that the distribution profile of branch lengths is strongly polymodal, and distinctly different from that of the amylopectin of storage starches. The mutation at the r locus results in the appearance of an amylopectin-like glucan of low molecular weight in the starch. The absorbance of the iodine complex of the amylopectin and analysis by HPAEC both indicate that the mutation causes an increase in the average branch length of the amylopectin but does not affect the polymodal nature of the distribution of branch lengths. The extent to which these effects of the mutation are specifically due to the loss of SBE A is discussed. It is suggested that differences in properties between isoforms of SBE are not the main factors that determine the polymodal distribution of branch lengths in amylopectin.     

Relationship between the distribution of the chain length of amylopectin and the crystalline structure of starch granules

The distributions of chain lengths in the amylopectins of starches from 20 species (11 A-, 6 B-, and 3 C-type) were characterised by h.p.l.c. in terms of the relationship between the molecular structure of the amylopectin and the crystalline structure of the starch granule. The weight-average chain-lengths of the amylopectins of the A-, B-, and C-type starches were in the ranges 23–29, 30–44, and 26–29, respectively. Gel-permeation chromatograms of the amylopectins debranched with isoamylase showed bimodal distributions of fractions containing long and short chains for 17 specimens (including corn, rice, potato, etc.) and trimodal distributions, of which the fraction containing short chains had twin peaks, for wheat, tapioca, and tulip amylopectins. The correlation coefficients between the average chain-lengths of amylopectins and the fractions of long and short chains and the ratio of the fractions of short and long chains by weight were 0.90, 0.69, and ?0.95, respectively. In general, amylopectin molecules of A-type starches have shorter chains in both the long- and short-chain fractions and larger amounts of the short-chain fractions than those of the B-type starches. The chain lengths of amylopectins of the C-type starches were intermediate and it is inferred that these starches possibly yield any type of crystalline structure depending on the environmental temperature and other factors, whereas the A- and B-type starches are insensitive to temperature.     

Starch degradation by the mould Trichoderma viride I. The mechanism of starch degradation

The mechanism of starch degradation by the fungus Trichoderma viride was studied in strain CBS 354.44, which utilizes glucose, starch and dextrins but is unable to assimilate maltose. It was shown that the amylolytic enzyme system is completely extracellular, equally well induced by starch, amylose or amylopectin and that it consists mainly of enzymes of the glucoamylase type which yield glucose as the main product of starch hydrolysis. Small amounts of -amylase are produced also. The enzymes produced in starch cultures degrade starch, amylose and amylopectin equally well.Enzyme synthesis in starch media takes place to a considerable extent after exhaustion of the carbon source when maximum growth has been attained.Low-molecular dextrins are degraded by extracellular enzymes of the glucoamylase type. These enzymes are produced in media containing starch or dextrins. Maltotriose is consumed for only one third leaving maltose in the culture filtrate. Maltose is hardly attacked and hardly induces any amylolytic enzyme activity. No stable -glucosidase appears to be produced.     

Amylopectin aggregation as a function of starch phosphate content studied by size exclusion chromatography and on-line refractive index and light scattering

Starches with a natural 65-fold span in covalently bound phosphate content were prepared from five different crops including sorghum, cassava, three potato varieties and an exotic ginger plant, Curcuma zedoaria, with extreme starch phosphate content. These starches were subjected to size exclusion chromatography with refractive index detection (SEC/RI). A simple and rapid method for starch solubilisation was used. The conditions during solubilisation (2 M NaOH) and separation (10 mM NaOH, 50°C) were such as enabling >94% recovery of the starch without detectable degradation. The aggregation properties of the starch was investigated using on line refractive index/multi angle laser light scattering (RI/MALLS) detection. Three major regions in the SEC profile were identified, consisting of large amylopectin aggregates, amylopectin particles with radius of gyration (R_g) of approx 200 nm (400 nm blocklets) and amylose. A procedure for correction of light scattering signals spread over the SEC profile as a result of aggregate tailing was developed. The significance of the relative amounts of these three molecular species on standard starch pasting parameters, as measured by a Rapid Visco Analyzer (RVA), was investigated. Starches with a high amount of amylopectin aggregates showed high peak viscosities. Moreover, very high amounts of starch bound phosphate or amylose appears to suppress the content of large aggregates resulting in low viscosity.     

Structural characterization of wheat starch granules differing in amylose content and functional characteristics

Small-angle X-ray scattering (SAXS) together with several complementary techniques, such as differential scanning calorimetry and X-ray diffraction, have been employed to investigate the structural features that give diverse functional properties to wheat starches (Triticum aestivum L.) within a narrow range of enriched amylose content (36–43%). For these starches, which come from a heterogeneous genetic background, SAXS analysis of duplicate samples enabled structural information to be obtained about their lamellar architecture where differences in lamellar spacing among samples were only several tenths of nanometer. The SAXS analysis of these wheat starches with increased amylose content has shown that amylose accumulates in both crystalline and amorphous parts of the lamella. Using waxy starch as a distinctive comparison with the other samples confirmed a general trend of increasing amylose content being linked with the accumulation of defects within crystalline lamellae. We conclude that amylose content directly influences the architecture of semi-crystalline lamellae, whereas thermodynamic and functional properties are brought about by the interplay of amylose content and amylopectin architecture.     

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.     

Cyclodextrin glycosyltransferase from Bacillus circulans DF 9R: Activity and kinetic studies

Activity characteristics and kinetic aspects of a cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans DF 9R were studied. A mixture of α-, β- and γ-cyclodextrins (CDs), glucose, maltose and negligible amounts of longer linear dextrins were produced from gelatinized amylose, amylopectin and starch from different sources. In the coupling reaction, CDs were the substrates in the presence of acceptors such as maltose and/or longer oligosaccharides. From oligosaccharides formed by three or more glucose units, this enzyme produced linear chains of several lengths which were then cyclized. CGTase catalytic efficiency was compared employing an analytical grade starch and cassava starch for food use. Since the results obtained were similar for both starches, the use of an economic starch is an advantage. CGTase was inhibited by the substrate and its own products. Starch concentrations over 20 mg/mL inhibited the cyclizing activity. CDs behaved as competitive inhibitors and maltose as an uncompetitive inhibitor while maltotriose showed a mixed inhibition pattern. Limit dextrins showed a scarce inhibitory effect on enzyme activity. CD production could be improved with an ultrafiltration membrane reactor for continuous removal of the products; the starch concentration should be maintained below an inhibitory concentration and limit dextrins would remain in the reactor without affecting enzyme activity.     

Genotype-specific spatial distribution of starch molecules in the starch granule: a combined CLSM and SEM approach

Starch granule types from a variety of botanical sources were selected to represent differences in crystalline polymorph, amylose and phosphate content, and amylopectin chain length distribution. Equimolar labeling of starch molecules with the fluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) was used to construct a detailed map of the distribution of amylose and amylopectin within the granule by confocal laser scanning microscopy (CLSM) analysis. Medium- and high-resolution scanning electron microscopy (SEM) were used to provide detailed images of granule surface structures. By using a combined surface and internal imaging approach, interpretations of a number of previous structural observations is presented. In particular, internal images of high amylose maize and potato suggest that multiple initiations of new granules are responsible for the compound or elongated structures observed in these starches. CLSM optical sections of rice granules revealed an apparent altered distribution of amylose in relation to the proposed growth ring structure, hinting at a novel mechanism of starch molecule deposition. Well-described granule features, such as equatorial grooves, channels, cracks, and growth rings were documented and related to both the internal and external observations. A new method for probing the phosphate distribution in native granules was developed using a phosphate-binding fluorescent dye and CLSM.     

Bioconversion of unmodified native starches by Pseudomonas stutzeri maltotetraohydrolase: effect of starch type

Maltooligosaccharides including maltotetraose (G₄) have unique uses in biochemical, clinical, pharmaceutical, and food applications. G₄ productivity utilizing a G₄-producing amylase from Pseudomonas stutzeri in a membrane recycle bioreactor (MRB) was compared using four kinds of unmodified starches differing in the ration of amylose to amylopectin and the amylopectin chain lengths, and also one modified soluble starch. The specific activity of the enzyme used for the MRB system was 21.5 units/mg protein. The product purity using a native corn starch was 20.0% higher than that using a modified soluble starch in an MRB of bench-top scale. Four kinds of unmodified native corn starches were shown to be better substrates than the modified soluble starch considering all the criteria of product purity, production rate, and total product output. Correspondence to: Gun-Jo Woo