Β-limit Dextrin Research Articles

Maize Starch-Branching Enzyme Isoforms and Amylopectin Structure. In the Absence of Starch-Branching Enzyme IIb, the Further Absence of Starch-Branching Enzyme Ia Leads to Increased Branching

Previous studies indicated that the deficiency of starch-branching enzyme (SBE) Ia in the single mutant sbe1a::Mu (sbe1a) has no impact on endosperm starch structure, whereas the deficiency of SBEIIb in the ae mutant is well known to reduce the branching of starch. We hypothesized that in maize (Zea mays) endosperm, the function of SBEIIb is predominant to that of SBEIa, and SBEIa would have an observable effect only on amylopectin structure in the absence of SBEIIb. To test this hypothesis, the mutant sbe1a was introgressed into lines containing either wx (lacking the granule-bound starch synthase GBSSI) or ae wx (lacking both SBEIIb and GBSSI) in the W64A background. Both western blotting and zymogram analysis confirmed the SBEIa deficiency in sbe1a wx and sbe1a ae wx, and the SBEIIb deficiency in ae wx and sbe1a ae wx. Using zymogram analysis, no pleiotropic effects of sbe1a genes on SBEIIa, starch synthase, or starch-debranching enzyme isoforms were observed. High-performance size exclusion chromatography analysis shows that the chain-length profiles of amylopectin as well as beta-limit dextrin were indistinguishable between wx and sbe1a wx, whereas significant differences for both were observed between ae wx and sbe1a ae wx, suggesting an effect of SBEIa on amylopectin biosynthesis that is observable only in the absence of SBEIIb. The amylopectin branch density and the average number of branches per cluster were both higher in endosperm starch from sbe1a ae wx than from ae wx. These results indicate possible functional interactions between SBE isoforms that may involve enzymatic inhibition. Both the cluster repeat distance and the distance between branch points on the short intracluster chains were similar for all genotypes however, suggesting a similar pattern of individual SBE isoforms in cluster initiation and the determination of branch point location.

Purification and properties of apoplastic amylase from oat (Avena sativa) seedlings.

The protein fraction extracted with a high ionic strength buffer from the cell wall preparation of oat (Avena sativa L.) coleoptiles and first leaves contained starch-degrading (amylase) activity. The activity of apoplastic amylase in the coleoptiles and first leaves continued to increase in parallel with organ growth. One of the apoplastic amylases recovered from shoot cell wall preparations was purified by sequential ion exchange and gel filtration chromatography, and the catalytic properties of the enzyme were analysed. The purified enzyme gave a single 25 kDa protein band on SDS-PAGE. The enzyme exhibited maximum activity at pH 5.0 against maltooligosaccharides. The purified enzyme hydrolysed soluble starch and maltooligosaccharides larger than tetraose at maltose unit, but did not hydrolyse beta-limit dextrin or p-nitrophenyl-alpha-d-glucopyranoside. These results as well as the findings that the molecular size and the catalytic properties of the purified enzyme are different from those of known amylases obtained from Gramineae caryopses suggest that this enzyme is a novel type of beta-amylase present in cell walls of vigorously elongating Gramineae shoot organs.

Lintnerization of Two Amylose‐free Starches of A‐ and B‐Crystalline Types, Respectively

AbstractWaxy maize starch (WMS) and potato amylopectin starch (PAPS), representing amylose‐free A‐ and B‐crystalline granules, respectively, were subjected to hydrolysis in diluted hydrochloric acid (lintnerization). The solubilization rate of the granules was dependent on the temperature, but there were only small differences between the samples. The compositions of the lintners obtained at 29°C were also similar, though the sensitivity to enzymatic debranching was different. The degree of debranching increased significantly if pullulanase and isoamylase were used successively. Because some branches remained resistant to enzymatic attack, the degree of scattered branching was difficult to estimate. The amount and composition of β‐limit dextrins suggested, however, that both starches contained scattered branches. Long chains and the shortest chains in the amylopectin molecules were sensitive to lintnerization and therefore probably located outside the crystallites. The composition of the PAPS lintners was dependent on the temperature of the lintnerization, whereas that of WMS lintners was not. The composition of a lintner from normal potato starch was compared with PAPS, and it is concluded that the presence of amylose has a higher effect than the type of crystallinity on the lintnerization results.

Natural variation in rice starch synthase IIa affects enzyme and starch properties.

The natural variation in starch synthase IIa (SSIIa) of rice (Oryza sativa L.) was characterised using near-isogenic lines (NILs). SSIIa is a candidate for the alk gene regulating the alkali disintegration of rice grains, since both genes are genetically mapped at the same position on chromosome 6 and related to starch properties. In this study, we report that the alkali-susceptible cultivar Nipponbare lacked SSIIa activity in endosperm. However, the activity was detected with NILs having the alk allele of alkali-tolerant Kasalath. SSIIa protein was present even in Nipponbare endosperm, but it was not associated with starch granules at the milky stage of endosperm. Three single-nucleotide polymorphisms (SNPs) predicting amino acid substitutions existed between the cDNA sequences of SSIIa of Nipponbare and Kasalath were genotyped with 65 rice cultivars and four wild relatives of cultivated rice. The results obtained explain the potential importance of two of the amino acid residues for starch association of rice SSIIa. An analysis of the chain-length distribution of β-limit dextrin of amylopectin showed that without SSIIa activity, the relative number of A-chains (the short chains without branches) increased and that of B1-chains (the short chains with branches) decreased. This suggests that, given the SSIIa defect, short A-chains could not reach a sufficient length for branching enzymes to act on them to produce B1-chains.

Composition and properties of starches extracted from tubers of different potato varieties grown under the same environmental conditions

Twelve commercially relevant potato cultivars were grown in a field plot in Perthshire (UK) in 1996. Starches were extracted from these tubers and their physico-chemical properties were investigated with a particular focus on aspects of granule order and corresponding gelatinisation characteristics. There was little variation in (average) granule diameter (21.4±1.6 μm), amylose content (28.7±1.9%) or phosphorus (0.54±0.16 μg mg −1) content. Structural elements of amylopectin were essentially constant in terms of unit distributions and chain lengths (average DP 28±2) with long ( F 1) and short ( F 2) chains with average DP of 56±2 and 19±1, respectively. The structures of the (debranched) β-limit dextrins of the amylopectin molecules were also very similar with the average DP of the two major fractions ( F 1β and F 2β) being 48±3 and 37±2, respectively. Peak gelatinisation temperatures ( T p) and enthalpy (Δ H) were also correspondingly similar (63.2 °C±1.2 and 18.6 °C±0.8, respectively). The constancy of physico-chemical properties for these starches is in marked contrast to variation in different parameters induced by environmental factors, especially growth temperature.

Molecular Basis of the Gelatinisation and Swelling Characteristics of Waxy Rice Starches Grown in the Same Location During the Same Season

Six waxy rice samples (grown at the same site during the same season) were investigated to identify those aspects of amylopectin structure and granule composition which differentiate the starches into high (Tp 77·8 °C on average) or low (Tp 66·2 °C on average) gelatinisation temperature. All starches contained less than 2·5% amylose with negligible lipid. Granule dimensions were similar (mean diameter 4·9–5·7 μm) as was the β-amylolysis limit (59·9–63·0%). However, the structural elements of amylopectins within the two groups varied. The low gelatinisation temperature starches contained two major chain fractions upon debranching with iso-amylase, F3 (DP 16) and F1 (DP 51) on average while the high gelatinisation temperature starches contained three fractions, F3 (DP 16), F2 (DP 19) and F1 (DP 40). The proportion of F1/(F2+F3) for the low and high gelatinisation starches representing on average 20·2 and 10·5% respectively. Hence, the higher proportion of exterior chains and especially those with DP ∼ 19 appear to confer the higher gelatinisation temperature characteristic of these starches. Debranched β-limit dextrins were prepared from these starches. Regardless of their origin, (high or low gelatinisation temperature), they contained three fractions: F3β (DP 5), F2β (DP 13) and F1β (DP 19). Hence, the high versus low gelatinisation characteristic was imparted by the exterior chains. Using 13-C CP-MAS/NMR it was confirmed that the number of double helices within the starches were approximately constant although small differences in crystallinity were identified by X-ray diffraction. Retrogradation of the solubilised native high and low gelatinisation temperature amylopectin molecules confirmed that the ‘high temperature gelatinisation amylopectin’ molecules form higher dissociation temperature and enthalpy crystalline domains. This confirms that the higher proportion of short and especially relatively longer short chains promotes crystallite formation.

Enzymatic properties and regulation of ZPU1, the maize pullulanase-type starch debranching enzyme

Starch debranching enzymes (DBE) are required for mobilization of carbohydrate reserves and for the normal structural organization of storage glucan polymers. Two isoforms, the pullulanase-type DBEs and the isoamylase-type DBEs, are both highly conserved in plants. To address DBE functions in starch assembly and breakdown, this study characterized the biochemical activity of ZPU1, a pullulanase-type DBE that is the product of the maize Zpu1 gene. Assays showed directly that recombinant ZPU1 (ZPU1r) expressed in Escherichia coli functions as a pullulanase-type enzyme, and 1H-NMR spectroscopy demonstrated that ZPU1r specifically hydrolyzes α(1→6) branch linkages. Preferred substrates for ZPU1r hydrolytic activity were determined, as were pH, temperature, and thermal stability optima. Kinetic properties of ZPU1r with respect to two substrates, β-limit dextrin and pullulan, were determined. ZPU1 activity was increased by incubation with thioredoxin h, and native activity was decreased in mutants that accumulate soluble sugars, suggesting potential regulatory mechanisms.

Characterisation of the in vitro products of potato starch branching enzymes I and II

An enzyme substrate obtained by ethanol fractionation of linear dextrins was branched by potato starch branching enzymes (SBE). During the branching process, samples were withdrawn from the incubation mixture and the chain length distributions were analysed by high performance anion exchange chromatography (HPAEC). The relative composition of chains with a degree of polymerisation of 9–35 was essentially constant throughout the branching process for both SBEI and SBEII. This showed that the change in size and structure of the substrate during the branching reaction did not considerably affect the patterns of chains produced by the enzymes. Chain length patterns as well as branching rates were different for the two SBE isoforms, supporting the theory that they have different roles in amylopectin synthesis. Despite the differences in chain length profiles, the degree of branching was found to be 3.7% for both SBE products when analysed by NMR. Using proton 2D-NMR, the structural differences between intact branching products and their β-limit dextrins were determined.

New Sweet Potato Line having low Gelatinization Temperature and altered Starch Structure

A new sweet potato breeding line, Kanto 116, was developed, featuring low gelatinization temperature and an altered starch fine structure. Starch granules from Kanto 116 showed an abnormal morphology characterized by cracking into granules. Starch content, amylose content and tuberous root appearance of Kanto 116 were similar to those of the control and the parents. Pasting temperatures of Kanto 116 starch determined by the Rapid Visco Analyser were 51.4 — 52.6 °C, approximately 20 °C lower than those of the control and parents starches. Onset, peak, and conclusion temperature of gelatinization, and gelatinization enthalpy of Kanto 116 starch determined by differential scanning calorimetry were 39.0 °C, 46.9 °C, 64.8 °C, and 8.8 J/g, respectively, and much lower than those of the control and parents starches. The chain-length distribution of the amylopectin molecules, determined by high-performance anion-exchange chromatography, showed that Kanto 116 starch had a higher proportion of short chains (DP 6 — 11) and a lower proportion of chains between DP 12 — 28 than control and parent starches. The debranched β-limit dextrin of Kanto 116 starch also showed that the proportion of both short and long B1 chains was different from those of the control and parents starches.

Effect of Starch Hydrolysis Products on the Determination of Limit Dextrinase and Limit Dextrinase Inhibitors in Barley and Malt

Abstract The activity of purified malt limit dextrinase was apparently increased by maltodextrins when enzyme activity was assayed by dyed pullulan substrates. At higher maltodextrin levels (different for each maltodextrin), the enzyme was inhibited. The apparent enzyme activation was not detected when assays were carried out with amylopectin β-limit dextrin, a more natural substrate for limit dextrinase, as substrate. Pullulanase (Aerobacter aerogenes) activity, when assayed with dyed pullulan, was not activated in the presence of maltodextrins. Barley extracts contained material that appeared to increase the activity of limit dextrinase. Results indicated that this material was a mixture of starch dextrins, which lost their activating ability when treated with amyloglucosidase. Treatment of malt extracts with amyloglucosidase prior to assaying for limit dextrinase significantly lowered the apparent limit dextrinase activities (when measured by dyed pullulan) and allowed for a more accurate estimate of both limit dextrinase and limit dextrinase inhibitor levels in the extracts. Enzyme and inhibitor levels in malt extracts, obtained using amyloglucosidase pre-treatments, showed that there was a strong relationship between inhibitor disappearance in malt extracts and the appearance of fully active limit dextrinase. These results confirm earlier conclusions that limit dextrinase is largely ineffective during the mashing stage of brewing because it is inhibited by two barley proteins still present in malt.

Microdialysis clean-up and sampling in enzyme-based methods for the characterisation of starch

Microdialysis was used for sampling enzyme hydrolysis products of starch hydrolysed with β-amylase, pullulanase, and/or isoamylase, to obtain information about the molecular structure of starch. Starches from waxy, normal, and high amylose maize, and from normal and genetically modified potato (amylose deficient) were used, and also commercial potato amyloses. The hydrolysis products were analysed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Simultaneous sampling and sample clean-up were achieved with microdialysis, thus enabling on-line injection into the liquid chromatographic system. The molecular weight cut-off of the membrane allowed for diffusion of small molecules such as oligosaccharides through the membrane, but hindered large molecules, e.g. enzymes and large polysaccharides, from entering the chromatographic system. With microdialysis sampling, it was possible to investigate the short chain fractions of debranched starch in the presence of amylose without pre-fractionation. The microdialysis–HPAEC-PAD system was also used for determination of the A:B chain ratio and the β-limit value. After β-amylolysis, only liberated maltose diffused through the dialysis membrane, which resulted in on-line sample clean-up from branched β-limit dextrin as well as from the enzyme. The proposed method is fast and easy to handle since clean-up of the hydrolysate is achieved on-line with the chromatographic system.

Structural heterogeneity in waxy-rice starch

Alpha-amylase of B. amyloliquefaciens was used for the structural characterization of the amylopectin from waxy-rice starch. Fractions of α-dextrins with a degree of polymerization (d.p.) <5000 were isolated from amylopectin hydrolysates after 1 and 3 h. φ, β-Limit dextrins were prepared by successive phosphorolysis and beta-amylolysis of the fractions and these were analysed by a second alpha-amylolysis. Based on the hydrolysis pattern, the limit dextrins were divided into two major groups, A and B, which possessed units of clusters of d.p. 100–200 and 90–130, respectively. An extensive alpha-amylolysis resulted in characteristic distributions of dextrins with d.p. <80 which represented branched building blocks. Type A dextrins possessed more larger building blocks with d.p. ∼40, but less intermediate and small blocks, than type B. The φ, β-limit dextrin of the original amylopectin had a distinct distribution enriched in small building blocks. A model is proposed in which the two types of dextrins originate from regular and less regular structural domains of the amylopectin fraction within the starch granules.

Enzymatic Characterisation of Novamyl®, a Thermostable α-Amylase

The thermostable -amylase Novamyl® is used in the baking industry as an antistaling agent due to its ability to reduce retrogradation of amylopectin. We have studied its enzymatic properties at pH 5.0. We make two main conclusions: (1) Novamyl® shows sequence homology to cycloglycosyl transferases (CGTases); like these enzymes, Novamyl® cleaves cyclodextrins, forms transglycosylation products and is subject to product inhibition by maltose. Novamyl® has 5 subsites in the active site and is also subject to substrate inhibition. (2) Novamyl® is clearly different from exoglucanases like β-amylase and glucoamylase. It is able to hydrolyse a pentasaccharide with bulky substituents at both ends (INdp5) and is inhibited by the α-amylase inhibitor Trestatin A. Although Novamyl® appears unable to hydrolyse α-1,6-linkages, it is able to degrade amylopectin to a greater extent than β-amylase as well as β-limit dextrins. Novamyl® degrades amylose in such a manner that initially the molecular weight is drastically reduced while β-amylase does not show any detectable effect on the molecular weight of this substrate. Products of the degradation of amylopectin and amylose by Novamyl® are maltose and oligosaccharides, whereas β-amylase and glucoamylase produce only maltose and glucose, respectively. This was shown in baking experiments as well. The new data presented here clearly show that unlike exoamylases, Novamyl® does not require a non-reducing end and attacks amylose, Indp5 and cyclodextrins in an endo-like manner. Based on these results Novamyl® should be reclassified.

Effects of enzymically modified amylopectin on the rheological properties of amylose-amylopectin mixed gels

Branched α-dextrins with different molecular weights were prepared from waxy maize. A series of β-limit dextrins was prepared from α-dextrins and native amylopectin. The fine structure of the dextrin samples was investigated by debranching, and was found to be similar to the unit chain distribution of native amylopectin. The absolute molecular weights of α- and β-limit dextrins and commercial potato amy lose were determined by gel-permeation chromatography (GPC) and with a dual light-scattering detector. Solubilized potato amy lose and α- and β-limit dextrins were mixed at different ratios to give a total concentration of 8%. Dynamic viscoelastic measurements showed that gel formation of amylose was highly dependent both on the ratio of amylose to α-dextrin and on the molecular weight of α-dextrin. α-Dextrin caused an increase of storage modulus, G, when the amylose: α-dextrin ratio was low and the molecular weight of α-dextrin was high. The high-molecular-weight α-dextrin influenced amylose gelation in the same way as native waxy maize starch, but the medium- and low-molecular-weight α-dextrins weakened the gel formation, especially at a ratio of 25:75 (amylose: α-dextrin). When low-molecular-weight β-limit dextrins were mixed with amylose, the resulting gels were more rigid than those in which amylose was mixed with corresponding α-dextrins. When high-molecular-weight β-limit dextrins were mixed with amylose, the resulting gels were weaker.

Immobilization of rice limit dextrinase on γ-alumina beads and its possible use in starch processing

Limit dextrinase (EC 3·2·1·41) partially purified from rice seeds was immobilized by adsorption on γ-alumina beads. Binding and activity yields were 88 and 52%, respectively. The adsorbed enzyme showed a broader pH optimum and an increased thermal stability compared to the free one. Optimum temperature was between 50 and 60°C and the enzyme retained about 70% of the initial activity after 7 days of incubation at 40°C in the presence of Ca 2+. The immobilized biocatalyst degraded pullulan completely to maltotriose and effectively converted both acid hydrolysed amylodextrins and β-limit dextrins into maltose and maltotriose. The possibility of using a debranching enzyme of plant origin in starch processing can be considered to be a new approach for the improvement of the quality of sugar syrups used in the food industry.

Studies on sweet potatoes—III. Distribution of unit chains of branched and unbranched molecules of starch

Freshly prepared sweet potato (SP) starch was separated quantitatively into amylose and amylopectin by a concanavalin A precipitation method. Amylose was further fractionated into two subfractions containing essentially linear (Am) and moderately branched (Ax) molecules, by hot-butanol extraction. The molecular weight determined by GPC of Am was 3 × 10 6 Da, the values for Ap and Ax were 1.5 × 10 7 and 5 × 10 6 Da, respectively. The β-amylolysis limit values were 57, 70 and 92%, respectively, for Ap, Ax and Am. The native, as well as the derived β-limit dextrins of Ap and Ax, were debranched with isoamylase and their unit chain lengths, measured by GPC and SE-HPLC methods, showed Ap (C̄L, 21.0) to have a trimodal distribution of unit chains with D̄P values of 52, 37 and 20, whereas Ax had tetramodal unit chains with D̄P values of 40, 30, 21 and 9. Am was more or less a linear molecule. In vitro SP starch was more digestible than legume and cereal starches.

The chemical structure of amylose and amylopectin fractions of starch from tobacco leaves during development and diurnally-nocturnally

Starches, isolated from rapidly expanding tobacco leaves four times during the day and night and once from fully expanded leaves, were fractionated with concanavalin A. From an examination of the amounts and properties of amylose, the diurnal decrease in iodine absorption of the starches on illumination appeared to be due to an increase in its branched character, and possibly the presence of unbranched polymer of low dp, combined with a decrease in the proportion of amylose fraction. The increase in apparent amylose content with age was due to an increase in the proportion of amylose. The amylose fractions at different times had only small differences in average mol size in solution and relative mol wt ( M r near 4 × 10 5) which were lower than those of storage organs. The average mol size in solution and relative mol wt of the amylopectins decreased during illumination, increased in darkness, and were lower ( M r 2–2.7 × 10 6) at all times than those from storage organs. Debranching followed by size-exclusion chromatography [on Fractogel TSK 50(S)] gave similar proportions of long, medium, and short chains for all amylopectin samples, and these proportions differed from those for debranched amylopectin from n-maize seed starch. On debranching and chromatography of the amylopectin β-limit dextrins (which gives an estimate of the proportions of core chains) differences persisted. Structural characteristics of amylopectin from tobacco leaf starch were similar to those of normal genotypes from storage organs. The proportion of glucosyl units in core chains, the external-to-core chain ratio, and indices of compactness were calculated for a number of (1 → 4)(1 → 6) α-glucans. A plot of the index of compactness for glycogens and amylopectins showed that the decrease in compactness and the increase in total average chain length that occurs from glycogen to normal and then to amylose extender amylopectins involves a proportionate increase in average internal, external, and core chain lengths and not a selective increase in one type of chain.

Effect of Potassium, Magnesium, and Calcium Deficiencies on Carbohydrate Pools and Metabolism in Citrus Leaves

Carbohydrate content and related enzyme activities were determined in leaves of rough-lemon (Citrus volkameriana Ten. &amp; Pasq) plants grown under K, Mg, and Ca deficiencies. Starch content was lower and soluble sugar significantly higher in K-deficient than in control leaves. Magnesium- and Ca-deficient leaves, on the other hand, accumulated large amounts of starch. Electron micrographs also showed a reduction in the number and size of starch grains in chloroplasts of K-deficient leaves, while those of Mg- and Ca-deficient leaves were filled with large starch grains. Total amylase activity increased 3- to 7-fold in K-deficient leaves, with maltose as the major product and small amounts of maltotriose. Electrophoretic separation of amylase isozymes on native gels containing starch or amylopectin showed higher band intensities in K-deficient leaves. Examination of the capacity of leaf extracts to use amylopectin vs. β-limit dextrin indicated that mainly 8-amylase was involved. Acid invertase activity increased 7-fold in K-deficient leaves, while alkaline invertase showed little change.

Fine structure of maize starches from four wx-containing genotypes of the W64A inbred line in relation to gelatinization and retrogradation

Maize starches from four wx-containing genotypes ( wx, du wx, ae wx, and ae du wx) of the W64A inbred line were examined to determine their gelatinization and retrogradation properties and the fine structure of their amylopectins (AP). Chain-length distribution profiles of the APs showed distinct patterns according to genotype. The wx and du wx starches had a large proportion of short chains and showed A-type X-ray patterns, whereas the ae wx and ae du wx starches had a large proportion of long chains and showed B-type X-ray patterns. The ae wx starch had the highest values of onset melting temperature ( T o) and heat uptake (Δ H), both for gelatinization and after retrogradation, which were attributed to a greater proportion of long chains (degree of polymerization > 16) in the AP. Debranching of β-limit dextrins also revealed that the interior chain-length distribution of AP from the ae wx genotype constituted a large proportion of long chains. The extent of retrogradation increased in the order wx, du wx, ae wx and ae du wx starches and appeared to be proportional to the level of unit chains with DP 16–30 and inversely proportional to the level of short chains with DP 6–11.

Sub- Tg thermal properties of amorphous waxy starch and its derivatives

The sub- T g thermal properties of aged amorphous waxy starch, β-limit dextrin, and amylopectin triacetate at low moisture contents were studied by differential scanning calorimetry (DSC). When T g was above 50°C (∼5–14% moisture), a 50°C endothermic peak was observed in waxy starch and β-limit dextrin samples. The ΔH of the 50°C peak was proportional to the moisture content. At a similar moisture content the ΔH of β-limit dextrin was greater than that of waxy starch. No 50°C peak was observed in the amylopectin triacetate samples at the moisture contents studied (∼0·5-5% moisture). All amylopectin triacetate samples showed an endothermic overshoot associated with the glass transition. It is suggested that water-hydroxyl group interactions may be required for the formation of the sub- T g peak. The 50°C endotherm is a separate event different from the aging peak associated with the glass transition seen in many polymers.