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PubMed1996

Starch degradation in chloroplasts isolated from C3 or CAM (crassulacean acid metabolism)-induced Mesembryanthemum crystallinum L.

C3 or crassulacean acid metabolism (CAM)-induced Mesembryanthemum crystallinum plants perform nocturnal starch degradation which is linear with time. To analyse the composition of metabolites released by isolated leaf chloroplasts during starch degradation we developed a protocol for the purification of starch-containing plastids. Isolated chloroplasts from C3 or CAM-induced M. crystallinum plants are also able to degrade starch. With respect to the endogenous starch content of isolated plastids the rate of starch degradation in intact leaves. The combined presence of Pi, ATP, and oxaloacetate is identified to be the most positive effector combination to induce starch mobilization. The metabolic flux through the oxidative pentose-phosphate pathway in chloroplasts isolated from CAM-induced M. crystallinum is less than 3.5% compared with other metabolic routes of starch degradation. Here we report that starch-degrading chloroplasts isolated from CAM-induced M. crystallinum plants use exogenously supplied oxaloacetate for the synthesis of malate. The main products of starch degradation exported into the incubation medium by these chloroplasts are glucose 6-phosphate, 3-phosphoglyceric acid, dihydroxyacetone phosphate and glucose. The identification of glucose 6-phosphate as an important metabolite released during starch degradation is in contrast to the observations made on all other types of plastids analysed so far, including chloroplasts isolated from M. crystallinum in the C3 state. Therefore, we analysed the transport properties of isolated chloroplasts from M. crystallinum. Surprisingly, both types of chloroplasts, isolated from either C3 or CAM-induced plants, are able to transport glucose 6-phosphate in counter exchange with endogenous Pi, indicating the presence of a glucose 6-phosphate translocator as recently demonstrated to occur in other types of plastids. The composition of metabolites released and the stimulatory effect of oxaloacetate on the rate of starch degradation are discussed with respect to the acidification observed for CAM leaves during the night.

Biological Transport, Active↗
PubMed2005

Postprandial glycaemic, lipaemic and haemostatic responses to ingestion of rapidly and slowly digested starches in healthy young women.

The objective of the present study was to investigate the postprandial metabolism of two starches with contrasting rates of hydrolysis in vitro. Characterized using the Englyst method of in vitro starch classification, C*Set 06 598 contained predominantly rapidly digestible starch and C*Gel 04 201 contained predominantly slowly digestible starch. Each test starch, naturally enriched with 13C, was fed to ten healthy female volunteers as part of a moderate fat test meal (containing 75 g test starch and 21 g fat), in a double-blind randomized crossover design. The metabolic response to each starch was measured after an overnight fast, in an acute 6 h study, before and after 14 d of daily consumption of 75 g test starch. During each acute study, blood samples were taken at 15 min intervals for the first 2 h and at 30 min intervals for the remaining 4 h. Breath 13CO2 enrichment was measured at the same time points and indirect calorimetry was performed for 20 min every 40 min immediately before and throughout the study. Significantly more rapid, greater changes in postprandial plasma glucose, NEFA and serum insulin concentrations were observed after consumption of the rapidly digestible starch. Breath 13CO2 output over the first 3-4 h rose rapidly then began to decline following consumption of the rapidly digestible starch, but plateaued for the slowly digestible starch. The 14 d adaptation period did not affect any of the glycaemic or lipaemic variables but there was a reduction in postprandial plasminogen activator inhibitor-1 concentrations. These data confirm that starches characterized as predominantly rapidly digestible versus slowly digestible by the Englyst procedure provoke distinctly different patterns of metabolism postprandially.

Adult↗
PubMed2000

Waxy corn starch: a potent cofiller in pellets produced by extrusion-spheronization.

The purpose of this study was to assess the usefulness of waxy corn (maize) starch as a cofiller and diluent in pellets produced by aqueous extrusion-spheronization. Waxy corn starch was combined with microcrystalline cellulose (MCC) in the range of 20-50% of the entire composition. Pellets containing ordinary corn starch or lactose with MCC were used as reference. The shape of pellets was characterized using an optical microscopic image analysis system. The surface and cross-sectional structure were investigated by means of scanning electron microscopy (SEM). The replacement of ordinary corn starch by waxy corn starch made it possible to increase the amount of starch from 20 to 40%. The pellets containing 50% waxy corn starch were of poorer quality but superior to those containing 30% corn starch. The surface structure became slightly more irregular with respect to the amount of either starch, and a cavity was formed inside the pellet during the spheronization. The origin of starch did not affect the surface structure of the pellets. Waxy corn starch is a potential cofiller: the amount of MCC can be reduced in pellets produced by extrusion-spheronization by using waxy corn starch as a cofiller. This enables the reduction of the manufacturing cost of pellets with low drug load.

Cellulose↗
PubMed2000

Fermentation of resistant rice starch produces propionate reducing serum and hepatic cholesterol in rats.

This study was designed to investigate the effects of different proportions of rice starch and cornstarch on lipid metabolism in rats fed high dietary cholesterol. Male Wistar rats were fed a 10 g/100 g fat diet containing 1 g/100 g cholesterol with 0 (control diet), 15, 30, 45 or 63 g/100 g rice starch with an enzyme resistant starch concentration of 1.26, 1.39, 1.52, 1.65 or 1.80 g/100 g, respectively, for 4 wk. Groups fed diets with < 63 g/100 g rice starch were supplemented with cornstarch to 63 g/100 g. The two kinds of starch had different structures as seen using scanning electron microscopy (SEM). The rice starch was an aggregation (n = 20-60) of smaller granules (3-8 microm in diameter), whereas the cornstarch was composed of larger (5-15 microm in diameter), single granules. The compound rice starch (0.99 kg/L) was larger in size and denser in structure than cornstarch (0.63 kg/L). Serum total cholesterol concentrations in rats fed both the 45 and 63 g/100 g rice starch diets were significantly lower than in all other groups (P < 0.05). The serum propionate concentration in the rats fed 63 g/100 g rice starch diets was significantly higher than that of other groups. Hepatic triglyceride and total cholesterol concentrations in rats fed 63 g/100 g rice starch diets were significantly lower than in the control group. These results suggest that, because the compound rice starch was an aggregation of smaller granules, larger in size and denser in structure than cornstarch, it was digested more slowly and altered lipid metabolism. Resistant rice starch may be fermented to produce propionate, which reduces serum and hepatic cholesterol.

Animals↗
PubMed2001

Starch and fiber fractions in selected food and feed ingredients affect their small intestinal digestibility and fermentability and their large bowel fermentability in vitro in a canine model.

The digestion of legumes, cereal grains, cereal and potato flours and grain-based foods in dogs was studied using two in vitro model systems. The first simulated the stomach and small intestine through the additions of acid and enzymes and large bowel fermentation through use of fecal inocula from dogs, and the second simulated small intestinal fermentation using canine ileal chyme as the bacterial source. All substrates were analyzed for total dietary fiber (TDF) including insoluble and soluble components, and starch fractions: rapidly digestible starch, slowly digestible starch, resistant starch (RS) and total starch. Legumes had high TDF and RS concentrations (mean 36.5 and 24.7%, respectively), resulting in lower ileal digestible starch and total digestible starch concentrations (mean 21 and 31%, respectively). Seventy-four percent of the TS in the cereal grains group was rapidly digestible starch plus slowly digestible starch compared with the flour group, where the corresponding value was 95%. This related to the processing of cereals to flours, in which TDF and RS concentrations were reduced markedly. This increased ileal digestible starch concentrations in the flour group (65%) versus the cereal grains group (60%). Ileal digestion of starch in grain-based food products like macaroni and spaghetti was high (96 and 92%, expressed as a percentage of TS, respectively). Fermentation of substrates with ileal microflora was influenced by substrate chemical composition, with the flour group exhibiting the highest organic matter disappearance values. The legume group had a high total short-chain fatty acid concentration (7.8 mmol/g organic matter fermented), perhaps as a result of fermentation of TDF as well as starch components. A database such as this one provides information about utilization of foods and feeds in the dog and potentially in humans.

Animals↗
PubMed2004

Phosphorylation of transitory starch is increased during degradation.

The starch excess phenotype of Arabidopsis mutants defective in the starch phosphorylating enzyme glucan, water dikinase (EC 2.7.9.4) indicates that phosphorylation of starch is required for its degradation. However, the underlying mechanism has not yet been elucidated. In this study, two in vivo systems have been established that allow the analysis of phosphorylation of transitory starch during both biosynthesis in the light and degradation in darkness. First, a photoautotrophic culture of the unicellular green alga Chlamydomonas reinhardtii was used to monitor the incorporation of exogenously supplied (32)P orthophosphate into starch. Illuminated cells incorporated (32)P into starch with a constant rate during 2 h. By contrast, starch phosphorylation in darkened cells exceeded that in illuminated cells within the first 30 min, but subsequently phosphate incorporation declined. Pulse-chase experiments performed with (32)P/(31)P orthophosphate revealed a high turnover of the starch-bound phosphate esters in darkened cells but no detectable turnover in illuminated cells. Secondly, leaf starch granules were isolated from potato (Solanum tuberosum) plants grown under controlled conditions and glucan chains from the outer granule layer were released by isoamylase. Phosphorylated chains were purified and analyzed using high performance anion-exchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry. Glucans released from the surface of starch granules that had been isolated from darkened leaves possessed a considerably higher degree of phosphorylation than those prepared from leaves harvested during the light period. Thus, in the unicellular alga as well as in potato leaves, net starch degradation is accompanied with an increased phosphorylation of starch.

Animals↗
PubMed1996

Contribution of a neopullulanase, a pullulanase, and an alpha-glucosidase to growth of Bacteroides thetaiotaomicron on starch.

Bacteroides thetaiotaomicron, a gram-negative colonic anaerobe, can utilize three forms of starch: amylose, amylopectin, and pullulan. Previously, a neopullulanase, a pullulanase, and an alpha-glucosidase from B. thetaiotaomicron had been purified and characterized biochemically. The neopullulanase and alpha-glucosidase appeared to be the main enzymes involved in the breakdown of starch, because they were responsible for most of the starch-degrading activity detected in B. thetaiotaomicron cell extracts. To determine the importance of these enzymes in the starch utilization pathway, we cloned the genes encoding the neopullulanase and alpha-glucosidase. The gene encoding the neopullulanase (susA) was located upstream of the gene encoding the alpha-glucosidase (susB). Both genes were closely linked to another starch utilization gene, susC, which encodes a 115-kDa outer membrane protein that is essential for growth on starch. The gene encoding the pullulanase, pulI, was not located in this region in the chromosome. Disruption of the neopullulanase gene, susA, reduced the rate of growth on starch by about 30%. Elimination of susA in this strain allowed us to detect a low residual level of enzyme activity, which was localized to the membrane fraction. Previously, we had shown that a disruption in the pulI gene did not affect the rate of growth on pullulan. We have now shown that a double mutant, with a disruption in susA and in the pullulanase gene, pulI, was also able to grow on pullulan. Thus, there is at least one other starch-degrading enzyme besides the neopullulanase and the pullulanase. Disruption of the alpha-glucosidase gene, susB, reduced the rate of growth on starch only slightly. No residual alpha-glucosidase activity was detectable in extracts from this strain. Since this strain could still grow on maltose, maltotriose, and starch, there must be at least one other enzyme capable of degrading the small oligomers produced by the starch-degrading enzymes. Our results show that the starch utilization system of B. thetaiotaomicron is quite complex and contains a number of apparently redundant degradative enzymes.

Amino Acid Sequence↗
PubMed1991

Estimation of the amount of dietary starch available to different populations.

The daily per capita availability of dietary starch was estimated for 38 selected countries using food disappearance data from the FAO provisional food balance sheets (1972-1974 average); starch availability was also estimated for Canada from 1960 to 1987 using Agriculture Canada disappearance data. Total starch availability varies fourfold among the difference countries; the availability of different sources of starch vary up to 80-fold. Wheat and roots-tubers were the major sources of starch in countries with low total starch availability. Either corn and pulses, rice and pulses, or wheat and potatoes were the major sources of starch in those countries with high total starch availability. Starch availability in Canada, both total and by source, has been relatively constant over the 27 years examined. Slight increases in total starch and starch from cereals and pulses in 1987 will need to be followed to determine whether these represent the beginning of a real change in per capita starch availability.

Canada↗
PubMed1991

Measurement of starch fermentation in the human large intestine.

Starch, not fibre, is probably the major substrate for fermentation in the human colon. However, quantitating the amount of starch that resists pancreatic amylase and thus escapes digestion in the small bowel is difficult. A number of techniques have been employed in man and are reviewed here, including direct intubation of the ileum, the ileostomy model, and breath studies. The results of a series of studies of the digestion of starch from potato and banana are reported. When fed to ileostomy patients, 3% of hot potato starch and 12% of cold potato starch were resistant to digestion, as was 75% of banana starch. In feeding experiments with healthy volunteers none of the starch was recoverable in faces, indicating its complete fermentation in the colon. Breath H2 measurements after test meals of these starches indicated that only 2-5% of potato starch and 7-12% of banana starch was fermented. A single blood acetate measurement timed to coincide with peak breath H2 was not useful. However, a number of problems with breath H2 studies are discussed, and it is suggested that either ileal intubation or the ileostomy model are the most reliable techniques presently available, with serial blood acetate determinations also potentially valuable. Overall on Western diets, approximately 10% of all starch is probably resistant starch.

Acetates↗
PubMed2005

Effects of plantain and corn starches on the mechanical and disintegration properties of paracetamol tablets.

The effects of plantain starch obtained from the unripe fruit of the plant Musa paradisiaca L. (Musaceae) on the mechanical and disintegration properties of paracetamol tablets have been investigated in comparison with the effects of corn starch BP using a 2(3) factorial experimental design. The individual and combined effects of nature of starch binder (N), concentration of starch binder (C), and the relative density of tablet (RD) on the tensile strength (TS), brittle fracture index (BFI), and disintegration time (DT) of the tablets were investigated. The ranking of the individual effects on TS was RD > C >> N, on BFI was C >> RD > N and on DT was N > C > RD. The ranking for the interaction effects on TS and DT was N-C >> N-RD > C-RD, while that on BFI was N-C >> C-RD > N-RD. Changing nature of starch from a "low" (plantain starch) to a "high" (corn starch) level, increasing the concentration of starch binding agent from 2.5% to 10.0% wt/wt, and increasing relative density of the tablet from 0.80 to 0.90, led to increase in the values of TS and DT, but a decrease in BFI. Thus, tablets containing plantain starch had lower tensile strength and disintegration time values than those containing corn starch, but showed better ability to reduce the lamination and capping tendency in paracetamol tablet formulation. The interaction between N and C was significantly (P < .001) higher than those between N and RD and between C and RD. There is therefore the need to carefully choose the nature (N) and concentration (C) of starch used as binding agent in tablet formulations to obtain tablets of desired bond strength and disintegration properties. Furthermore, plantain starch could be useful as an alternative binding agent to cornstarch, especially where faster disintegration is required and the problems of lamination and capping are of particular concern.

Acetaminophen↗
PubMed2006

Increased puroindoline levels slow ruminal digestion of wheat (Triticum aestivum L.) starch by cattle.

Starch is the primary nutrient in ruminant diets used to promote high levels of performance. The site of starch digestion alters the nature of digestive end products (VFA in the rumen vs. glucose in the small intestine) and the efficiency of use. Cereal grain endosperm texture plays a major role in the rate and extent of starch degradation in ruminants. Wheat grain texture is regulated by the starch surface protein complex friabilin that consists primarily of puroindoline (PIN) A and B. Soft kernel texture in wheat is a result of both PIN genes being in the wild type active form and bound to starch. The objective of this study was to investigate the effect of varying PIN content in wheat on the rate of starch digestion in the rumen of beef cattle. In Exp. 1, 6 transgenic soft pin a/b isolines created in a hard wheat background, and 2 hard wheat controls were milled to yield a wide range of mean particle sizes across all lines. Milled samples were incubated in situ for 3 h. Increased expression of both PINA and PINB decreased DM digestibility (DMD) by 29.2% (P < 0.05) and decreased starch digestibility by 30.8% (P < 0.05). Experiment 2 separated the effects of particle size and total PIN content on digestion by milling the hardest and softest lines such that the mean particle size was nearly identical. Increased PIN decreased DMD by 21.7% (P < 0.05) and starch digestibility by 19.9% (P < 0.05) across particle sizes smaller than whole kernel. Experiment 3 addressed the time course of PIN effects in the rumen by observing ground samples of the hardest and softest lines over a 12-h in situ period. Increased PIN decreased DMD by 10.4% (P < 0.05) and starch digestibility by 11.0% (P < 0.05) across all time points. Dry matter and starch digestibility results demonstrated that increased expression of PIN was associated with a decreased rate of ruminal digestion independent of particle size. Puroindolines seem to aid in the protection of starch molecules from microbial digestion in the rumen, potentially increasing the amount of starch entering the small intestine.

Animals↗
PubMed2001

In vitro mixed ruminal microorganism fermentation of whole cottonseed coated with gelatinized corn starch and urea.

We conducted an in vitro mixed ruminal microorganism fermentation study to determine the effect of coating whole cottonseed with gelatinized corn starch and feed grade urea. Treatments were arranged as a 3 x 4 factorial to provide three concentrations of starch (0.0, 2.5, and 5.0%) and four concentrations of urea (0.0, 0.25, 0.5, and 1.0%). All treatments were prepared from one lot of whole cottonseed. Batch culture fermentations were conducted using anaerobic medium that contained 20% (vol/vol) ruminal fluid in 160-ml serum bottles. Whole cottonseed was ground to pass through a 6-mm screen and weighed amounts (0, 0.4, 0.8, and 1.2 g) were added to the serum bottles. As starch increased, H2, CH4, total volatile fatty acids, and molar proportions of propionate increased linearly, whereas pH, molar proportions of acetate, and the acetate to propionate ratio decreased linearly. L-Lactate concentrations were highest with 2.5% starch compared with 0 or 5.0%. As the amount of urea in the coating increased, pH and CH4 increased linearly, whereas H2 concentrations decreased linearly. Ammonia concentrations exhibited a quadratic response due to moderate increases with the addition of 0.25 and 0.5% urea, and a greater increase was observed with 1.0% urea. Interactions between starch and urea were observed for H2, CH4, NH3, and L-lactate. Concentrations of H2 decreased and CH4 was relatively constant as urea increased in the presence of 0 and 2.5% starch, but increased with 5% starch. L-Lactate concentrations were unchanged (0% starch), higher (2.5% starch), or lower (5.0% starch) as urea increased. Ammonia concentrations increased after urea exceeded 10% of the starch concentration. The addition of urea did not prevent the decline in pH, but did reduce H2 and CH4 accumulation with 2.5% starch.

Animals↗
PubMedDate not supplied

A comparative study of modified starches in direct compression of a water soluble drug-chloroquine phosphate.

Some in vitro properties of chloroquine phosphate tablets formulated with four modified starches were investigated. The drug was formulated as tablets containing 250 mg of chloroquine phosphate and produced by the direct compression technique. The starches were isolated from maize, zea mays, rice, Oryza sativa, cassava, Manihot esculenta and cocoyam, Zanthosoma sagittifolium. They were modified through physicochemical process, Sta-Rx 1500, a directly compressible starch was used as basis for comparison. The hardness of the chloroquine tablets generally decreased to a minimum with all the modified starches at concentration level of 40% and with maximum hardness obtained when their concentrations were increased to 80%. The least hardness values were obtained with modified cocoa yam starch while the highest hardness values were obtained with modified rice starch. Modified rice and cassava starches produced chloroquine tablets that exhibited higher mechanical properties than those of modified maize starch, cocoayam starch and Sta-Rx 1500. On the basis of dissolution profile of chloroquine phosphate tablets, the modified starch samples were ranked in order of increasing dissolution as modified cocoayam maize Sta-Rx 1500 cassava rice starch sample. The release rate of chloroquine was found to be dependent on the physico-chemical properties of the individual modified starch granules such as particle size and degree of gelatinization.

Antimalarials↗
PubMed2026

Resistant starch types 2 and 4 induce distinct and reversible changes in the human gut microbiome.

Resistant starch (RS) can confer benefits for the gut microbiome and host cardiometabolic health. However, different types of resistant starch can differentially affect gut microbiome composition and functional capacity, especially given interindividual variability in responses, thus limiting the application of resistant starch in dietary strategies. We used shotgun metagenomics to perform a secondary analysis of samples collected during a previously reported randomized clinical trial to determine the effects of dietary supplementation with two types of resistant starch (RS2 and RS4) and a digestible starch (control) on the gut microbiome. Both resistant starch types induced distinct but transient alterations in the gut microbial community. RS2 enriched the keystone degrader, Ruminococcus bromii, and Blautia glucerasea, whereas RS4 favored Parabacteroides distasonis and known but uncharacterized microbial species such as a Lachnospiraceae bacterium. Moreover, we detected strain-level differences in the response of Bifidobacterium adolescentis to resistant starch. Microbial functional profiling revealed an enhanced capacity for complex carbohydrate utilization following resistant starch intake, including increased abundance of specific &#x3b1;-amylases, glycoside hydrolases, starch utilization systems, and other currently uncharacterized genes. Identifying the bacterial strains and genes that respond to different RS types will help to more accurately predict who will benefit from a given RS type. Our findings demonstrate that RS2 and RS4 differentially shape microbial ecology and metabolic capacity and provide a foundation for microbiome-informed personalization of resistant starch-based dietary interventions.IMPORTANCEDietary intake influences human health by modulating metabolism, partly by shaping the microbiota inhabiting the gut. Resistant starch (RS), a dietary fiber, is associated with metabolic improvements. While previous research has explored how RS alters the gut microbiome, RS comprises five types with differing physical and chemical characteristics, and the distinct impacts of each type on the microbiome and host health have not been fully characterized, particularly using high-resolution approaches such as shotgun metagenomics. In this secondary analysis of samples from a longitudinal crossover intervention study, we link dietary supplementation with RS2 and RS4 with distinct and transient changes in the composition and functional potential of the human gut microbiome. Specifically, we identify species that increase in abundance with each RS type, accompanied by increases in genes and pathways involved in complex carbohydrate utilization. The findings support the development of precision nutrition strategies utilizing RS supplementation to improve metabolic health.This study is registered with ClinicalTrials.gov as NCT05743790.

Humans↗
PubMed1977

Role of orthophosphate and other factors in the regulation of starch formation in leaves and isolated chloroplasts.

Starch synthesis in leaves was increased by phosphate starvation or by treatments which decreased cytoplasmic orthophosphate levels (such as mannose feeding). Usually less than 30% of the total carbon fixed during CO(2) assimilation was incorporated into starch in spinach (Spinacia oleracea L.), spinach beet (Beta vulgaris), and tobacco (Nicotiana tabacum) leaves.In isolated spinach chloroplasts, formation of starch from CO(2) was usually less than in leaves. In the absence of significant levels of 3-phosphoglycerate, concentrations of phosphate as low as 1 mm (in the medium) or 10 mm (in the stroma) almost completely inhibited starch synthesis. The inhibitory action of phosphate could be overcome by 3-phosphoglycerate. The controlling factor of starch synthesis appeared to be the ratio of phosphoglycerate to orthophosphate rather than the stromal hexose monophosphate concentration, and it is suggested that this control is exerted via the phosphate translocator and the known allosteric regulation of ADP-glucose pyrophosphorylase. Starch synthesis was also favored by the presence of dihydroxyacetone phosphate and by high light and high temperature. Oxygen was inhibitory, probably owing to carbon drain into glycolate. Starch formation by intact chloroplasts could not be promoted by added glucose or glucose 6-phosphate.Starch mobilization in the dark was promoted by orthophosphate and phosphate-dependent mobilization was inhibited by phosphoglycerate. The principal products of starch breakdown in the presence of phosphate were the transport metabolites dihydroxyacetone phosphate and 3-phosphoglycerate. Formation of these compounds from starch was stimulated by ATP or oxaloacetate. In a phosphate-independent reaction, starch was also converted to neutral products such as maltose and glucose. The rates of phosphate-dependent starch degradation phosphorolysis were very much higher than those of starch hydrolysis for which there was no phosphate requirement.

Journal Article↗
PubMed1981

Evidence for independent genetic control of the multiple forms of maize endosperm branching enzymes and starch synthases.

Soluble starch synthase and starch-branching enzymes in extracts from kernels of four maize genotypes were compared. Extracts from normal (nonmutant) maize were found to contain two starch synthases and three branching enzyme fractions. The different fractions could be distinguished by chromatographic properties and kinetic properties under various assay conditions. Kernels homozygous for the recessive amylose-extender (ae) allele were missing branching enzyme IIb. In addition, the citrate-stimulated activity of starch synthase I was reduced. This activity could be regenerated by the addition of branching enzyme to this fraction. No other starch synthase fractions were different from normal enzymes. Extracts from kernels homozygous for the recessive dull (du) allele were found to contain lower branching enzyme IIa and starch synthase II activities. Other fractions were not different from the normal enzymes. Analysis of extracts from kernels of the double mutant ae du indicated that the two mutants act independently. Branching enzyme IIb was absent and the citrate-stimulated reaction of starch synthase I was reduced but could be regenerated by the addition of branching enzyme (ae properties) and both branching enzyme IIa and starch synthase II were greatly reduced (du properties). Starch from ae and du endosperms contains higher amylose (66 and 42%, respectively) than normal endosperm (26%). In addition, the amylopectin fraction of ae starch is less highly branched than amylopectin from normal or du starch. The above observations suggest that the alterations of the starch may be accounted for by changes in the soluble synthase and branching enzyme fractions.

Journal Article↗
PubMed1984

Use of polyethylene glycol in isolation and assay of stable, enzymically active starch granules from developing wheat endosperms.

A procedure using polyethylene glycol (PEG), molecular weight 1000, was developed for the isolation of starch granules from wheat endosperm. Immature endosperm tissue was cut repeatedly in 300 millimolar PEG 1000 and filtered through Miracloth. Centrifugation separated a pellet from a supernatant with inhibitory activity. The pellet contained several enzyme activities, including soluble and bound components of starch synthase, starch phosphorylase, and sucrose synthase activities. The starch phosphorylase activity was unaffected by several washings with 300 millimolar PEG 1000 but was lost when the granules were washed once without PEG or washed with sucrose, glycerol, or sorbitol (up to 30%, w/v). The fraction of starch synthase, remaining on the granules after a wash without PEG (the ;bound' activity) was not affected by the addition of 30% sorbitol to the wash buffer. This fraction became larger with grain development (0.2-0.7).To obtain high activity, PEG was required not only during isolation of granules but also in the assay of both starch phosphorylase and starch synthase giving optimum activity at 225 to 255 millimolar. PEG reduced the requirement for glycogen as primer with soluble starch synthase. However, the ;bound' starch synthase activity was unaffected by PEG. PEG of different size were compared by their effects in the assay of starch granules: with increase in molecular size, the same effect was obtained at ever lower polymer concentration (w/v) down to a limit.Treatment of granules with Triton X-100 did not affect their starch synthase activity, but it removed the capacity to incorporate label from UDP [(14)C]G into non-starch polymers.It is concluded that PEG, like some other active compounds (ethanol Na(3)-citrate, and Ficoll) could mediate enzyme-primer interaction by exclusion.

Journal Article↗
PubMed1983

Production and Characteristics of Raw Starch-Digesting Glucoamylase O from a Protease-Negative, Glycosidase-Negative Aspergillus awamori var. kawachi Mutant.

Production of a raw starch-digesting glucoamylase O (GA O) by protease-negative, glycosidase-negative mutant strain HF-15 of Aspergillus awamori var. kawachi was undertaken under submerged culture conditions. The purified GA O was electrophoretically homogeneous and similar to the parent glucoamylase I (GA I) in the hydrolysis curves toward gelatinized potato starch, raw starch, and glycogen and in its thermostability and pH stability, but it was different in molecular weight and carbohydrate content (250,000 and 24.3% for GA O, 90,000 and ca. 7% for GA I, respectively). The chitin-bound GA O hydrolyzed raw starch but the chitin-bound GA I failed to digest raw starch because chitin was adsorbed at the raw starch affinity site of the GA I molecule. The removal of the raw starch affinity site of GA O with subtilisin led to the formation of a modified GA O (molecular weight, 170,000), which hydrolyzed glycogen 100%, similar to GA O and GA I, and was adsorbed onto chitin and fungal cell wall but not onto raw starch, Avicel, or chitosan. The modified GA I (molecular weight, 83,000) derived by treatment with substilisin hydrolyzed glycogen up to only 80% and failed to be adsorbed onto any of the above polysaccharides. The N-bromosuccinimide-oxidized GA O lost its activity toward gelatinized and raw starches, but the abilities to be adsorbed onto raw starch and chitin were preserved. It was thus suggested that both the raw starch affinity site essential for raw starch digestion and the chitin-binding site specific for the binding with chitin in the cell wall could be different from the active site, located in the three respective positions in the GA O molecule.

Journal Article↗