An enzymic microassay for starch

Abstract Conditions are described for measuring the starch content of plant tissues or extracts as glucose over the range from 10^?7 mol to 10^?14 mol. The method is based on the hydrolysis of gelatinized starch by amyloglucosidase; the glucose released is measured by reduction of NADP⁺ by coupled enzymic reactions. The NADPH is determined directly either spectrophotometrically or fluorimetrically, or after enzymic amplification. Amyloglucosidases were tested for contaminating enzymes which might degrade glucans other than starch, and a commercial preparation from Rhizopus niveus was found to be suitable for use without pretreatments. Glucose present in tissues and extracts may be measured and subtracted from starch values using appropriate blanks, or first destroyed by dilute alkali and heat. Addition of α-amylase to amyloglucosidase during starch hydrolysis was not found to increase percentage hydrolysis from the normal range of 86–99% from starches of different sources. The procedures described are rapid and several orders of magnitude more sensitive than current methods, and can be used to measure the starch content of single cells.     

Increases in 1,5-anhydroglucitol levels in germinating amaranth seeds and in ripening banana

To examine whether 1,5-anhydroglucitol (AG) is derived from starch degradation in plant tissues, we colorimetrically measured AG contents of germinating amaranth seeds and ripening banana pulp. In both cases, as starch degradation proceeded, AG levels were significantly increased, but were 1,700-5,000 times lower than those of total soluble carbohydrates. alpha-1,4-Glucan lyase activity, which is measured by the 1,5-anhydrofructose (AF) liberated from non-reducing glucose residues of starch or glycogen, was too low to be detected in amaranth or banana by the 3,5-dinitrosalicylic acid method. On the other hand, AF reductase, which reduces AF to AG, was detected in germinating amaranth seeds and banana pulp. Thus, the increases in AG levels are conceived to be derived from starch breakdown, although further investigation is needed to answer whether the starch degradation pathway via alpha-1,4-glucan lyase/AF reductase exists in plant tissues.     

Increases in 1,5-Anhydroglucitol Levels in Germinating Amaranth Seeds and in Ripening Banana

To examine whether 1,5-anhydroglucitol (AG) is derived from starch degradation in plant tissues, we colorimetrically measured AG contents of germinating amaranth seeds and ripening banana pulp. In both cases, as starch degradation proceeded, AG levels were significantly increased, but were 1,700-5,000 times lower than those of total soluble carbohydrates. α-1,4-Glucan lyase activity, which is measured by the 1,5- anhydrofructose (AF) liberated from non-reducing glucose residues of starch or glycogen, was too low to be detected in amaranth or banana by the 3,5-dinitrosalicylic acid method. On the other hand, AF reductase, which reduces AF to AG, was detected in germinating amaranth seeds and banana pulp. Thus, the increases in AG levels are conceived to be derived from starch breakdown, although further investigation is needed to answer whether the starch degradation pathway via α-1,4-glucan lyase/AF reductase exists in plant tissues.     

Extraction of starch by dimethyl sulfoxide and quantitation by enzymatic assay

A method for the rapid, sensitive, and specific determination of starch in plant tissues is described. Starch from a variety of plant tissues is solubilized by stirring for 24 h or by sonication for 40 min in dimethyl sulfoxide. Dilution of this extract to less than 20% dimethyl sulfoxide permits a nearly complete hydrolysis of the starch in less than 3 h with glucoamylase from Rhizopus niveus. Quantitation of liberated glucose by a coupled hexokinase and glucose-6-phosphate dehydrogenase method provides an additional degree of specificity.     

Enzymic mechanism of starch breakdown in germinating rice seeds I. An analytical study

Time-sequence analyses of carbohydrate breakdown in germinating rice seeds shows that a rapid breakdown of starch reserve in endosperm starts after about 4 days of germination. Although the major soluble carbohydrate in the dry seed is sucrose, a marked increase in the production of glucose and maltooligosaccharides accompanies the breakdown of starch. Maltotriose was found to constitute the greatest portion of the oligosaccharides throughout the germination stage. α-Amylase activities were found to parallel the pattern of starch breakdown. Assays for phosphorylase activity showed that this enzyme may account for much smaller amounts of starch breakdown per grain, as compared to the amounts hydrolyzed by α-amylase. There was a transient decline in the content of sucrose in the initial 4 days of seed germination, followed by the gradual increase in later germination stages. During the entire germination stage, sucrose synthetase activity was not detected in the endosperm, although appreciable enzyme activity was present in the growing shoot tissues as well as in the frozen rice seeds harvested at the mid-milky stage. We propose the predominant formation of glucose from starch reserves in the endosperm by the action of α-amylase and accompanying hydrolytic enzyme(s) and that this sugar is eventually mobilized to the growing tissues, shoots or roots.     

Kojic acid production byAspergillus flavus using gelatinized and hydrolyzed sago starch as carbon sources

Direct conversion of gelatinized sago starch into kojic acid byAspergillus flavus strain having amylolytic enzymes was carried out at two different scales of submerged batch fermentation in a 250-mL shake flask and in a 50-L stirred-tank fermentor. For comparison, fermentations were also carried out using glucose and glucose hydrolyzate from enzymic hydrolysis of sago starch as carbon sources. During kojic acid fermentation of starch, starch was first hydrolyzed to glucose by the action of α-amylase and glucoamylase during active growth phase. The glucose remaining during the production phase (non-growing phase) was then converted to kojic acid. Kojic acid production (23.5g/L) using 100 g/L sago starch in a shake flask was comparable to fermentation of glucose (31.5 g/L) and glucose hydrolyzate (27.9 g/L) but in the 50-L fermentor was greatly reduced due to non-optimal aeration conditions. Kojic acid production using glucose was higher in the 50-L fermentor than in the shake flask.     

Starch Phosphorylation in Potato Tubers Proceeds Concurrently with de Novo Biosynthesis of Starch

The in vivo phosphorylation of starch was studied in Solanum tuberosum cv Dianella and Posmo. Small starch granules contain 25% more ester-bound phosphate per glucose residue than large starch granules. The degree of phosphorylation was found to be almost constant during tuber development. Isolated tuber discs synthesize starch from externally supplied glucose at a significant rate. Tuber discs supplied with glucose and [32P]orthophosphate incorporate radiolabeled phosphorus into the starch. The level of 32P incorporation is proportional to the amount of starch synthesized. The incorporation of 32P from orthophosphate is correlated to de novo synthesis of starch, since the incorporation of 32P is diminished upon inhibition of starch synthesis by fluoride. Based on the amount of [14C]glucose phosphate isolated after hydrolysis of purified starch from tuber discs incubated in the presence of [U-14C]glucose, approximately 0.5% of the glucose residues of the de novo-synthesized starch are phosphorylated. This value is in general agreement with the observed levels of phosphorus in starch accumulated during tuber development. Thus, the enzyme system responsible for starch phosphorylation is fully active in the isolated tuber discs, and the starch phosphorylation proceeds as an integrated part of de novo starch synthesis.     

Rethinking the starch digestion hypothesis for AMY1 copy number variation in humans

Alpha‐amylase exists across taxonomic kingdoms with a deep evolutionary history of gene duplications that resulted in several α‐amylase paralogs. Copy number variation (CNV) in the salivary α‐amylase gene (AMY1) exists in many taxa, but among primates, humans appear to have higher average AMY1 copies than nonhuman primates. Additionally, AMY1 CNV in humans has been associated with starch content of diets, and one known function of α‐amylase is its involvement in starch digestion. Thus high AMY1 CNV is considered to result from selection favoring more efficient starch digestion in the Homo lineage. Here, we present several lines of evidence that challenge the hypothesis that increased AMY1 CNV is an adaptation to starch consumption. We observe that α‐ amylase plays a very limited role in starch digestion, with additional steps required for starch digestion and glucose metabolism. Specifically, we note that α‐amylase hydrolysis only produces a minute amount of free glucose with further enzymatic digestion and glucose absorption being rate‐limiting steps for glucose availability. Indeed α‐amylase is nonessential for starch digestion since sucrase‐isomaltase and maltase‐glucoamylase can hydrolyze whole starch granules while releasing glucose. While higher AMY1 CN and CNV among human populations may result from natural selection, existing evidence does not support starch digestion as the major selective force. We report that in humans α‐amylase is expressed in several other tissues where it may have potential roles of evolutionary significance.     

Comparison of copper status in rats when dietary fructose is replaced by either cornstarch or glucose

The purpose of this study was to determine what levels of starch or glucose replacement for fructose in the copper-deficient diet (copper) can minimize the fructose-copper interaction. Experimental diets contained either 100% fructose as the carbohydrate source, or the fructose was partially replaced with 50% starch, 50% glucose, 75% starch, or 75% glucose. Diets were either copper adequate (7-8 ppm) or inadequate (less than 1 ppm). Male weanling rats were fed their respective diet for 5 weeks and then fasted overnight. After decapitation, blood was collected and liver and heart were removed. Plasma copper was significantly reduced and ceruloplasmin was not detected in all copper-deficient groups. Copper deficiency increased plasma cholesterol, as well as heart and liver weight in the glucose groups, but not in the starch groups. Those organ weights were heavier in glucose-copper than starch-copper rats. Erythrocyte copper-zinc-superoxide dismutase activity was greater in starch-copper rats. Erythrocyte copper-zinc-superoxide dismutase activity was greater in starch-copper than glucose-copper rats regardless of carbohydrate amount. Hepatic copper concentration of the group fed starch-copper was twice levels observed in glucose-copper. The 50% glucose rats had lower hepatic copper than the 75% glucose rats. Hepatic copper-zinc-superoxide dismutase activity showed patterns similar to hepatic copper. Cardiac copper was greater in starch-copper than glucose-copper rats. Cardiac copper-zinc-superoxide dismutase activity was equally reduced in all copper-deficient groups. The 50% starch-replaced diet was more effective in minimizing copper deficiency than the 75% glucose-replaced diet. This poorer improvement of copper deficiency by glucose than starch may partially be due to a more severe reduction of food intake in glucose than in starch diets.     

Starch Removal from Potato Tuber Sections

Heating plant sections at 90 C with 0.5% aqueous ammonium oxalate is required to remove pectins. When applied to tissues rich in starch such as potato, this step produces heavy dextrinization of the starch which hinders subsequent evaluation of the extinction values of the cell walls. To overcome this a method has been devised to brush away the starch granules from the sections with a thin paint brush, just after paraffin removal by xylene. The slide is then processed as usual: pectins are removed by heat treatment, cell walls are stained with PAS and the stain intensity can be evaluated by photometry.     

In situ analysis of plant tissue underivatized carbohydrates and on-probe enzymatic degraded starch by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry by using carbon nanotubes as matrix

Underivatized carbohydrates of tulip bulb and leaf tissues were characterized in situ by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) by using carbon nanotubes (CNTs) as matrix. Two sample preparation methods—(i) depositing CNTs on the fresh tissue slices placed on the probe and (ii) locating semitransparent tissues on a dried layer of CNTs on the probe—were examined. Furthermore, practicability of in situ starch analysis by MALDI-TOF MS was examined by detection of glucose originated from on-probe amyloglucosidase-catalyzed degradation of starch on the tissue surface. Besides, CNTs could efficiently desorb/ionize natural mono-, di-, and oligosaccharides extracted from tulip bulb tissues as well as glucose resulting from starch enzymatic degradation in vitro. These results were compared with those obtained by in situ MALDI-TOF MS analysis of similar tissues. Positive ion mode showed superior signal reproducibility. CNTs deposited under semitransparent tissue could also desorb/ionize neutral carbohydrates, leading to nearly complete elimination of matrix cluster signals but with an increase in tissue-originated signals. Furthermore, several experiments were carried out to compare the efficiency of 2,5-dihydroxybenzoic acid, nor-harmane, α-cyano-4-hydroxycinnamic acid, and CNTs as matrices for MALDI of neutral carbohydrates from the intact plant tissue surface and for enzymatic tissue starch degradation; these results are discussed in brief. Among matrices studied, the lowest laser power was needed to acquire carbohydrate signals with high signal-to-noise ratio and resolution when CNTs were used.     

Starch metabolism in leaves

Starch is the most abundant storage carbohydrate produced in plants. The initiation of transitory starch synthesis and degradation in plastids depends mainly on diurnal cycle, post-translational regulation of enzyme activity and starch phosphorylation. For the proper structure of starch granule the activities of all starch synthase isoenzymes, branching enzymes and debranching enzymes are needed. The intensity of starch biosynthesis depends mainly on the activity of AGPase (adenosine 5'-diphosphate glucose pyrophosphorylase). The key enzymes in starch degradation are beta-amylase, isoamylase 3 and disproportionating enzyme. However, it should be underlined that there are some crucial differences in starch metabolism between heterotrophic and autotrophic tissues, e.g. is the ability to build multiprotein complexes responsible for biosynthesis and degradation of starch granules in chloroplasts. The observed huge progress in understanding of starch metabolism was possible mainly due to analyses of the complete Arabidopsis and rice genomes and of numerous mutants with altered starch metabolism in leaves. The aim of this paper is to review current knowledge on transient starch metabolism in higher plants.     

Response of sugar interconversion, starch and protein accumulation to supplied metabolites during pod filling in chickpea

Detached chickpea inflorescences bearing pods at 20 days after flowering (DAF) were cultured for 5 days in complete liquid medium supplemented separately with asparate, myo-inositol, alpha-ketoglutarate and phytic acid. Effect of these metabolites on sugar interconvestion and starch and protein accumulation in developing pods was studied. Substituting asparate (62.5 mM) for glutamine in culture medium decreased relative proportion of sucrose in all pod tissues but increased the level of sugars, starch and protein in pod wall and cotyledons. In cotyledons, whereas myo-inositol (75 mM) reduced the accumulation of starch without affecting protein level, alpha-ketoglutarate (44 mM) increased both starch and protein accumulation. Both myo-inositol and alpha-ketoglutarate increased relative proportion of sucrose in cotyledons. Phytic acid (1 mM) decreased in cotyledons 14C incorporation from glucose into EtOH extract (principally constituted by sugars), amino acids and proteins but increased the same into starch. In cotyledons, phytic acid also increased 14C incorporation from glutamate into amino acids but this increase was negatively correlated with protein synthesis. Phytic acid decreased the relative distribution of 14C from glucose and glutamate into sucrose from pod wall but enhanced the same into EtOH extract from embryo. Based on the results, it is suggested that mode of metabolic response to exogenously supplied metabolites widely differs in pod tissues of chickpea.     

Differential amylosaccharide metabolism of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum.

Clostridium thermosulfurogenes displayed faster growth on either glucose, maltose, or starch than Clostridium thermohydrosulfuricum. Both species grew faster on glucose than on starch or maltose. The fermentation end product ratios were altered based on higher ethanol and lactate yields on starch than on glucose. In C. thermohydrosulfuricum, glucoamylase, pullulanase, and maltase were mainly responsible for conversion of starch and maltose into glucose, which was accumulated by a putative glucose permease. In C. thermosulfurogenes, beta-amylase was primarily responsible for degradation of starch to maltose, which was accumulated by a putative maltose permease and then hydrolyzed by glucoamylase. Regardless of the growth substrate, the rates of glucose, maltose, and starch transformation were higher in C. thermosulfurogenes than in C. thermohydrosulfuricum. Both species had a functional Embden-Meyerhof glycolytic pathway and displayed the following catabolic activities: ferredoxin-linked pyruvate dehydrogenase, acetate kinase, NAD(P)-ethanol dehydrogenase, NAD(P)-ferredoxin oxidoreductase, hydrogenase, and fructose-1,6-diphosphate-activated lactate dehydrogenase. Ferredoxin-NAD reductase activity was higher in C. thermohydrosulfuricum than NADH-ferredoxin oxidase activity, but the former activity was not detectable in C. thermosulfurogenes. Both NAD- and NADP-linked ethanol dehydrogenases were unidirectional in C. thermosulfurogenes but reversible in C. thermohydrosulfuricum. The ratio of hydrogen-producing hydrogenase to hydrogen-consuming hydrogenase was higher in C. thermosulfurogenes. Two biochemical models are proposed to explain the differential saccharide metabolism on the basis of species enzyme differences in relation to specific growth substrates.     

Seasonal Changes of the Metabolites in the Leaves, Bark and Xylem Tissues of Olive Tree (Olea europaea. L) II. Carbohydrates

Seasonal changes of starch and soluble carbohydrates in leaves,bark and xylem tissues of olive tree were examined during acomplete annual cycle. Leaf starch and soluble carbohydrateswere detected at high levels during the spring and autumn metabolicallyactive periods. The low level of leaf starch in combinationwith the drastic reduction of soluble carbohydrates and mannitol,defined the summer period of the low metabolic state of thetree. The low leaf starch level in conjunction with the risensoluble carbohydrate levels in leaves in winter were associatedwith cold acclimation processes. The bark and xylem tissueswere performing as starch deposition sites, and differencesin the extent of starch accumulation in these tissues were detectedduring the seasons. The starch fluctuations in bark and xylemwere discussed in relation to the translocation of metabolitesand other physiological processes. Mannitol, the most abundantleaf carbohydrate, was examined in relation to the reducingsugars exported to the bark. The bark mannitol was examinedin conjunction with the sucrose, glucose and starch levels inthe maturing bark tissues and was correlated to the low wintertemperatures. During the winter there was a drastic reductionof mannitol circulation from the bark to xylem. Olive tree, Olea europaea, L, carbohydrates     

Starch Biosynthesis in Developing Wheat Grain : Evidence against the Direct Involvement of Triose Phosphates in the Metabolic Pathway

We have used ¹³C-labeled sugars and nuclear magnetic resonance (NMR) spectrometry to study the metabolic pathway of starch biosynthesis in developing wheat grain (Triticum aestivum cv Mardler). Our aim was to examine the extent of redistribution of ¹³C between carbons atoms 1 and 6 of [1-¹³C] or [6-¹³C]glucose (or fructose) incorporated into starch, and hence provide evidence for or against the involvement of triose phosphates in the metabolic pathway. Starch synthesis in the endosperm tissue was studied in two experimental systems. First, the ¹³C sugars were supplied to isolated endosperm tissue incubated in vitro, and second the ¹³C sugars were supplied in vivo to the intact plant. The ¹³C starch produced by the endosperm tissue of the grain was isolated and enzymically degraded to glucose using amyloglucosidase, and the distribution of ¹³C in all glucosyl carbons was quantified by ¹³C-NMR spectrometry. In all of the experiments, irrespective of the incubation time or incubation conditions, there was a similar pattern of partial (between 15 and 20%) redistribution of label between carbons 1 and 6 of glucose recovered from starch. There was no detectable increase over background ¹³C incidence in carbons 2 to 5. Within each experiment, the same pattern of partial redistribution of label was found in the glucosyl and fructosyl moieties of sucrose extracted from the tissue. Since it is unlikely that sucrose is present in the amyloplast, we suggest that the observed redistribution of label occurred in the cytosolic compartment of the endosperm cells and that both sucrose and starch are synthesized from a common pool of intermediates, such as hexose phosphate. We suggest that redistribution of label occurs via a cytosolic pathway cycle involving conversion of hexose phosphate to triose phosphate, interconversion of triose phosphate by triose phosphate isomerase, and resynthesis of hexose phosphate in the cytosol. A further round of triose phosphate interconversion in the amyloplast could not be detected. These data seriously weaken the argument for the selective uptake of triose phosphates by the amyloplast as part of the pathway of starch biosynthesis from sucrose in plant storage tissues. Instead, we suggest that a hexose phosphate such as glucose 1-phosphate, glucose 6-phosphate, or fructose 6-phosphate is the most likely candidate for entry into the amyloplast. A pathway of starch biosynthesis is presented, which is consistent with our data and with the current information on the intracellular distribution of enzymes in plant storage tissues.     

Seasonal Variations in the Carbohydrates in the Xylem of Antiaris africana

The xylem of Antiaris africana contains sucrose, starch, glucose,fructose, maltose, and raffinose. Sucrose and starch are themost abundant carbohydrates. Glucose and fructose occur in relativelyequal amounts while maltose and raffinose are the least abundant.Raffinose disappears from the xylem during the dry season, justbefore leaf fall. The pattern of seasonal variation in the individualsugars and starch is similar. There is generally a peak at leaffall and a depletion of these reserves at new flush. Accumulationof carbohydrates during leaf fall occurs first in the youngestxylem (i.e. the 0–2-cm segment). The youngest xylem alsoaccumulates the greatest amount of reserve sugars and starch.The concentrations of the sugars decrease inwards until theybegin to rise after the 4–6-cm segment. There is, however,no such rise in the concentration of starch. The dry-mattercontents increase inwards from the youngest xylem until theylevel out after the 4–6-cm segment. There is a rapid fallin the sucrose and starch contents of felled A. africana. Sucrosedropped by about 65 per cent and starch by about 73 per centin the first 10 days after felling. The levels of other sugarsdecreased gradually except for glucose and fructose which initiallyrose and then fell. Glucose and maltose could still be detectedon the 68th day after felling.     

综合利用薯芋淀粉生产肌苷的研究

本文报道综合利用野生薯芋属植物盾叶薯芋(Dioscorea zingibensisC.H.wright)和穿龙薯芋(Dioscorea nipponica Makino)中的淀粉生产肌苷(Inosine)的方法。将薯芋原料带水磨碎,在水中筛分得到皂甙淀粉浆,用稀酸水解该物质使淀粉糖化,分离后得到糖液和糖渣。糖液加氮源和无机盐,发酵生产肌苷。糖渣再水解提取薯芋皂甙元。     

Starch synthesis by isolated amyloplasts from wheat endosperm

The aim of this work was to discover which compound(s) cross the amyloplast envelope to supply the carbon for starch synthesis in grains of Triticum aestivum L. Amyloplasts were isolated, on a continuous gradient of Nycodenz, from lysates of protoplasts of endosperm of developing grains, and then incubated in solutions of ¹⁴C-labelled: glucose, glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate and glycerol 3-phosphate. Only glucose 1-phosphate gave appreciable labelling of starch that was dependent upon the integrity of the amyloplasts. Incorporation into starch was linear with respect to time for 2 h. At the end of the incubations, 98% of the ¹⁴C in the soluble fraction of the incubation mixture was recovered as [¹⁴C]glucose 1-phosphate. Thus it is unlikely that the added [¹⁴C glucose 1-phosphate was extensively metabolized prior to uptake by the amyloplasts. It is argued that the behaviour of the isolated amyloplasts, and previously published data on the labelling of starch by [¹³C]glucose, are consistent with the view that in wheat grains it is a C-6, not a C-3, compound that enters the amyloplast to provide the carbon for starch synthesis.Abbreviations PPase alkaline inorganic pyrophosphatase - UDPglucose uridine 5-diphosphoglucose     

Reappraisal of the currently prevailing model of starch biosynthesis in photosynthetic tissues: a proposal involving the cytosolic production of ADP-glucose by sucrose synthase and occurrence of cyclic turnover of starch in the chloroplast.

A vast amount of information has accumulated which supports the view that sucrose and starch are end-products of two segregated, yet highly interconnected, gluconeogenic pathways taking place in the cytosol and chloroplast, respectively. However, several lines of experimental evidences indicate that, essentially identical to the case of heterotrophic tissues, starch formation in the photosynthetic tissues may involve the direct import to the chloroplast of cytosolic hexose (C6) units derived from the sucrose breakdown. This evidence is consistent with the idea that synthesis of a sizable pool of ADP-glucose takes place in the cytosol by means of sucrose synthase whereas, basically in agreement with recent investigations dealing with glycogen biosynthesis in bacteria and animals, chloroplastic phosphoglucomutase and ADP-glucose pyrophosphorylase are most likely playing a role in channelling of glucose units derived from the starch breakdown in the chloroplast, thus making up a regulatory starch turnover cycle. According to this new view, we propose that starch production in the chloroplast is the result of a flexible and dynamic mechanism wherein both catabolic and anabolic reactions take place simultaneously in a highly interactive manner. Starch is seen as an intermediate component of a cyclic gluconeogenic pathway which, in turn, is connected with other metabolic pathways. The possible importance of metabolic turnover as a way to control starch production is exemplified with the recently discovered ADP-glucose pyrophosphatase, an enzyme likely having a dual role in controlling levels of ADP-glucose linked to starch biosynthesis and diverting carbon flow towards other metabolic pathways.