β-amylase in developing apple fruits: activities, amounts and subcellular localization

Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuberous roots, and fleshy fruit development. Based on previously reported in vitro assays, β-amylase is considered one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was shown often extrachloroplastic in living cells. The present experiment showed that β-amylase activity was progressively increasing concomitantly with decreasing starch concentrations during apple (Malus domestica Borkh cv. Starkrimson) fruit development. The apparent amount of β-amylase assessed by Western blotting also increased during the fruit development, which is consistent with the seasonal changes in the enzyme activity. The subcellular-localization studies via immunogold electron-microscopy technique showed that β-amylase visualized by gold particles was predominantly located in plastids especially at periphery of starch granules, but the gold particles were scarcely found in other subcellular compartments. These data proved for the first time that the enzyme is compartmented in its functional sites in plant living cells. The predominantly plastid-distributed pattern of β-amylase in cells was shown unchanged throughout the fruit development. The density of gold particles (β-amylase) in plastids was increasing during the fruit development, which is consistent with the results of Western blotting. So it is considered that β-amylase is involved in starch hydrolysis in plastids of the fruit cells.     

Starch degradation in the cotyledons of germinating lentils

Starch, total amylolytic and phosphorylase activities were determined in lentil cotyledons during the first days of germination. Several independent criteria show that the amylolytic activity is due mainly to an amylase of the α type. Starch is degraded slowly in the first days; during this time, α- and β-amylase activity are very low, while phosphorylase increases and reach a peak on the 3rd day. On the 4th day, there is a more rapid depletion of starch which coincides with an increase in α-amylase activity. By polyacrylamide gel electrophoresis of the crude starch-degrading enzyme, five bands were obtained: one phosphorylase, three α-amylases, and one β-amylase. Based on their heat lability or heat stability, two sets of α-amylase seem to exist in lentil cotyledons.     

Leaf starch degradation comes out of the shadows

During the day, plants accumulate starch in their leaves as an energy source for the coming night. Based on recent findings, the prevailing view of how the transitory starch is remobilized needs considerable revision. Analyses of transgenic and mutant plants demonstrate that plastidic glucan phosphorylase is not required for normal starch breakdown and cast doubt on the presumed essential role of alpha-amylase but do show that beta-amylase is important. Repression of the activity of a plastidic beta-amylase, the export of its product (maltose) or further metabolism of maltose by a newly identified transglucosidase impairs starch degradation. Breakdown of particulate starch also depends on the activity of glucan-water dikinase, which phosphorylates glucosyl residues within the polymer.     

Beta-amylase-resistant amylose. Effect of urea on the limited hydrolysis of amylose by beta-amylase.

Amylose prepared from starch dispersed in 10M-urea, pH6.2, was found to be resistant to the action of beta-amylase and phosphorylase, though it was degraded by alpha-amylase. Amylose isolated by conventional methods was similarly refractory after urea treatment, and was hydrolysed by beta-amylase to the extent of 32-35%; it had no inhibitory effect towards beta-amylase. The physical and chemical properties of the modified amylose were in general comparable with those of normal amylose with a beta-amylolysis limit of 94-98%. Starch and amylopectin were unaffected by urea treatment, i.e. the presence of amylopectin protected amylose against changes induced in it by urea. It is speculated that urea treatment "freezes" amylose molecules in a conformation that renders non-reducing termini inaccessible to the active site of the exo-enzymes. Such changes may limit the degradative action of beta-amylase and phosphorylase.     

Carbohydrate metabolism in growing rice seedlings under arsenic toxicity

We studied in the seedlings of two rice cultivars (Malviya-36 and Pant-12) the effect of increasing levels of arsenic in situ on the content of sugars and the activity of several enzymes of starch and sucrose metabolism: alpha-amylase (EC 3.2.1.1), beta-amylase (EC 3.2.1.2), starch phosphorylase (EC 2.4.1.1), acid invertase (EC 3.2.1.26), sucrose synthase (EC 2.4.1.13) and sucrose phosphate synthase (EC 2.4.1.14). During a growth period of 10-20 d As2O3 at 25 and 50 microM in the growth medium caused an increase in reducing, non-reducing and total soluble sugars. An increased conversion of non-reducing to reducing sugars was observed concomitant with As toxicity. The activities of alpha-amylase, beta-amylase and sucrose phosphate synthase declined, whereas starch phosphorylase, acid invertase and sucrose synthase were found to be elevated. Results indicate that in rice seedlings arsenic toxicity causes perturbations in carbohydrate metabolism leading to the accumulation of soluble sugars by altering enzyme activity. Sucrose synthase possibly plays a positive role in synthesis of sucrose under As-toxicity.     

Biochemical Changes in the Rice Grain during Germination

Changes in the content of starch, protein, and RNA and in the activity of their hydrolases in the rice endosperm (Oryza sativa L., variety IR8) were determined during the first week of germination without added nutrient both in the dark and in the light. Changes were generally more rapid in the dark than in the light. Oxygen uptake and RNase activity started to increase and the root protruded on the second day, followed by the coleoptile on the third day, and the primary leaf on the fourth day. ATP level was at a maximum on the fourth day. The activity of amylases and R enzyme increased progressively, but that of phosphorylase tended to decrease during starch degradation. A new α amylase isozyme band appeared during germination. Glucose was the major product of starch degradation. Sucrose, maltose, maltotriose, raffinose, and fructose were also detected. Protease activity reached a maximum on the fifth or sixth day and closely paralleled the increase in soluble amino N and soluble protein.     

Pathway of starch breakdown in photosynthetic tissues of Pisum sativum.

1. The aim of this work was to discover the pathway of starch breakdown in the photosynthetic tissues of Pisum sativum. 2. Measurements of the starch in the leaves of plants grown in photoperiods of 12 or 18 h showed that starch, synthesized in the light, was rapidly metabolized in the dark at rates of 0.04--0.06 mumol glucose/min per g fresh weight. 3. The maximum catalytic activities of alpha-amylase, beta-amylase, hexokinase, alpha-glucan phosphorylase and phosphoglucomutase in extracts of leaves showed no diurnal variation in either photoperiod, and exceeded estimates of the rate of net starch breakdown in the dark. 4. Studies with intact chloroplasts, isolated from young shoots and from leaves, indicated that pea chloroplasts do not contain significant activities of alpha-amylase, beta-amylase and hexokinase, although some of the latter may be attached to the outside of the chloroplast envelope. These studies also showed that pea chloroplasts contained sufficient alpha-glucan phosphorylase and phosphoglucomutase to mediate the observed rates of starch breakdown. 5. It is proposed that starch breakdown in pea chloroplasts is phosphorolytic.     

Light dependency of the cytokinin-induced bud initiation in protonemata of the moss Funaria hygrometrica

The activities of starch synthesizing enzymes were investigated in wheat grains ( Triticum aestivum L. cv. Kolibri) throughout the grain development period. Starch phosphorylase (E.C. 2.4.1.1.) activity was especially high during the early period of grain development, while starch synthase I (ADP glucose α-glucan 4-α-glucosyl-transferase, E.C. 2.4.1.21) had a maximum activity during the later stage of grain filling. The synthetic potential of starch phosphorylase measured in vitro was about 16 times higher than the quantity of starch actually produced. It is therefore suggested that starch phosphorylase is of substantial importance in grain starch synthesis, particularly in the early period of grain growth. The synthetic potential of starch synthase I measured in vitro made up 25 to 50% of the starch production and the synthetic potential of starch synthase II (UDP glucose α-glucan 4α-glucosyl-transferase. E.C. 2.4.1.11) only about 5%.
Reducing light intensity (shading) during the grain filling period depressed grain growth and starch production by about 20%. Starch phosphorylase was not significantly affected by the reduced light intensity if enzyme activity is calculated on unit grain weight and not as activity per grain. Starch synthase I activity, however, was depressed by shading during the later stage of grain development. The depressed starch production found under low light conditions, however, cannot only be explained by an affected starch synthase I activity, but probably was also related to other still unknown factors limiting grain growth under low light conditions. The poor starch production in the shaded plants was not due to an insufficient supply of assimilates.     

Polymorphism in rice amylases at an early stage of seed germination

A polymorphism in rice amylases at an early stage of seed germination is analyzed by zymogram. In non-glutinous cultivars of rice, alpha-amylase isozymes are mainly confirmed in germinating seeds. However, in glutinous cultivars, beta-amylase isozymes, which are not confirmed in nonglutinous cultivars, make up the major part of the total amylase activity and the expression of alpha-amylases are repressed.     

Starch phosphorylase: Role in starch metabolism and biotechnological applications

The α-glucan phosphorylases of the glycosyltransferase family are important enzymes of carbohydrate metabolism in prokaryotes and eukaryotes. The plant α-glucan phosphorylase, commonly called starch phosphorylase (EC 2.4.1.1), is largely known for the phosphorolytic degradation of starch. Starch phosphorylase catalyzes the reversible transfer of glucosyl units from glucose-1-phosphate to the nonreducing end of α-1,4-d-glucan chains with the release of phosphate. Two distinct forms of starch phosphorylase, plastidic phosphorylase and cytosolic phosphorylase, have been consistently observed in higher plants. Starch phosphorylase is industrially useful and a preferred enzyme among all glucan phosphorylases for phosphorolytic reactions for the production of glucose-1-phosphate and for the development of engineered varieties of glucans and starch. Despite several investigations, the precise functional mechanisms of its characteristic multiple forms and the structural details are still eluding us. Recent discoveries have shed some light on their physiological substrates, precise biological functions, and regulatory aspects. In this review, we have highlighted important developments in understanding the role of starch phosphorylases and their emerging applications in industry.     

Partial purification and properties of thermostable intracellular amylases from a thermophilic Bacillus sp. AK-2

Intracellular thermostable amylases from a thermophilic Baccilus sp. AK-2 have been isolated and purified. The crude enzyme, having pH optimum at 6.5. and temperature optimum at 68 degrees C was purified by DEAE-cellulose column chromatography. Three separable enzyme fractions having starch hydrolyzing property were eluted by lowering the pH from 8.5 to 7.0. Electrophoretic mobility of these fractions showed a single band. Calcium ion up to a concentration of 20 mM had an activating effect on the three fractions. The optimum temperature for the three fractions (FI, FII and FIII) was 65 degrees C and the pH optimum for each was 6.0, 6.5 and 6.0, respectively. The -SH group in the amylase molecule was essential for enzyme activity. Except for Ca2+, Mg2+, Sr2+ and Mn2+ all other metal ions studied inhibited both alpha and beta-amylase activities. EDTA showed dose dependent non-competitive inhibition. Product formation studies proved FI and FIII to be of the alpha-amylase type and FII of the beta-amylase type. The Km for the substrate (starch) in the presence or absence of EDTA was 0.8 X 10(-3) and 1.13 X 10(-3) g/ml for alpha-amylase and beta-amylase, respectively.     

Starch degradation during germination of Civer arietinum L. seeds.

The variations in starch and soluble sugar content, in phosphorylase and amylase activities in cotyledons of germinating seeds of Cicer arietinum L. are determined. Results from various experiments prove that the alpha-amylases are chiefly responsible for amylase activity. Phosphorylase plays an important r?le during the first two days of germination, but it is relegated to a secondary position as the amylase activity increases. Disc electrophoresis on polyacrylamide gel shows the existence of a phosphorylase throughout germination, and detects two alpha-amylases after 48 and 96 h germination respectively. The increase in alpha-amylase activity during germination is due to de novo synthesis of the two isoenzymes, since both are inhibited by cycloheximide and actinomyces D. This de novo synthesis depends on some embryo produced factor, unreplaceable either by giberellic acid or by kinetin.     

Enzymic mechanisms of starch breakdown in germinating rice seeds: 7. Amylase formation in the epithelium

The time sequence analysis of the starch digestion pattern of the thin sectioned germinating rice (Oryza sativa L.) seed specimens using the starch film method showed that at the initial stage amylase activity was almost exclusively localized in the epithelium septum between the scutellum and endosperm. Starch breakdown in the endosperm tissues began afterward; amylase activity in the aleurone layers was detectable only after 2 days. Polyacrylamide gel electrofocusing (pH 4 to 6) revealed nearly the same zymogram patterns between endosperm and scutellum extracts, although additional amylase bands appeared in the endosperm extracts at later germination stages (4 to 6 days). These are presumably attributable to the newly synthesized enzyme molecules in the aleurone cells.     

Amylolytic enzymes: molecular aspects of their properties

The present review describes the structural features of alpha-amylase, beta-amylase and glucoamylase that are the best known amylolytic enzymes. Although they show similar function, i.e. catalysis of hydrolysis of alpha-glucosidic bonds in starch and related saccharides, they are quite different. alpha-Amylase is the alpha --> alpha retaining glycosidase (it uses the retaining mechanism), and beta-amylase together with glucoamylase are the alpha --> beta inverting glycosidases (they use the inverting mechanism). While beta-amylase and glucoamylase form their own families 14 and 15, respectively, in the sequence-based classification of glycoside hydrolases, alpha-amylase belongs to a large clan of three families 13, 70 and 77 consisting of almost 30 different specificities. Structurally both alpha-amylase and beta-amylase rank among the parallel (beta/alpha)8-barrel enzymes, glucoamylase adopts the helical (alpha/alpha)6-barrel fold. The catalytic (beta/alpha)8-barrels of alpha-amylase and beta-amylase differ from each other. The only common sequence-structural feature is the presence of the starch-binding domain responsible for the binding and ability to digest raw starch. It is, however, present in about 10% of amylases and behaves as an independent evolutionary module. A brief discussion on structure-function and structure-stability relationships of alpha-amylases and related enzymes is also provided.     

Effect of growth temperature on reserve mobilization and hydrolytic enzyme activities in Vigna mungo cotyledons

Seeds of Vigna mungo were allowed to germinate at 27, 18 and15°C, and time-course changes of hydrolytic enzyme activitiesand the mobilization rate of reserve components in cotyledonswere studied. The seeds germinated at 27 and 18°C grew normally,whereas the growth at 15°C was markedly retarded. In cotyledonsof seedlings grown at 27 and 18°C, amylolytic and proteolyticenzyme activities increased at early stages of growth and therates of starch and protein mobilization changed correspondingto the hydrolytic enzyme activities. At 15°C the enzymeactivities increased gradually during the experimental periodof 16 days, but the reserves in cotyledons remained almost unchangeduntil the end of the experimental period. Changes of zymogram patterns of amylolytic and proteolytic activitiesin cotyledons of seedlings grownat 27, 18 and 15°C wereexamined using polyacrylamide gel electrophoresis. The intensitiesof a main band of a-amylase and at least two bands of protease(gelatin-hydrolyzing activity) increased concurrently with invitro activities of amylolytic and proteolytic enzymes. At leastthree bands of starch phosphorylase were present in cotyledonsat early stages of germination and their intensities decreasedduring the growth of seedlings at 27, 18 and 15°C. (Received June 4, 1980; )     

General Biochemical Characterization of Thermostable Extracellular beta-Amylase from Clostridium thermosulfurogenes

Clostridium thermosulfurogenes, an anaerobic bacterium which ferments starch into ethanol at 62 degrees C, produced an active extracellular amylase and contained intracellular glucoamylase but not pullulanase activity. The extracellular amylase was purified 2.4-fold, and its general physicochemical and catalytic properties were examined. The extracellular amylase was characterized as a beta-amylase (1,4-alpha-d-glucan maltohydrolase) based on demonstration of exocleavage activity and the production of maltose with a beta-anomeric configuration from starch. The beta-amylase activity was stable and optimally active at 80 and 75 degrees C, respectively. The pH optimum for activity and the pH stability range was 5.5 to 6 and 3.5 to 6.5, respectively. The apparent [S](0.5V) and V(max) for beta-amylase activity on starch was 1 mg/ml and 60 U/mg of protein. Similar to described beta-amylase, the enzyme was inhibited by p-chloromercuribenzoate, Cu, and Hg; however, alpha- and beta-cyclodextrins were not competitive inhibitors. The beta-amylase was active and stable in the presence of air or 10% (vol/vol) ethanol. The beta-amylase and glucoamylase activities enabled the organism to actively ferment raw starch in the absence of significant pullulanase or alpha-amylase activity.     

Phosphorylases I and II of Maize Endosperm

Two phosphorylases have been found in the endosperm of Zea mays. Phosphorylase I is found through all stages of endosperm development and seed germination investigated. The other enzyme, phosphorylase II appears only at the stage of rapid starch biosynthesis and is not found during germination. At 22 days after pollination, the activity of phosphorylase II is 10 times that of phosphorylase I. These 2 phosphorylases are separable by column chromatography and behave differently in several respects.     

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.     

Light-induced degradation of storage starch in turions of Spirodela polyrhiza depends on nitrate

Light induces both the germination of turions of the duckweed Spirodela polyrhiza and the degradation of the reserve starch stored in the turions. The germination photoresponse requires nitrate, and we show here that nitrate is also needed for the light-induced degradation of the turion starch. Ammonium cannot substitute for nitrate in this regard, and nitrate thus acts specifically as signal to promote starch degradation in the turions. Irradiation with continuous red light leads to starch degradation via auto-phosphorylation of starch-associated glucan, water dikinase (GWD), phosphorylation of the turion starch and enhanced binding of alpha-amylase to starch granules. The present study shows that all of these processes require the presence of nitrate, and that nitrate exerts its effect on starch degradation at a point between the absorption of light by phytochrome and the auto-phosphorylation of the GWD. Nitrate acts to coordinate carbon and nitrogen metabolism in germinating turions: starch will only be broken down when sufficient nitrogen is present to ensure appropriate utilization of the released carbohydrate. These data constitute the first report of control over the initiation of reserve starch degradation by nitrate.