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PubMed1989

Altered regulation of beta-amylase activity in mutants of Arabidopsis with lesions in starch metabolism.

Three classes of mutants of Arabidopsis thaliana (L.) Heynhold with alterations in starch metabolism were found to have higher levels of leaf amylase activity than the wild type when grown in a 12-hr photoperiod. This effect was dependent upon the developmental stage of the plants and was largely suppressed during growth in continuous light. The various amylolytic activities in crude extracts were separated by electrophoresis in nondenaturing polyacrylamide gels and visualized by activity staining. The increased amylase activity in the mutants was due to an up to 40-fold increase in the activity of an extrachloroplast beta-amylase (EC 3.2.1.2). These observations indicate the existence of a regulatory mechanism that controls the amount of beta-amylase activity in response to fluctuations in photosynthetic carbohydrate metabolism. It is paradoxical that beta-amylase appears to be a highly regulated enzyme, but as yet no physiologically relevant function can be assigned to this enzyme due to the absence of starch in the cytoplasmic compartment of leaf cells.

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PubMed1971

Amylases in developing barley seeds.

The amylases of developing barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated by colorimetric and electrophoretic methods. Maxima of amylolytic activity appeared in the aleurone layers and starchy endosperm at 5 and 20 days after anthesis. Amylase from 5-day-old aleurone layers could be separated into four rapidly moving bands with alpha-amylase activity. By 20 days the four bands had been replaced by seven bands of medium mobility. These seven bands of amylase were electrophoretically identical to those observed when mature aleurone layers are treated with gibberellic acid. Immature aleurone layers failed to respond to exogenous gibberellic acid. In the starchy endosperm the seven bands of medium mobility were also present. Calcium-dependent alterations in the electrophoretic mobility and activity of particular bands occurred during the maturation of the starchy endosperm. Treatment of the immature starchy endosperm with papain yielded four forms of beta-amylase.

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PubMed1979

Synthesis of a possible precursor of alpha-amylase in wheat aleurone cells.

alpha-Amylase from wheat aleurone (Triticum aestivum) was synthesized in a S-150 wheat germ readout system using polysomes, and a messenger RNA-dependent reticulocyte lysate system using polyadenylic acid [poly(A)]-enriched RNA. The product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, precipitation by specific lambda-globulin for alpha-amylase, and proteolysis. Two immunoprecipitated products were synthesized from the readout system, the predominant species migrating coincidentally with authentic alpha-amylase on sodium dodecyl sulfate-polyacrylamide gels. A putative precursor, 1,500 daltons larger, was evident but was less abundant. The relationship between the two polypeptides was established by proteolytic analysis using Staphylococcus aureus V8 protease. At least nine fragments were generated and were identical in both species. The poly(A)-enriched RNA synthesized only the putative precursor in the reticulocyte lysate system. Attempts to process the precursor to the mature size of alpha-amylase failed. These findings are discussed in connection with the signal hypothesis (proposed for the transport of proteins across membranes) and the mode of secretion of alpha-amylase in aleurone cells.

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PubMed1979

Influence of Cotyledons upon alpha-Amylase Activity in Pea Embryonic Axes.

alpha-Amylase activity remained relatively low in the axes of intact etiolated pea seedlings; the activity was predominantly confined to the epicotyl. Starch accumulated slightly. When the cotyledons were removed and the axes cultured on medium containing no carbon source, the starch reserve in the axes disappeared within a few days. This was accompanied by a 10- to 15-fold increase in alpha-amylase activity, in the absence of additional epicotyl growth. The phenonemon was observed for axes throughout early growth, although the relative accumulation of alpha-amylase activity in cultured axes was less for older seedlings. This change was attributed to a reduced response by nongrowing tissues. There was no corresponding change in beta-amylase activity. These observations, described for several varieties of peas, demonstrate the control of cotyledons upon the utilization of stored reserves within the axis, with alpha-amylase as a key enzyme.

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PubMed1980

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 9. DE NOVO SYNTHESIS OF beta-AMYLASE.

Germinating rice seeds were fed with [(35)S]methionine and the incorporation of (35)S into beta-amylase demonstrated by quantitative immunoprecipitation using rabbit anti-beta-amylase immunoglobulin G fraction. Separation of the antigen-antibody complex by Na-dodecylsulfate gel electrophoresis and subsequent radioautography clearly showed the radioactive labeling of the beta-amylase molecule. The specific radioactivity of beta-amylase derived from scutellum by immunoprecipitation was significantly greater than that of the endosperm. The results strongly indicate that at the onset of germination of rice seeds beta-amylase is synthesized de novo in the scutellum and that in later stages there occurs activation of an inactive, latent form of the enzyme associated with starch granules in the endosperm. In later stages of germination this activated form of the enzyme becomes dominant.

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PubMed1984

Starch Degradation in Synchronously Grown Chlamydomonas reinhardtii and Characterization of the Amylase.

The activities of amylase and phosphorylase were monitored during the 12-hour light/dark synchronous cell cycle of autotrophically grown Chlamydomonas reinhardtii 11-32/90. The activity of amylase increased from 7.3 to 42 micromole reducing equivalents per 10(9) cells per hour while phosphorylase increased from 43 to 214 micromole glucose 1-phosphate released per 10(9) cells per hour between the midlight and middark periods. Cellular fractionation indicated that both enzymes were localized solely within the chloroplast. The pH optima for amylase and phosphorylase were 6.7 to 7.6 and 6.0 to 7.4, respectively. The amylase is a heat-labile alpha-amylase which is insensitive to ethylenetetraaecetate but inhibited by N-ethylmaleimide.

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PubMed1984

Changes in Levels of alpha-Amylase Components in Barley Tissues during Germination and Early Seedling Growth.

Kernels of Klages barley (Hordeum vulgare L.) were germinated for 1 to 4 days on moist sand at 18 degrees C. Representative kernels from each time period were dissected to give the following fractions: scutellum, subscutellar endosperm, aleurone-scutellum interface, remaining aleurone, subaleurone endosperm, and core endosperm. These tissues were analyzed for alpha-amylase components by isoelectric focusing and rocket-line immunoelectrophoresis. Although aleurone and scutellar tissues appeared to synthesize the same alpha-amylase components, enzyme was detected first in the scutellum. A larger proportion of scutellar alpha-amylase was excreted into the endosperm compared to aleurone synthesized alpha-amylase. Aleurone cells appeared to synthesize appreciably more alpha-amylase than did scutellar tissue.

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PubMed1986

The Multiple Forms of alpha-Amylase Enzyme of the Araucaria Species of South America: A. araucana (Mol.) Koch and A. angustifolia (Bert.) O. Kutz : A Comparative Study.

alpha-Amylase is one of the major enzymes present in the seeds of both Araucaria species of South America and it initiates starch hydrolysis during germination and early seedling growth. The pattern of the multiple forms of alpha-amylase of the two Araucaria species was investigated by electrophoresis and isoelectrofocusing of the native enzyme in polyacrylamide gels. The enzyme forms were compared in the embryo and megagametophyte of quiescent seeds and of seeds imbibed for 18, 48, and 90 hours. Specific alpha-amylase enzyme forms appear and disappear during these imbibition periods showing both similarities and differences between tissues and species. Before imbibition, there are five alpha-amylase forms identical in both tissues, but different between species. After 18 hours of imbibition, there are two enzyme forms in both tissues of Araucaria araucana seeds, only one form in the embryo of Araucaria angustifolia but two forms in the megagametophyte of this specie. After 48 hours of seed imbibition, most of the enzyme forms present in quiescent seeds reappear. At 90 hours of imbibition different enzyme forms are detected in the embryo with respect to the gametophyte. The changes in form patterns of alpha-amylase are discussed according to a possible regulation of gene expression by endogenous gibberellins.

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PubMed1988

Localization of alpha-Amylase in the Apoplast of Pea (Pisum sativum L.) Stems.

Most of the activity of an alpha-amylase present in crude pea (Pisum sativum L. cv Laxton's Progress No. 9) leaf preparations cannot be found in isolated pea leaf protoplasts. The same extrachloroplastic alpha-amylase is present in pea stems, representing approximately 6% of total stem amylolytic activity and virtually all of the alpha-amylase activity. By a simple infiltration-extraction procedure, the majority (87%) of this alpha-amylase activity was recovered from the pea stem apoplast without significantly disrupting the symplastic component of the tissue. Only 3% of the beta-amylase activity and less than 2% of other cellular marker enzymes were removed during infiltration-extraction.

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PubMed2004

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.

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PubMed1980

Kinetics of Growth and Amylase Production of Saccharomycopsis fibuligera on Potato Processing Wastewater.

The kinetics of growth and amylase production of Saccharomycopsis fibuligera were studied in a chemostat on a synthetic potato processing blancher water. Dilution rates (D) from 0.101 to 0.480 h were examined. A mathematical model based on the Monod equation was developed. The yield of cell mass from carbohydrates was constant and equal to 0.84. The maximum specific growth rate and the Monod constant were determined to be 0.596 h and 0.226 mg/ml, respectively. An equation for the steady-state starch concentrations was empirically derived. The steady-state noncarbohydrate carbon levels rose linearly with D. Reducing sugars were the growth-limiting substrate, and their steady-state levels conformed to Monod kinetics. The yield of amylase from the cell mass (Y(z)) declined as D rose and was described by the equation Y(z) = (-8.005D + 4.076). The model predicted that the maximum production of cell mass should occur at D = 0.35 h and the maximum production of amylase should occur at D = 0.22 h. The mathematical model presented agreed with the experimental results in its prediction of the steady-state level of reducing sugar, starch, cell mass, and amylase concentrations as well as the productivity of amylase.

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PubMed1989

Cloning and Expression of a Schwanniomyces occidentalis alpha-Amylase Gene in Saccharomyces cerevisiae.

An alpha-amylase gene (AMY) was cloned from Schwanniomyces occidentalis CCRC 21164 into Saccharomyces cerevisiae AH22 by inserting Sau3AI-generated DNA fragments into the BamHI site of YEp16. The 5-kilobase insert was shown to direct the synthesis of alpha-amylase. After subclones containing various lengths of restricted fragments were screened, a 3.4-kilobase fragment of the donor strain DNA was found to be sufficient for alpha-amylase synthesis. The concentration of alpha-amylase in culture broth produced by the S. cerevisiae transformants was about 1.5 times higher than that of the gene donor strain. The secreted alpha-amylase was shown to be indistinguishable from that of Schwanniomyces occidentalis on the basis of molecular weight and enzyme properties.

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PubMed1989

Continuous Production of Thermostable beta-Amylase with Clostridium thermosulfurogenes: Effect of Culture Conditions and Metabolite Levels on Enzyme Synthesis and Activity.

A beta-amylase-overproducing mutant of Clostridium thermosulfurogenes was grown in continuous culture on soluble starch to produce thermostable beta-amylase. Enzyme productivity was reasonably stable over periods of weeks to months. The pH and temperature optima for beta-amylase production were pH 6.0 and 60 degrees C, respectively. Enzyme concentration was maximized by increasing biomass concentration by using high substrate concentrations and by maintaining a low growth rate. beta-Amylase concentration reached 90 U ml at a dilution rate of 0.07 h in a 3% starch medium. A further increase in enzyme activity levels was limited by acetic acid inhibition of growth and low beta-amylase productivity at low growth rates.

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PubMed1995

Purification and Properties of Extracellular Amylase from the Hyperthermophilic Archaeon Thermococcus profundus DT5432.

A hyperthermophilic archaeon, Thermococcus profundus DT5432, produced extracellular thermostable amylases. One of the amylases (amylase S) was purified to homogeneity by ammonium sulfate precipitation, DEAE-Toyopearl chromatography, and gel filtration on Superdex 200HR. The molecular weight of the enzyme was estimated to be 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 5.5 to 6.0 and was stable in the range of pH 5.9 to 9.8. The optimum temperature for the activity was 80(deg)C. Half-life of the enzyme was 3 h at 80(deg)C and 15 min at 90(deg)C. Thermostability of the enzyme was enhanced in the presence of 5 mM Ca(sup2+) or 0.5% soluble starch at temperatures above 80(deg)C. The enzyme activity was inhibited in the presence of 5 mM iodoacetic acid or 1 mM N-bromosuccinimide, suggesting that cysteine and tryptophan residues play an important role in the catalytic action. The amylase hydrolyzed soluble starch, amylose, amylopectin, and glycogen to produce maltose and maltotriose of (alpha)-configuration as the main products. Smaller amounts of larger maltooligosaccharides were also produced with a trace amount of glucose. Pullulan; (alpha)-, (beta)-, and (gamma)-cyclodextrins; maltose; and maltotriose were not hydrolyzed.

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PubMed1982

Evidence of duplication of the human salivary amylase gene.

Isoelectric focusing of human parotid saliva reveals different alpha-amylase patterns reflecting qualitative and quantitative variations. A puzzling pattern, which shows three different amylase gene products, was found in four individuals. Based on this observation a model is presented in which the salivary amylase gene is duplicated. Family studies show that the AMY1*A2 gene forms a haplotype with the normal gene, AMY1*A1, whereas the AMY1*A3 gene still exists in a single form. The absence of homozygote 2-2 in offspring of 1-2 X 1-2 marriages and in population material, and the fact that the variant protein makes up about only 20-30% of the total amylase protein in heterozygotes can be considered as additional evidence supporting the hypothesis. The possibility that cis-acting regulatory variants are involved in the patterns with quantitative variation is discussed.

Amylases↗

PubMed1981

Ontogenesis of alpha-amylase in rat parotid gland during postnatal development.

Changes in alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) of parotid gland were investigated during postnatal development of the rat. Modifications in amylase activity after birth allow the distinction of three stages which can be correlated with the morphologic development of the parotid gland. Significant sexual differences in the evolution of alpha-amylase activity were found. During the first stage (from birth to the 20th day) there is a higher increase in females, while males have a more pronounced increment in the second stage (from the 20th to the 30th day). By means of gel electrophoresis of parotid extracts, four molecular forms of amylase can be separated. The slowest migrating band (Form 1) is not detected at the initial stage.

Amylases↗

PubMed1989

Changes in carbohydrate and nitrogenous components and amylase activities during germination of grain amaranth.

Grain amaranth (Amaranthus hypochondriacus), Yercaud local variety, was soaked overnight and germinated for 192 h taking the soaked grains as the zero time (0 h) sample. The changes in the activities of alpha- and beta-amylases, starch, sugar, protein and lysine contents during germination are reported. Activity of alpha-amylase was high in the 0 h soaked grains, while beta-amylase activity was high in 72 h germinated grains. The joint action of the amylases resulted in a decrease of starch content from 0 to 192 h in germinated grains and an increase in total sugars during the initial period of germination. Protein nitrogen was found to decrease from 48 h to 192 h accompanied by an increase in free amino acid and non-protein nitrogen contents. Total lysine content was found to be increased by 31% in 24 h germinated grain amaranth. Protein fractionation of raw, soaked and 24 h germinated grain amaranth showed that the distribution of different types of proteins varied during germination of the grains. An increase of water soluble protein content was noticed in 24 h germinated grains.

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PubMed1983

Automated assay of alpha-amylase inhibitor proteins by continuous flow analysis.

A procedure for the automated assay of purified amylase inhibitors was developed. Samples were analyzed at a rate of 60/h using ferricyanide reagent to monitor the suppression of the release of reducing groups from a solution of starch by a calibrated alpha-amylase reagent. In addition to the sequential analysis of individual samples, the use of gradients permitted the continuous analysis of the effect of substrate and of inhibitor concentration. Also described are some of the effects of starch and inhibitor concentration and time of preincubation on the amylase-inhibitor reaction. The procedure was also suitable for the assay of samples of amylase and should be applicable to the determination of the effects of inhibitors on other enzymes which release reducing sugars.

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