Peroxidases in Relation to Removal of Dormancy and Germination of Apple Embryos

The changes in germination, peroxidase activity and isoperoxidase spectrum have been studied in apple embryos at 5°C (stratification) and at 20°C in the presence or absence of seed coats. The embryo dormancy is progressively released at 5°C, but not at 20°C. The peroxidase activity in embryos covered with seed coats is very low at 5°C as well as at 20°C which corresponds to a restricted number of isoenzymes. In isolated embryos the peroxidase activity increases significantly. This is due to an increase in both the number and the activity of the isoperoxidases and it is more pronounced at 20°C than at 5°C. The obtained results suggest that the soluble peroxidases are not involved in the process of the release of embryo dormancy. The variations observed are attributed to the growth process following germination, which can occur even at low temperature.     

Heat stress negatively affects physiology and morphology during germination of Ormosia coarctata (Fabaceae,Papilionoideae)

Research on the morphophysiological behavior of forest seeds during germination with respect to climate change is scarce. To date, there have been no studies on the biochemical or morphological aspects of Ormosia spp. In this study, we subjected Ormosia coarctata seeds to various temperature conditions to investigate temperature-dependent impacts on morphology, reactive oxygen species (ROS) generation, antioxidant systems, and storage systems. Analyses were performed on seeds exposed to 25, 35, and 40 °C for 48, 96, and 144 h. The morphology was evaluated by radiation using a Faxitron MX-20 device. ROS production (superoxide anion and hydrogen peroxide), malonaldehyde (MDA), carbonylated proteins, antioxidant enzyme activity (superoxide dismutase [SOD], ascorbate peroxidase [APX], catalase [CAT], and peroxidase [POX]), β-carotene, lycopene, glucose, and reserve enzyme activity (α- and β-amylase, lipase, and protease) were analyzed by spectrophotometry. Heat stress (40 °C) decreased germination by 76.2% and 78.1% (compared to 25 and 35 °C, respectively), caused damage to the external morphology of the seed, increased the content of ROS, MDA, and carbonylated proteins, and reduced APX, CAT, and POX activity. Furthermore, heat stress decreased glucose content and α-amylase activity. These results suggest that an increase of 5 °C in temperature negatively affects germination, promotes oxidative stress, and induces deterioration in O. coarctata seeds.

     

Adjustment of the activity of α-amylase extracted from the style of the black mussel Choromytilus meridionalis (Krauss) in response to thermal acclimation

The mussel Choromytilus meridionalis (Krauss) is a common inhabitant of the intertidal zone on the west coast of the Cape Peninsula, South Africa, and experiences temperatures of between 8°C when immersed by the tide and at least 25°C on exposure to air. The activity of α-amylase extracted from the crystalline style of freshly-collected mussels has a low temperature coefficient of ≈ 1.12 over much of the temperature range experienced in the natural environment. Warm acclimation results in an increase in the α-amylase activity, despite the fact that individual rate: temperature curves for extracts from mussels acclimated to 8, 15 and 22°C have rather low temperature coefficients of 1.14–1.17 between 10 and 20°C. The increase of activity of the α-amylase following warm acclimation may form an integral part of the improved filtration, ingestion and assimilation which is necessary to offset increased metabolic losses during the warm conditions of the summer months.     

α- and β-Amylases in seed germination

During the first 24 h of germination of wheat seeds, starch is hydrolysed by free β-amylase. In the next 24 h, some amount of inactive form of β-amylase is converted into active form and this together with α-amylase synthesizedde novo brings about the hydrolysis of starch. The amount of α-amylase is greater in seeds with embryo intact than with embryo excised after 24 h hydration. However, at later stages of seed germination α-amylase becomes predominant and the activity of β-amylase steadily diminishes.     

Properties of the isoforms of α-amylase from kilned and unkilned malted sorghum (Sorghum bicolor)

We have previously reported the presence of a relatively heat-stable α-amylase with a low K_m for starch in kilned malted sorghum. In order to establish the industrially useful and more efficient isoforms, we have separated this α-amylase into different isoforms using both cation and anion-exchange chromatographies. Unkilned malted α-amylase crude was separated into three different isoforms (a1, a2 and a3) whereas kilned samples were separated into two (a1 and a2). Apparently one isoform (a3) was lost during kilning due to heat lability. a1 isoform which appears to have a neutral pI and constitute about 60% of the total α-amylases protein that were induced during germination, have the lowest K_m for starch. They are more generally stable than other isoforms at all the temperatures studied. These isoforms lost only 10% activity at 80 °C for 30 min and still had some residual activity at 100 °C incubation for 30 min. a1 isoform could therefore be adapted for industrial starch conversion processes which are carried out within this range of gelatinizing temperatures because of its properties.     

Studies on α-amylase production by Bacillus licheniformis MIR-61

An acid α-amylase hyperproducing strain, designated as MIR-61, was isolated in a screening procedure from South American soil samples. MIR-61, a 60°C thermoresistant strain, was identified using 98 biochemical and morphological tests and characterized as Bacillus licheniformis by numerical taxonomy. Batch cultures of B. licheniformis MIR-61 showed extracellular α-amylase and α-glucosidase activities during the exponential growth phase. The production of α-amylase was studied at free and constant pH values at 37 and 45°C. Maximum α-amylase activity (4,767 kU/dm³ in a liquid medium) was detected at 45°C at a constant pH (7.0) in the late exponential phase. The α-amylase production by B. licheniformis MIR-61 is 10 to 300 times higher than the enzyme production reported in strains of the same species. Optimum α-amylase activity was found at 50 to 67°C in an acid pH range from 5.5 to 6.0. These properties would allow its use in starch industry processes.     

Dark-mediated dormancy release in stratified Lolium rigidum seeds is associated with higher activities of cell wall-modifying enzymes and an apparent increase in gibberellin sensitivity

Dormancy release in freshly matured, imbibed annual ryegrass (Lolium rigidum) seeds is inhibited by light and involves a decrease in seed sensitivity to abscisic acid. Other processes involved in dormancy release in the dark were investigated by measuring seed storage compound mobilisation and the activity of cell wall-degrading enzymes. Activities of endo-β-mannanase and total peroxidase were higher in dark-stratified compared to light-stratified seeds, indicating that weakening of the structures constraining the embryo was accelerated in the dark. A dramatic degradation of storage proteins in light-stratified seeds, accompanied by induction of a high molecular mass protease, suggests that maintenance of storage(-like) proteins is also important in dark-mediated dormancy release. α-Amylase activity was induced in dark-stratified seeds at least 48 h prior to radicle emergence upon transfer to conditions permitting germination, or in light-stratified seeds supplied with exogenous gibberellin A₄. This suggests that (a) α-amylase is involved in stimulation of germination of non-dormant L. rigidum seeds, and (b) dark-stratified seeds have an increased sensitivity to gibberellins which permits the rapid induction of α-amylase activity upon exposure to germination conditions. Overall, it appears that a number of processes, although possibly minor in themselves, occur in concert during dark-stratification to contribute to dormancy release.     

Investigation of α-amylase and proteinase of culture filtrate from bacillus licheniformis MB 80 strain

At the investigation of some properties of the α-amylase and proteinase in the culture filtrat from Bacillus licheniformis MB 80 strain it has been established that the α-amylase activity is the highest at pH 6.0 to 6.5 and at 90°C, that the proteolytic activity is the highest at pH 9.5 to 10.0 and at 70°C and that the proteinase is inactivated at temperatures over 70°C.     

α-isoamylases of rye seeds

Rye seeds contained 5 α-type amylases. Three behaved like typical α-amylases and were called Aα-amylases. Two showed chemical activity like α-amylase, but behaved differently physically and were called Bα-amylases. The latter were partly inactivated at pH 3·3 and at 70°. They were more resistant to EDTA than were the Aα-amylases. Barley and wheat seeds contained amylases behaving like Bα-amylases. Aleurone layers contained relatively large amounts of Aα-amylases. Relative amounts of Aα-and Bα-amylases depended on temperature during germination. Bα-amylases remained active for a longer time after germination than Aα-amylases.     

α-Amylase in the Barley Grain Influenced by the Auxin-Cytokinin Interaction in the Coleoptile

Two phases are distinguished in the α-amylase production in barley (Hordeum vulgare) grains. There is an increase in activity extended to the third or fourth day of germination, then a slight decrease follows. This decrease is accelerated by kinetin while it is prevented by IAA applied at the top of the embryo coleoptile. IAA reverses partially the kinetin action. IAA applied in the germination medium has practically no effect. Removal of the coleoptile stops further increase in α-amylase activity and induces complete insensitivity to hormone treatment. The results indicate that auxin metabolism in the coleoptile participates in the control of α-amylase evolution in the barley grain and that kinetin could act through auxin metabolism in this coleoptile.     

High temperature — induced changes in germination,seedling vigour and the metabolic activities in rice seeds

The effects of temperature treatments on rice seeds in the early imbibitional phase were studied with respect to changes in germination percentage, seedling growth in terms of root and shoot lengths, water uptake, respiration and hydrolytic enzymes,viz. α-amylase, adenosine triphosphatase (ATPase) and phytase. As compared with the control, the treatment for 30 min at 50 °C caused a variable degree of increase in all these characters and a positive correlation existed between seedling vigour and hydrolase activities. The treatment for 30 min at 60 °C, on the other hand, elicited a retarding influence on these characters. As regards the enzyme activities, the damaging effect of 60 °C could, however, be visualized only after 72 h of germination which was preceded by an enhancement during the early hours.     

A novel cold-active and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda: molecular cloning,heterologous expression and biochemical characterization

A novel gene (amyZ) encoding a cold-active and salt-tolerant α-amylase (AmyZ) was cloned from marine bacterium Zunongwangia profunda (MCCC 1A01486) and the protein was expressed in Escherichia coli. The gene has a length of 1785 bp and encodes an α-amylase of 594 amino acids with an estimated molecular mass of 66 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 13 and shows the highest identity (25 %) to the characterized α-amylase TVA II from thermoactinomyces vulgaris R-47. The recombinant α-amylase showed the maximum activity at 35 °C and pH 7.0, and retained about 39 % activity at 0 °C. AmyZ displayed extreme salt tolerance, with the highest activity at 1.5 M NaCl and 93 % activity even at 4 M NaCl. The catalytic efficiency (k _cat/K _m) of AmyZ increased from 115.51 (with 0 M NaCl) to 143.30 ml mg^?1 s^?1 (with 1.5 M NaCl) at 35 °C and pH 7.0, using soluble starch as substrate. Besides, the thermostability of the enzyme was significantly improved in the presence of 1.5 M NaCl or 1 mM CaCl₂. AmyZ is one of the very few α-amylases that tolerate both high salinity and low temperatures, making it a potential candidate for research in basic and applied biology.     

Hydrolysis of starches by the action of an α-amylase from Bacillus subtilis

A moderately thermophilic Bacillus subtilis strain, isolated from fresh sheep’s milk, produced extracellular thermostable α-amylase. Maximum amylase production was obtained at 40 °C in a medium containing low starch concentrations. The enzyme displayed maximal activity at 135 °C and pH 6.5 and its thermostability was enhanced in the presence of either calcium or starch. This thermostable α-amylase was used for the hydrolysis of various starches. An ammonium sulphate crude enzyme preparation as well as the cell-free supernatant efficiently degraded the starches tested. The use of the clear supernatant as enzyme source is highly advantageous mainly because it decreases the cost of the hydrolysis. Upon increase of reaction temperature to 70 °C, all substrates exhibited higher hydrolysis rates. Potato starch hydrolysis resulted in a higher yield of reducing sugars in comparison to the other starches at all temperatures tested. Soluble and rice starch took, respectively, the second and third position regarding reducing sugars liberation, while the α-amylase studied showed slightly lower affinity for corn starch and oat starch.     

Trehalose production with a new enzymatic system from Sulfolobus solfataricus KM1

An amylolytic activity that converts soluble starch to α,α-trehalose (trehalose) was found in the cell homogenate of the hyperthermophilic, acidophilic archaeum Sulfolobus solfataricus KM1. Two enzymes, a glycosyltransferase and an α-amylase, which are essential for this activity, were purified to homogeneity. A glycosyltransferase catalyzed the conversion of maltooligosaccharides to glycosyltrehaloses and an α-amylase catalyzed the hydrolysis of glycosyltrehaloses to trehalose. The glycosyltransferase transferred an oligomer segment of maltooligosaccharide to the C1–OH position of glucose, located at the reducing end of the maltooligosaccharide, to produce a glycosyltrehalose having an α-1,1 linkage. The α-amylase hydrolyzed only the α-1,4 glucosidic linkage adjacent to the trehalose unit of the glycosyltrehaloses. Their activities were maximal at 70–80°C and 70–85°C, with high thermostability, respectively. The genes encoding for both enzymes were cloned and expressed in Escherichia coli. The regions highly conserved in α-amylase family exist in the amino acid sequences of these enzymes. A new process for trehalose production from starch was developed using the purified enzymes. The yield of trehalose from starch was 81.5% using these two enzymes. This review describes our efforts to reveal in detail the characters of these enzymes involved in practical trehalose production.     

The Role of Endogenous Ethylene in Carbohydrate Metabolism of Medicago sativa L. Somatic Embryos in Relation to Their Regenerative Ability

Effects of the ethylene biosynthesis inhibitors salicylic acid (SA) and aminoethoxyvinylglycine (AVG) on germination of Medicago sativa L. somatic embryos and their conversion to seedlings in relation to carbohydrate content and α-amylase activity were studied. Both SA, an inhibitor of ACC oxidase, and AVG, an inhibitor of ACC synthase, when present in the regeneration medium (0.1 and 1 μM) were found to drastically reduce the embryo germination rate. In addition, SA and AVG were found to almost completely or completely, respectively, arrest the process of embryo conversion to seedlings. The inhibitory effects of SA and AVG on germination and conversion may indicate that the processes required endogenous ethylene. AVG and SA clearly slowed down starch disappearance during the 48-h imbibition in the regeneration medium prior to radicle elongation, which was correlated with inhibition of the activity of α-amylase, an enzyme responsible for starch hydrolysis. It is probable that ethylene may activate α-amylase in the germinating alfalfa somatic embryos. In contrast, the disappearance of soluble sugars in the embryos in the presence of the inhibitors tested was accelerated. The disappearance of soluble sugars (to null or almost null) in embryos was faster in the presence of SA in the regeneration medium after 24 and 48 h compared to the disappearance rate with AVG present in the medium. Only glucose was present after a 48-h incubation in the regeneration medium in the presence of the two ethylene biosynthesis inhibitors, in contrast to the control embryos in which glucose was not detected.     

Viability of onion (Allium cega) seed as influenced by temperature during seed growth

The yield and performance of seeds from crops of winter-hardy, bolting-resistant onion grown at temperatures of 15–16, 18–19 and 22–23°C in 1979, 1980 and 1982 were compared. Yields of seed from crops grown at 22–23°C were lower than those from crops grown at lower temperatures but the seeds ripened between 11 and 32 days earlier. Seeds from crops grown at mean temperatures of above 18°C gave higher percentage germination when imbibed at 30°C than 20°C and they also gave higher percentage seedling emergence than those from crops grown at lower temperatures. Seedlings from seeds produced at mean temperatures above 18°C were heavier than those from seeds of a similar weight but produced at lower temperatures. None of these differences were associated with differences in seed weight, embryo weight or seed dormancy but were positively correlated with differences in seed N-concentration. The differences were also associated with the rate of imbibition of water as high germination, high N-content seeds had a slower rate of imbibition than low germination, low N-content seeds of the same weight.     

Characterization of α-amylase inhibitor from rice bean with inhibitory activity against midgut α-amylases from Spodoptera litura

The α-amylase inhibitor (α-AI) activity varied from 7.529 to 10.766 (IU/g) in 13 rice bean with different genotypes. BRS-2 exhibited the highest α-AI activity (55.3%). Rice bean α-AI was purified to homogeneity by 80% ammonium sulfate precipitation, dialysis, ion exchange chromatography on DEAE-Sepharose and gel filtration through Superdex-75. Its homogeneity was confirmed by SDS-PAGE under reducing conditions showing a single band protein of molecular weight 25 kDa. The inhibitor was purified to 75.9 fold with final yield of 28.0% with specific activity of 660.2 IU. Inhibition studies carried out at pH from 2.2 to 9.0 revealed pH optimum at pH 6.9 (69.3%). The maximum α-AI activity was found at 37°C (68.8 %) and the lowest was revealed at 100°C (37.0%). Optimum inhibitory activity was expressed during pre-incubation of enzyme with inhibitor at pH 6.9 and 37°C. Isoelectric focusing of purified inhibitor showed a single band near pH 4.7. The first 6 amino acids in the N-terminus were recorded as Ala-Ser-Ser-Arg-Phe-Cys (ASSRFC). The purified inhibitor inhibited the α-amylase from the larval midgut of Spodoptera litura up to 86.6%. The α-amylase inhibitors are important seed storage proteins because of their potentiality for exploitation in pest control and crop defense against insect infestation. Their expression at high levels can confer resistance in transgenic legumes, which could be exploited for crop improvement.     

Assay of α-amylase in soil and river sediments: Its use to determine the effects of heavy metals on starch degradation

α-Amylase (1,4-α-d-glucan glucanohydrolase EC 3.2.1.1) activity was assayed and characterized in soil and in starch amended river sediment. Zero order reactions between initial activity; length of incubation; soil or sediment weight; and substrate concentration were demonstrated. Optimum pH for α-amylase activity in soil was 5.0 and in sediment 5.9–6.2, while the temperature optima were 37°C and 45°C respectively. The addition of starch to samples of sediment led to a marked increase in α-amylase and numbers of amylolytic bacteria. An example is also given of how the assay can be used to determine the effects of heavy metals on α-amylase activity in a river sediment.     

Saccharification of cassava (manihot esculenta) with millet amylase

Millet amylase formed during malting was used for teh saccharification of cassava (Manihot esculenta) into glucose syrup. The resulting syrup was also fermented to ethanol and citric acid. The maximum α-amylase activity occurred within 30 hours of millet germination at an optimum pH 8.0 and 40°C. The T. reesei enzymes enhanced the rate of cassava hydrolysis and paper chromatography revealed the presence of maltose, glucose and galactose in the cassava hydrolysate. The low cost of millet coupled with the fact that it is readily available makes it an economic source of amylase.     

Digestive amylase and pectinase activity in the larvae of alfalfa weevil Hypera postica (Coleoptera: Curculionidae)

The alfalfa weevil Hypera postica is a serious economic pest in most alfalfa grown in many countries worldwide. Digestive α-amylase and pectinase activities of larvae were investigated using general substrates. Midgut extracts from larvae showed an optimum activity for α-amylase against starch at acidic pH (pH 5.0). α-Amylase from larval midgut was more stable at mildly acidic pH (pH 5–6) than highly acidic and alkaline pH. The enzyme showed its maximum activity at 35°C. α-Amylase activity was significantly decreased in the presence of Ca²⁺, Mg²⁺ and sodium dodecylsulfate. On the contrary, K⁺ and Na⁺ did not significantly affect the enzyme activity. Zymogram analysis revealed the presence of one band of α-amylase activity in in-gel assays. Pectinase activity was assayed using agarose plate and colorimetric assays. Optimal pH for pectinase activity in the larval midgut was determined to be pH 5.0. Pectinase enzyme is more stable at pH 4.0–7.0 than highly acidic and alkaline pH. However, the enzyme was more stable at slightly acidic pH (pH 6.0) when incubation time increased. Maximum activity for the enzyme incubated at different temperatures was observed to be 40°C. Optimum pH activity for α-amylase and pectinase is not completely consistent with the pH prevailing in the larval midgut. This is the first report of the presence of pectinase activity in H. postica.