Increased Α-amylase Activity Research Articles

Improved thermostable α-amylase activity of Bacillus amyloliquefaciens by low-energy ion implantation

Thermostable α-amylase is of great importance in the starch fermentation industry; it is extensively used in the manufacture of beverages, baby foods, medicines, and pharmaceuticals. Bacillus amyloliquefaciens produces thermostable α-amylase; however, production of thermostable α-amylase is limited. Ion-beam implantation is an effective method for mutation breeding in microbes. We conducted ion-beam implantation experiments using two different ions, Ar(+) and N(+), to determine the survival rate of and dose effect on a high α-amylase activity strain of B. amyloliquefaciens that had been isolated from soil samples. N(+) implantation resulted in a higher survival rate than Ar(+) implantation. The optimum implantation dose was 2.08 × 10(15) ions/cm(2). Under this implantation condition, we obtained a thermally and genetically stable mutant α-amylase strain (RL-1) with high enzyme activity for degrading α-amylase. Compared to the parental strain (RL), the RL-1 strain had a 57.1% increase in α-amylase activity. We conclude that ion implantation in B. amyloliquefaciens can produce strains with increased production of thermostable α-amylase.

Efeito da espermina na qualidade fisiológica de sementes de alface

O objetivo nesse estudo foi investigar o desempenho dos reguladores do crescimento: ácido giberélico, espermina e ácido abscísico na germinação e no crescimento inicial de plântulas de alface cv. Grand Rapids. Para isso, as sementes foram tratadas com soluções de ácido giberélico, espermina, e ácido abscísico na concentração de 0,5 mM; 0,05 mM e 0,04 mM respectivamente e avaliadas por meio dos testes de germinação, primeira contagem da germinação, emergência de plântulas, índice de velocidade de emergência de plântulas, comprimento de parte aérea e raiz das plântulas, massas fresca e seca total de plântulas, teste de condutividade elétrica e atividade total da enzima da α- amilase. A espermina aplicada isoladamente embora tenha induzido aumento da atividade da enzima α-amilase teve pouco efeito na indução da germinação de sementes de alface, sendo que reduziu de maneira geral a viabilidade e vigor das sementes. No entanto, quando foi combinada com o ácido giberélico ou abscísico induziu o crescimento da parte aérea superando quando presente a ação inibitória do ácido abscísico. O ácido abscísico apresenta efeito positivo na reorganização de membranas. Com base nos resultados, pode-se inferir que os fitorreguladores não afetam a viabilidade das sementes e que sementes tratadas com espermina apresentam pior desempenho no vigor.

Long-Term Storage-Dependent Changes in Seed Physiological Aspects of Hyoscyamus Niger from a Cold Desert Region of Trans-Himalaya

Henbane (Hyoscyamus niger L.), an economically important medicinal plant, has an endangered status in Himachal Pradesh (India) that needs appropriate conservation interventions. We have examined seed physiological aspects of H. niger from Lahaul (Himachal Pradesh, India), a cold desert region. The freshly harvested seeds exhibited complete dormancy. Gibberellic acid (GA3) and chilling treatment strongly promoted seed germination which was accompanied by increased α-amylase activity. KNO3, NaN3 and sodium nitroprusside (SNP), an NO donor, also promoted germination. During storage, the seeds retained high viability even after a storage of 72 months under ambient conditions. However, they remained dormant during the entire storage period. The responsiveness of seeds to GA3 and chilling treatment gradually declined with progression of storage period. Concomitantly, the triphenyl tetrazolium chloride (TTC) reduction ability of seeds was lowered. The seed responsiveness to KNO3, NaN3 and SNP during storage increased until one year and decreased thereafter. With the progression of the storage period, seeds exhibited elevated lipid peroxidation and reduced catalase activity implying a role of oxidative stress in observed changes. The involvement of phenolics in seed dormancy of H. niger was not evident. The findings are of significance for conservation and cultivation of H. niger through seeds in the arid mountain region.

Mutagenesis and genetic characterisation of amylolytic Aspergillus niger

Aspergillus niger FCBP-198 was genetically modified for its ability to reveal extra cellular α-amylase enzyme activity. From 76 efficient mutants isolated after ultraviolet (UV) irradiation, An-UV-5.6 was selected as the most efficient UV mutant, with 76.41 units mL−1 of α-amylase activity compared to wild (34.45 units mL−1). In case of ethyl methane sulphonate (EMS), among 242 survivors, 74 were assayed quantitatively and An-Ch-4.7 was found to be the most competent, as it exhibited a three-fold increase in α-amylase activity (89.38 units mL−1) than the parental strain. Genetic relationships of the mutants of A. niger FCBP-198 were analysed with a randomly amplified polymorphic DNA–polymerase chain reaction (RAPD–PCR). Results obtained from the comparison between genotypes of A. niger FCBP-198 showed differences in the sizes and numbers of amplified fragments per primer for each isolate. The dendrogram showed that genotypes An-Ch-4.7 and An-Ch-4.2 were distinctly classified into one category, while the isolates An-UV-5.6, An-UV-5.1 and A. niger FCBP-198 have the nearest genetic relationship. The five isolates from A. niger FCBP-198 genotypes shared an average of 65% bands.

Properties of field-sprouted sorghum and its performance in ethanol production

The objective of this research was to investigate physicochemical and biochemical characteristics of field-sprouted grain sorghum and its fermentation performance in ethanol production. Five field-sprouted grain sorghum varieties, which received abnormally high rainfall during harvest, were used in this study. Enzyme activities, microstructure, flour pasting properties, kernel hardness, kernel weight, kernel size, flour size and particle distribution of field-sprouted grain sorghum were analyzed. The effect of germination (i.e., sprouting) on conversion of grain sorghum to ethanol was determined by using a laboratory dry-grind ethanol fermentation procedure. Sprouted sorghum had increased α-amylase activity; degraded starch granules and endosperm cell walls; decreased kernel hardness, kernel weight, kernel size, and particle size; and decreased pasting temperature and peak and final viscosities compared with non-sprouted grain sorghum. The major finding is that the time required for sprouted sorghum to complete fermentation was only about half that of non-sprouted sorghum. Also, ethanol yield from sprouted sorghum was higher (416–423 L/ton) than that from non-sprouted sorghum (409 L/ton) on a 14% moisture basis.

Germination, α-, β-Amylase and Total Dehydrogenase Activities of Amaranthus Caudatus Seeds Under Water Stress in the Presence of Ethephon or Gibberellin A3

Amaranthus caudatus L. seed germination was studied under different levels of water deficit induced by PEG 6000 in laboratory conditions. PEG at osmotic potentials -0.2 to -0.3 MPa at 24°C in darkness delayed germination and reduced final germination percentage. PEG solutions at osmotic potential lower than -0.3 MPa almost totally blocked seed germination. Ethephon was much more effective than GA₃ in reversing PEG-caused inhibition of A. caudatus seed germination. PEG decreased α-amylase activity after 14 h incubation. It decreased β-amylase activity after 14 and 20 h, and caused an increase in total dehydrogenase activity only after 20 h of incubation. Unlike GA₃, ethephon increased α-amylase activity in seeds after 12 and 14 h of incubation under water deficit. After 20 h of incubation there was no difference in α-amylase activity in any of the treatments. Neither ethephon nor GA₃ affected the activity of β-amylase and dehydrogenase.

Variation in Endogenous Gibberellins, Abscisic Acid, and Carbohydrate Content During the Growth Cycle of Colored Zantedeschia spp., a Tuberous Geophyte

Phenologic changes and variation in the level of endogenous gibberellins (GAs), abscisic acid (ABA), carbohydrate content, and α-amylase activity were examined in colored Zantedeschia spp. cv. Cala Gold. These changes were examined in the primary bud tissues and in the attached tuber tissue during the growth cycle. Dormant tubers were dry-stored at 20°C for 3 months, planted in a phytotron, and grown under 22/16 ± 1°C. Plant development was monitored under continued irrigation until leaf senescence and tuber dormancy. GAs and ABA were extracted from the primary bud tissues, fractionated by HPLC, and analyzed using GC-SIM. Starch, glucose, soluble protein, and α-amylase activity were monitored in the tuber tissue attached to the primary bud. Endogenous changes in GAs and ABA in the primary bud were correlated with endogenous changes in carbohydrate content and α-amylase activity in the attached tuber tissue. These correlations were observed during the rest and the growth periods and were associated with developmental changes in the plant, that is, bud dormancy relaxation, bud growth, and inflorescence differentiation. ABA content decreased and a transient pulse of GA was measured in the primary bud concomitantly with the onset of shoot elongation in dry tubers during storage, before planting. The sharp increase of GAs in the bud preceded inflorescence differentiation as observed in dissected apices by about 15 days, as well as the increase in α-amylase activity in the attached tuber tissue. A steep decrease in starch level was measured in the tuber after planting, concomitantly with massive plant growth. These findings suggest a possible involvement of gibberellin in the initiation of α-amylase activity during dormancy relaxation in colored Zantedeschia and in the autonomous induction of flowering.

Expression and functional analysis of β-adrenoceptor subtypes in rabbit submandibular gland

β-Adrenoceptors (β-ARs) mediate important physiological functions in salivary glands. Here we investigated the expression and function of β-AR subtypes in rabbit submandibular gland (SMG). Both β 1- and β 2-ARs, but not β 3-AR, were strongly expressed in rabbit SMG. β 1-AR proteins were widely expressed in acinar and ductal cells whereas β 2-AR proteins were mainly detected in ductal cells. A [ 3H]-dihydroalprenolol binding assay revealed that β-AR B max was 186 ± 11.9 fmol/mg protein and K d was 2.71 ± 0.23 nM. A competitive binding assay with CGP 20712A, a β 1-AR antagonist, indicated that the proportion of β 1-AR and β 2-AR was 71.9% and 28.1%, respectively. Gland perfusion with the β-AR agonist isoproterenol induced a significant increase in saliva secretion which was abolished by pretreatment with the non-selective β-AR antagonist propranolol. Pretreatment with β 1- or β 2-AR selective antagonists, CGP 20712A or ICI 118551, diminished isoproterenol-induced increase in saliva secretion by 71.2% and 28.8%, respectively. The expression of α-amylase mRNA was significantly stimulated by isoproterenol, which was eliminated by propranolol and CGP 20712A. Perfusion with isoproterenol decreased α-amylase protein storage in SMG and increased α-amylase activity in saliva. These alterations became less significant after pretreatment with propranolol and CGP 20712A. Our results suggest that both β 1- and β 2-ARs are expressed in rabbit SMG. β 1-AR is the predominant subtype and may play an important role in regulating saliva and α-amylase secretion.

α-Amylase and programmed cell death in aleurone of ripening wheat grains

Late maturity alpha-amylase (LMA) in wheat is a genetic defect that may result in the accumulation of unacceptable levels of high pI alpha-amylase in grain in the absence of germination or weather damage. During germination, gibberellin produced in the embryo triggers expression of alpha-Amy genes, the synthesis of alpha-amylase and, subsequently, cell death in the aleurone. LMA also involves the aleurone and whilst LMA appears to be independent of the embryo there is nevertheless some evidence that gibberellin is involved. The aim of this investigation was to determine whether the increase in alpha-amylase activity in LMA-prone genotypes, like alpha-amylase synthesis by aleurone cells in germinating or GA-challenged grains, is followed by aleurone cell death. Programmed cell death was seen in aleurone layers from developing, ripe and germinated grains using confocal microscopy and fluorescent probes specific for dead or living cells. Small pockets of dying cells were observed distributed at random throughout the aleurone of ripening LMA-affected grains and by harvest-ripeness these cells were clearly dead. The first appearance of dying cells, 35 d post-anthesis, coincided with the later part of the 'window of sensitivity' in grain development in LMA-prone wheat cultivars. No dead or dying cells were present in ripening or fully ripe grains of control cultivars. In germinating grains, dying cells were observed in the aleurone adjacent to the scutellum and, as germination progressed, the number of dead cells increased and the affected area extended further towards the distal end of the grain. Aside from the obvious differences in spatial distribution, dying cells in 20-24 h germinated grains were similar to dying cells in developing LMA-affected grains, consistent with previous measurements of alpha-amylase activity. The increase in high pI alpha-amylase activity in developing grains of LMA-prone cultivars, like alpha-amylase synthesis in germinating grains, is associated with cell death, providing further evidence for the involvement of gibberellin in the LMA response.

Effects of Chitosan and Lactic Acid on Enzymatic Activities and Bioactive Compounds during Germination of Black Rice

The effect of chitosan on enzymatic activities and on bioactive compounds was characterized during germination at 25℃ for 7 days to search for a method to produce a germinated black rice. The germination rate was reduced by the addition of lactate and chitosan. The rotting rate was greatly decreased by chitosan, suggesting that the addition of chitosan into a germination solution might be an effective method for controlling fungal contamination during the germination of cereals. The addition of 100 and 200 ppm chitosan increased α-amylase activity after 7 days by up to 152% and 197%, respectively. The activities of β-amylase and β-glucosidase were lower with 200 ppm chitosan than in distilled water and 100 ppm lactate. The amount of total soluble phenolics and total flavonoids decreased rapidly for four days and thereafter remained constant until the seventh day. The antioxidant activity of germinated black rice, in terms of hydrogen-donating activity, increased slowly and did not correspond to the changes of total soluble phenolics and total flavonoids. The amount of phytic acid was reduced by the addition of 200 ppm chitosan compared to distilled water, indicating that chitosan could be used as an elicitor for the increase of phytase activity during the germination of black rice.

Effect of Seed Hardening on the Seedling Emergence and .ALPHA.-amylase Activity in the Grains of Wheat and Rice Sown in Dry Soil.

Seed hardening, wetting and redrying of the seed before sowing, increases the rates of germination and seedling emergence of wheat and rice sown in dry soil. We examined the effect of the seed hardening on the water potential of seed, a driving force of water absorption from soil, and the activity of α-amylase, a key enzyme for soluble carbohydrate supply. In wheat variety Norin-61, the water and osmotic potentials at the time of sowing were -7.2 and -12.3 MPa, respectively, in the hardened seed, and was -4.8 and -9.9 MPa, respectively, in non-hardened seed. However, in rice variety Nipponbare, neither water nor osmotic potential was clearly affected by seed hardening. In hardened seeds, the activity of α-amylase at 12 hours after sowing in dry soil was 2.7 and 2.8 times as high as that in non-hardened seeds in both Norin-61 and Nipponbare, respectively. In several other Japanese varieties and Korean native varieties also, seedling emergence rate in dry soil and α-amylase activity before sowing were increased by hardening treatment, and α-amylase activity of hardened seedsbefore sowing was 1.9∼8.3 times as high as that of non-hardened seeds. It was assumed that the rates of germination and seedling emergence in dry soil were increased by seed hardening through the increase of soluble carbohydrate supply owing to increased α-amylase activity. However, hardening treatment did not decrease osmotic potential of rice seed. Redrying during the hardening treatment may have kept the activities of some enzymes in addition to α-amylase at the levels just before germination, and thus promoted germination.

Changes in enzyme activities and aflatoxin elaboration in sorghum genotypes followingAspergillus parasiticus infestation

Sorghum is a relatively poor substrate for aflatoxin production compared with high-risk agricultural commodities like maize and groundnut, even though it is susceptible to fungal attack. Fungal infestation of sorghum results in a varied biochemical composition of the deteriorated grain. In this study, six sorghum genotypes (red—AON 486, IS 620; yellow—LPJ, IS 17 779; white—SPV 86, SPV 462) were inoculated with a toxigenic strain of Aspergillus parasiticus (NRRL 2999) in order to evaluate the changes in the activities of various hydrolytic enzymes (α- and β-amylases, protease and lipase) in comparison with those in uninfected grains. Enzyme activities were measured at different times after fungal infestation, and the enzymatic activities were correlated with the aflatoxin production. Alpha-amylase activity was observed to be greater than β-amylase activity in all six genotypes under both healthy and infected conditions. The increase in α-amylase activity during the period of infection was higher in white genotypes than in red sorghum genotypes. Alpha-amylase activity in all the genotypes increased up to day 6 after fungal infection, but was significantly lower in infected grains than in healthy grains. The variability in the basal enzyme activities among the six sorghum genotypes was quite high compared with the amount of induction of each specific enzyme due to infection and germination. Higher protease activity was observed in the infected grains than in healthy grains. The enzyme activities in high tannin red genotypes were less than those in yellow and white genotypes. The α- and β-amylase activities were positively correlated (r = 0.406 and 0.436; P < 0.05) to aflatoxin production. Inherent lipase activity was highest (on day 0) in AON 486, SPV 462 and SPV 86, as compared with the activity in infected grains. The total aflatoxins produced (quantified by TLC-fluorodensitometry) were lower in red genotypes than in yellow and white genotypes, suggesting that red genotypes were least susceptible to aflatoxin elaboration among the various genotypes tested. All four aflatoxins, (B1, B2, G1 and G2) were present in five genotypes (IS 620, LPJ, IS 17 779, SPV 86 and SPV 462) at all the stages of infection, but, aflatoxin could not be detected in the red genotype AON 486 on day 3 after infection. White genotypes SPV 86 and SPV 462) showed maximal aflatoxin (total) production on day 6 after infection. © 2000 Society of Chemical Industry

Expression of α-amylase in cultured callus of french bean

α-Amylase (EC 3.2.1.1) expression was found in calli of French bean (Phaseolus vulgaris L. cv Goldstar). We examined enzyme activity in the calli to investigate influence of gibberellin and sugars on enzyme expression. After subculture of the calli, α-amylase activity decreased, and then increased at a stationary phase of callus growth. Exogenous application of gibberellin and an inhibitor of gibberellin synthesis, uniconazole, did not have any significant effects on the enzyme expression. Sugar starvation increased the activity, while addition of metabolizable sugars, such as sucrose, glucose and maltose, to the medium repressed expression. Addition of 6% mannitol, a non-metabolizable sugar, to the medium induced higher α-amylase expression as compared to addition of 3% mannitol. This result suggests that osmotic stress enhances α-amylase activity in the calli. Furthermore, high concentrations of agar in the medium increased α-amylase activity in the calli. It is probable that high concentrations of agar prevented incorporation of nutrient into the calli and induced the α-amylase activity in the calli.

Changes in α-amylase activity during plant regeneration from rice calli

Summary Changes in α-amylase activity in rice calli during organogenesis were investigated in 5 rice varieties that exhibit different abilities for plant regeneration. During organ differentiation in rice callus tissues, samples were taken at 5-day intervals up to 35 days and α-amylase (EC. 3.2.1.1) activities were measured. The activity in regenerative calli began to increase 15 to 20 days after transfer to the regeneration medium and were elevated 7–11 fold during the culture period, while the calli transferred to callus maintenance medium did not increase and maintained a stable state. The increase in α-amylase activity in regenerative calli was more rapid in the calli that showed higher regenerative abilities (Sasanishiki, Tadukan and Tetep) than in the calli that showed lower regenerative abilities (Fujisaka 5 and Nipponbare). Correlation coefficients between frequencies of organ differentiation and α-amylase activity in the calli 35 days after transfer to the regeneration medium was highly significant. Expression of a rice α-amylase gene ( RAmylA ) in callus tissues during organ differentiation was examined by Northern blot and Western blot analyses. Messenger RNA from the RAmylA gene, which is a main transcript in germinating rice seeds, was highly expressed in the regenerating calli 15 days after transfer. The translated product from the mRNA of the RAmylA gene and α-amylase isozyme band I (1A and 1B) were highly expressed 20 days after transfer, especially in the regeneration medium.

清酒醪の並行複発酵に及ぼすフィチン酸及びその関連化合物の影響

The influence of phytic acid and related compounds on the parallel fermentation in small scale sake brewing was investigated. It was revealed that the digestion of steamed rice was enhanced inithe sake brewing with the addition of phytic acid because of the release of adsorption of α-amylase onto rice protein, resulting in the increase of α-amylase activity in the liquid phase of moromi mash.On the other hand, it was found that α-amylase was inhibited by the degradation products of phytic acid which were cleaved by phytase and others in sake-koji.

Effects of Wounding, Ethylene and Abscisic Acid on the Development of α-Amylase Activity in Detached Cotyledons of Pisum sativum

Summary Alpha-amylase activity in detached cotyledons of Pisum sativum increased gradually during incubation with distilled water, in contrast to the absence of any increase in α-amylase activity in embryo-free halfgrains of barley during incubation. We examined whether the wounding caused by cutting away the embryonic axis influenced the development of α-amylase activity in the wounded cotyledonary tissue. The larger the amount of detached cotyledons that was removed with the axis, the greater was the increase in enzymatic activity during incubation with distilled water. The development of α-amylase activity was accelerated dramatically during incubation with auxins (Hirasawa, 1989). The development of activitiy in detached cotyledons of Pisum sativum during incubation with distilled water or with 2,4-dichlorophenoxyacetic acid was inhibited in the presence of ethylene or abscisic acid. Alpha-amylase activity in detached cotyledons of Phaseolus vulgaris and Azuki angularis increased during incubation with distilled water, but 2,4-dichlorophenoxyacetic acid, abscisic acid, and ethylene had no effect on the increase in the cotyledons of these Leguminosae. These results suggest that α-amylase activity in detached cotyledons during incubation with distilled water is induced by wounding caused by removal of the embryonic axis and/or by an endogenous auxin in the cotyledons.

Isolation and characterization of α-amylase derived from starchgrownClostridium acetobutylicum ATCC 824

An extracellular α-amylase was purified to homogeneity from the culture supernatant ofClostridium acetobutylicum ATCC 824 grown in synthetic medium containing starch by using a combination of ammonium sulfate fractionation, anion exchange chromatography and HPLC-gel filtration. The molecular weight of the 160-fold purified α-amylase was determined by SDS-PAGE to be 61 kDa. HPLC analysis of end-products of enzyme activity on various substrates indicated that the enzyme acted specifically in an endo-fashion on the α-1,4-glucosidic linkages. Enzyme activity was optimal over a pH range of 4.5–5.0 and temperature of 55°C, but was rapidly inactivated at higher temperatures. Addition of calcium chloride (2–5 mM) increased α-amylase activity by ca. 20%, while the addition of 19 μg ml−1 of acarbose (a differential inhibitor of amylases) resulted in 50% inhibition. TheV max andK m of α-amylase were 2.17 mg min−1 and 3.28 mg ml−1 on amylose, and 1.67 mg min−1 and 1.73 mg ml−1 on soluble starch, respectively.

Loss of Starch and Increase of α-Amylase Activity in Leaves of Flooded Tobacco Plants

Loss of Starch and Increase of α-Amylase Activity in Leaves of Flooded Tobacco Plants

Effect of gassing, agitation, substrate supplementation and dialysis on the growth of an extremely thermophilic archaeon Pyrococcus woesei

Growth of an extremely thermophilic archaeon, Pyrococcus woesei, at 90°C in a 2–1 fermentor was significantly enhanced by gassing with N2/CO2 (95%/5%). Both growth and α-amylase activity were also positively influenced by increasing the agitation speed up to 1200 rpm under continuous gassing at 0.2 vvm. However, increasing the agitation speed to 2400 rpm led to decreases in the maximum cell concentration and α-amylase activity. Fed-batch cultivation resulted in increases in the specific growth rate, maximum cell concentration and α-amylase activity. Although the latter two parameters were higher when the broth was supplemented with both starch and concentrated medium, the specific growth rate was relatively smaller. Cultivation in a dialysis reactor gave a cell concentration of 2 × 109 cells/ml, which represents a 2.8-fold increase over that obtained in ordinary batch cultivation. This increase in the cell concentration was accompanied by a 5.2-fold increase in α-amylase activity.

Mode and control of starch mobilization during germination of seeds of horse gram

Starch and total soluble carbohydrates declined while the reducing sugars increased in the cotyledons of horse gram ( Macrotyloma uniflorum (Lam.) cv. Co 1) during the 4-day period of germination. Starch-mobilizing enzymes of the amylolytic pathway namely, α-amylase, β-amylase, maltase and pullulanase were active, whereas starch phosphorylase declined from the beginning. Inhibition by cycloheximide was suggestive of the de novo synthesis of α-amylase. The increase in α-amylase activity, reducing sugars and starch degradation were at a lower rate upon axis removal. The effect of the axis on α-amylase development was not replaced by gibberellic acid or benzyladenine. The restricted development of α-amylase activity in the absence of the axis could be due to feedback inhibition by the total soluble carbohydrates, the absolute levels of which were higher in detached cotyledons. Lack of inhibition of α-amylase activity by glucose and maltose suggested that the non-reducing component(s) of the total soluble carbohydrates may play a significant role in this regulation.