Both β-mercaptoethanol and DTT enhanced α- amylase activity up to 3.0 and 3.8 fold, respectively (Fig 4 & 5); because thiol compounds such as β- mercaptoethanol and DTT caused stimulatory effect on amylase activity indicating that cysteine residue (s) do not take part in catalysis. In addition, this activation is attributed to the reduction in aggregate size by destroying the intermolecular disulfide linkages and protection of thiol groups that stabilize the three dimensional structure of enzyme 16 (Khedher, et al, 2008).
The relationship between maltose concentration and absorbance at 540 nm was determined by reacting various concentrations of maltose with DNS reagent under the conditions of the assay and recording the absorbance at 540 nm. The maltose standard curve shown in figure 4.2 was used to calculate amylase activities based on observed absorbance. A unit of amylase activity is defined as the amount that liberated 1 μmol of reducing sugar per minute under the conditions of the assay. A unit of amylase inhibitory activity is defined as the amount that prevented the liberation of 1 μmol of reducing sugar per minute under the conditions of the assay. Specific activity was calculated by dividing the units of activity by the amount of protein (mg) in the sample.
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activity assay was performed by measuring the amount of reducing sugar released from soluble starch. The amount of reducing sugar was determined using the 3,5-dinitro- salicylic acid (DNS) method . Briefly, a 2.0 mL assay mixture containing 0.1 mL of crude enzyme extract and 1.0 mL 1% soluble starch in 20 mM sodium phosphate buffer (0.9 mL with pH 6.0) was incubated at 70 °C for 5 min, at which point 3 mL of DNS solution was added to terminate the reaction. This reaction mixture was boiled for 7 min; then, 10 mL of deionized water was added, and the absorbance of the mixture at 540 nm was measured. One unit of α-amylase activity was defined as the amount of enzyme that released 1 μmol of reducing sugar per min from soluble starch under the assay conditions described above. Purification of the wild-type (AmyS) and mutant (AmySA) enzymes was performed according to the method described by Li et al. . The optimal tempera- tures of AmyS and AmySA were determined by assay- ing protein samples at 60–90 °C in 20 mM phosphate buffer (pH 6.0). The optimal pH of AmyS and AmySA were determined by measuring the enzyme activity at 70 °C in 20 mM citrate buffers (pH 4.5–6.0) and 20 mM phosphate buffers (pH 5.5–7.5), respectively. The enzyme activities at different temperatures and pH values were expressed as a percentage of the highest activity.
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Thermal stability is a desired characteristic of most of the industrial enzymes. The highest amylase activity was found at 37 0 C (383±3 U/ml for 3hrs) and the lowest at 60 0 C (37±1 U/ml for 10hrs). The highest stability of amylase was found at 4 0 C (307±7 U/ml for 9 hours) and the lowest was observed at 60 0 C (37±1 U/ml for 10hours). Alpha amylase has many applications in preparation of Bakery products, Chapatti preparation, Ethyl alcohol dual fermentation, Treatment of Sago and Rice effluent, Sewage water treatment, fodder production, Desizing in Textile industry, Glucose Industry, Chocolate Syrup industry, Building product industry, Unmalted cereal liquefaction industry, Manufacture of maltose, Manufacture of high fructose containing syrups, Manufacture of high molecular weight branched dextrin etc.
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Activity staining of amylase was done according to the method of Scandalios . The gel consisted of 1% agar in 0.4 M phosphate buffer of pH 7.5. The plant extracts (1 mg/mL) that were pre incubated with the enzyme were loaded in to different wells. Untreated enzyme served as a positive control in a separate well. The buffer used in the gel was also used in the electrode compartments. A stabilized current of 100 V was passed through the gel for 2 h at 4°C. For visualization of the amylase bands the tray was immersed in 0.5% soluble starch and incubated at 37°C for 30 min. The excess starch was then washed and the gel was flooded with iodide potassium iodide solution for 1 min. Colourless bands against a deep blue background indicated amylase activity.
The phytotoxic effect of mercuric chloride on seed germination and seedling growth of maize (var. Jaunpuri) was studied by seed soaking treatment with different concentrations of mercuric chloride (0 to 3.0 mM). Different physico-chemical parameters were studied, viz., seed germination, length of plumule and radicle, absolute water uptake (AWU%), relative water uptake (RWU%), amylase activity (endosperm) and proline content (seedling). Increase in concentration of mercuric chloride from 0.5 to 3.0 mM decreased seed germination significantly when compared with control. Similar trend was noticed in other parameters like radicle and plumule length, AWC%, RWU% and amylase activity. However, seedling proline content increased with increase in HgCl 2 concentration. In another experiment seed treatment for 48 h with mercuric chloride (0.5 to 1 mM), inhibited the no. of leaves, root length, shoot length, no. of adventitious root, leaf area, fresh weight of roots and shoot, chlorophyll and nitrogen contents and nitrate reductase activity while superoxide dismutase activity as well as membrane injury enhanced in 8 and 12 days old seedlings, respectively. However, values of no. of leaves, root length, shoot length and fresh weight of roots were not significant. Similarly, maize leaves collected from 12 days old control seedling when floated over mercuric chloride solution (0.5 to 1 mM) for 24 h a constant decline was noted in nitrate reductase activity whereas accelerations were recorded in superoxide dismutase activity and membrane injury. The finding suggested that the mercury has direct impact on membrane structure of the tissue and on the enzymatic activity of the plant system.
(De Coen and Janssen, 1997). Methanolic extract of Artemisia annua L., a weed collected around paddy fields in north of Iran, was investigated for its toxic effects on: feeding, growth, fecundity, fertility including the biochemical characteristics of elm leaf beetle Xanthogaleruca luteola, Mull. Twenty-four hours after treating 3rd instar larva with the extract the level ofα-amylasesignificantly changed.However, at 48 h the extract lost its potency (Shekari et al., 2008). Different other studieshave been done on the effects of pesticides on α- amylase activity on different groups of animals but studies on the effect of herbicides on the amylase activity of earthworms has not been reported so far.
were then separately presoaked in the two concentration grade leaf leachates and for control. Data on seed germination percentage, DNA and RNA levels, and activity of amylase enzyme in seeds were tested. Chlorophyll, DNA and RNA contents, as well as amylase activity, were recorded from 10 uniformly growing 30 days old plants raised from each leaf leachate treated seeds. The plants were grown in Vidyasagar University research field for these analyses. The percentage of seed germination can be analyzed from continuous treatment sets. Three groups of 100 fresh seeds (i.e., 300 fresh seeds) were transferred to separate Petri dishes containing filter paper moistened with
α -amylase production by Brevibacillus carrying the α -amylase-pHis1522 induced with 0,5% of xylose was compared to those obtained with the α -amylase-pNI vector. The influence of temperature on protein expression was assessed for all clones by western blot analysis and by measuring the amylase activity after 48 and 120 h of induction. Both α -amylase-pNI and α -amylase-pHis1522 Brevibacillus clones showed a maximum of total protein production at 25°C after about 120 h of culture (60 U/mL and 123 U/mL respectively) (Figure 2) while at 37°C and 30°C the performance of both inducible and non indu- cible vectors were reduced. Interestingly the expression in pHis1522 vector was more than 2 fold higher com- pared to those achievable with the GFP-pNI vector (Figure 2). No significant basal expression of α -amylase was detected growing Brevibacillus carrying GFP-pHis1522 for 120 h without induction, confirming that protein expression is repressed in absence of xylose (Figure 2A and 2B).
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gut no studies have been undertaken. If digestion of food in the aphid gut happens, then the aphids, like the other species of chewing insects, are prone to enzyme inhibitors. Using enzyme inhibitors to control insect pest has already been demonstrated to be an important system for the insect pest con- trol since these inhibitors have detrimental effects on the insect growth and development by interfer- ing in food digestion (Confalonieri et al. 1998). α-Amylase activity has been described from different species of several insect orders including Orthoptera, Coleoptera, Hymenoptera, Diptera, Lepidoptera, and Hemiptera (Baker & Woo 1985; Terra et al. 1988; Mendiola-Olaya et al. 2000; Zeng & Cohen 2000; Oliveira-Neto et al. 2003; Kazzazi et al. 2005; Safaei-Khorram et al. 2010; Darvishza- deh & Bandani 2012). α-Amylases are important enzymes involved in carbohydrate metabolism in insects, thus α-amylase inhibitors should be used in the control of agricultural pest. For example pea and azuki transgenic plants expressing α-amylase inhibitors from beans were completely resistant to Bruchus pisorum and Callosobrouchus chinensis which are two main pests of stored pulses (Svens- son et al. 1986; Morton et al. 2000; Carlini & Grossi-de-Sá 2002; Franco et al. 2002). Also, bioas- say results of a study using artificial diet showed that protease inhibitors in pea aphid (A. pisum), cotton aphid (A. gossypii), and peach potato aphid (Myzus persicae) (Rahbe et al. 2003; Ribeiro et al. 2006) can produce antimetabolic effects (detrimental effect on growth and development and reduced fecundity). The understanding of biochemistry and physiology of digestion is essential when developing methods of insect pest control using enzyme inhibitors. Thus, the aim of the current study was to extract α-amylase from the digestive system of two aphid species, A. fabae Scopoli and A. gossypii Glover, and determine its characteristics using a specific substrate for the enzyme. The knowledge thus achieved should lead to better understanding of digestive physiology of the two aphid species which could be used to devise new management strategies for their control.
The activities of total amylase, α-amylase, and α-amylase inhibitor in the albumin-globulin fractions of isogenic non transgenic control (CY 45) and ppt (phosphinothrichin) resistant transgenic spring wheat (triticum aestivum L.) lines (T106, T116, T117, T124, T128, T129) were studied in two subsequent years. The plants were either sprayed with a selective herbicide Granstar (G), a wide range herbicide Finale 14 SL (F), or were grown without spraying (Q). Samples were obtained from field trial experiments of the Cereal Research Non Profit Co (Szeged, Hungary). Our results showed an increased trend in total amylase activity of untreated transgenic wheat lines in comparison with non transgenic wheat. The herbicide treat- ments enhanced the total amylase activity in both transgenic and non transgenic wheat samples. The changes in α-amylase inhibitor activity showed the same trend as that observed in total amylase activity in transgenic lines.
Previous studies concerning other plants from Fabaceae family were screened for α-amylase activity and showed inhibitory activity. They been reported with α-amylase inhibitory activity, namely: Galega officinalis, Phaseolus vulgaris and Tamarindus indica which shown respectively 35, 45-75, 90% inhibition of α- amylase at concentration of 200 mg/mL (Sales et al., 2012). Extracts from O. angustissima are more potent inhibitors of α- amylase since they reached 77% at concentration of 3.3 mg/mL comparing to: Galega officinalis L. known for its antidiabetic property containing galegine (guanidine), as source of an oral antidiabetic drug, Metformine, acting by reduction of hepatic gluconeogenesis (Fabrican and Fransworth, 2001); while extract of Trigonella foenum- graecum have an IC 50 value of 1.92 mg/mL
An adapted α-amylase inhibition assay as described by Ali et al.  was utilised. The dried crude acetone extracts were re-dissolved in DMSO to a concentration of 20 mg/ ml and used for the α-amylase inhibition assay. Ice cold porcine pancreatic α -amylase solution (200 μ l) at 4 unit/ml (type VI) was pre-incubated with 40 μ l of crude acetone extracts and 160 μl of distilled water, and mixed in a screw-top plastic tube. The reaction was started by the addition of 400 μ l of potato starch (0.5%, w/v) in 20 mM phosphate buffer (pH 6.9) containing 6.7 mM sodium chloride, and thereafter incubated at 25°C for 3 min. Final concentrations in the incubation mixture were plant ex- tract (1 mg/ml), 0.25% (w/v) starch and 1 unit/ml enzyme. An aliquot of the mixture (200 μ l) was removed and placed into a separate tube containing 100 μ l DNS colour reagent solution (96 mM 3, 5-dinitrosalicylic acid, 5.31 M sodium potassium tartrate in 2 M NaOH) and placed into an 85°C water bath. After 15 min, this mixture was removed from the water bath, cooled and diluted with 900 μl distilled water. α -Amylase activity was determined by measuring the remaining starch content by measuring the absorbance of the mixture at 540 nm. Control incubations, repre- senting 100% enzyme activity were conducted by replacing the plant extract with DMSO (40 μ l). For the blanks (nega- tive controls) the enzyme solution was replaced with dis- tilled water and the same procedure was carried out as above. Acarbose was used as positive control (acarbose 1 mg/ml: α -amylase 1 unit/ml). The α -amylase inhib- ition activity was expressed as;
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α-Amylase, a salivary or pancreatic enzyme plays an important role in early breakdown of starch into glucose and maltose. Modulation of α-amylase activity affects the utilization of carbohydrates as an energy source and stronger is this modulation, more significant is the re- duction in the breakdown of complex carbohydrates. Some of the N-substituted phthalimide derivatives of coumarins and 1-azacoumarins have been subjected to α-amylase enzyme inhibition activity. Retro synthetic analysis for the target compounds (Figure 2) indicates that crucial C-N bond forming step can be achieved using
Some compounds like p-purothionins may cause inactivation of a -amylase activity ..!!!. � by controlling the availability of calcium to serve as a co-factor (Jones and Meredith, 1982). p -purothionin purified from wheat flour was shown to inhibit wheat a -amylase in enzyme assays, but when calcium chloride was included in the enzyme-inhibitor mixture, a -amylase activity was not inhibited. Similarly Abdul Hussain (1987) reported that proteins from sprouting resistant genotypes inhibited a -amylase in standard assays, but adding EDTA to chelate calcium induced inhibitory activity in extracts of all genotypes. He concluded that proteinaceous a -amylase inhibitors interact with calcium ions, but do not play a primary role in the control of sprouting, although they may have secondary effects on the process. In barley however, it has been shown that the a-amylase inhibitor was an ABA induced protein and that it functions as an active mediator of a -amylase activity during seed develop ment and germination (Mundy, 1984). In wheat, King (1976) has provided correlative evidence for a possible control of a -amylase production by ABA during grain development (Sec. 2.4.3).
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Amylases are widely distributed and are one of the most studied enzymes. These enzymes have wide scale application ranging from food to effluent treatment. Amylases are a class of enzymes (hydrolases) that are capable of digesting the glycosidic linkages found in starch or glycogen. Under aqueous conditions amylases act on glycosidic bonds present in starch. Starch degrading enzymes like amylase have received a great deal of attention because of their perceived technological significance and economic benefits. This enzyme is also useful for the commercial production of glucose. Nowadays, the renewed interest in the exploration of extracellular amylase production in bacteria and fungi is due to various industrial applications. Few attempts have been made to elucidate the control mechanism involved in the formation and secretion of the extracellular enzymes. The production of alpha amylase by moulds has been greatly reported. In present work Bacillus amyloliquefacienswas found as an effective enzymes producer though submerged fermentation process. In the present study the highest amount of amylase production was observed in lactose supplemented medium and conservation was that the production of amylase was stimulated by the presence of glucose, lactose and starch by Bacillus sp. Temperature is one of the important factors, which strongly affect the submerged fermentation (Vidyalakhsmiet al., 2009). Variation of the temperature brought about a change in metabolic pattern of the micro-organism; it exhibited its best amylase production in the mesophilic range (Vidyalakhsmiet al., 2009).
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Porcine pancreatic lipase (PPL, type II) activity was measured using p-nitrophenyl butyrate (p-NPB) as a substrate. The method used for measuring the pancreatic lipase activity was modified from that previously described by Kim et al., 2010. PPL stock solutions (1 mg/mL) were prepared in a 0.1 mM potassium phosphate buffer (pH 6.0) and the solutions were stored at −20 °C. To determine the lipase inhibitory activity, the extracts (final concentrations 100, 50, 25, 10, 5, 2.5 μg/mL) or Orlistat (at same concentrations) as a positive control were pre-incubated with PPL for 1 h in a potassium phosphate buffer (0.1 mM, pH 7.2, 0.1% Tween 80) at 30 °C before assaying the PPL activity. The reaction was then started by adding 0.1 μL NPB as a substrate, all in a final volume of 100 μL. After incubation at 30 °C for 5 min, the amount of p-nitrophenol released in the reaction was measured at 405 nm using a UV-Visible spectrophotometer (BioTek Synergy HT, Winooski, VT, USA). The activity of the negative control was also examined with and without an inhibitor. The inhibitory activity (I) was calculated according to the following formula:
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Patients of type-2 diabetes mellitus have high postprandial blood glucose level. One of the therapeutic approaches, therefore, is to reduce postprandial hyperglycemia. This can be achieved by inhibiting carbohydrate splitting enzymes. One such enzyme is alpha amylase which hydrolyses complex carbohydrates of food to free sugars. Inhibition in alpha amylase activity reduces hydrolysis of complex carbohydrate thereby postprandial hyperglycemia may be kept under control. Acarbose, one alpha amylase inhibitor, has already been included in the list of drugs of type - 2 diabetes mellitus. Still there is continuous search for alpha amylase inhibitors from different sources which even extended to the field of medicinal plants. Recently we have shown that ethanol extract of Murrya koenigii Linn. Spreng Wettst (M. koenigii L.) leaves has maximum in vitro alpha amylase inhibitory activity. Aim of the present work, therefore, was to isolate alpha amylase inhibitor from M. koenigii L. leaves. M. koenigii L. was collected from the local market and identified by the taxonomist. Ethanol extract of the plant leaves was processed for isolation work by standard methods. Solvent extraction and acid hydrolysis were done followed by solvent treatment and chromatographic experiments. Finally a compound was crystallized. In vitro alpha amylase inhibitory activity of the isolated compound was checked by standard method. Acarbose, an alpha amylase inhibitor, was used as control. Results showed that the isolated compound had strong alpha amylase inhibitory activity which was comparable to that of acarbose. The isolated compound may, therefore, be used in the management of diabetes.
Investigations on actinomycetes are profoundly significant area of research since they form major resource for bioactive compounds, antimicrobials, an- ticancer agents, immunosuppressants, and biological control agents. A total of 59 actinomycetes were isolated from the soil sample collected from Do- mang, Lachung, North Sikkim, Sikkim, India. Out of the total isolates, 26 iso- lates with unique and distinct characteristic features were selected and ana- lysed for antimicrobial activity as well as extracellular enzyme production. Out of 26 isolates, 17 (66%) isolates exhibited different level of growth inhibi- tion against the test microorganism. 12 (47%) isolates showed antifungal ac- tivity and six (23%) isolates showed antibacterial activity. Most of the isolates showed antifungal activity. Isolate RCS260 was found to exhibit maximum growth inhibition (60%) against Colletotrichum gloeosporioides MTCC 8021. Isolate RCS252 showed maximum growth inhibition (67%) against Bacillus subtilis MTCC 441. Out of 26 isolates, 14 (54%) isolates exhibited chitinase activity, 25 (96%) isolates showed cellulase production, 20 (77%) isolates produced amylase enzyme and 17 (65%) isolates showed positive for protease activity. Potential isolate RCS260 has been characterized and identified as Streptomyces vinaceus strain RCS260 while isolate RCS252 was identified as Kitasatospora aburavienis strain RCS252. The antagonistic profile of strain RCS260 highlights its potential as antifungal agent against phytopathogens.
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Aim of the present was to evaluate α- amylase inhibitory potential of marketed polyherbal formulation which consists of nineteen herbs namely Acacia arabica Wild. (Leguminosae), Asphaltum, Bombax ceiba Linn. (Bombacaceae), Butea monosperma Wild. (Fabaceae), Emblica officinalis Gaertn. (Euphorbiaceace), Eugenia jambolana Lam. (Myrtaceae), Ficus bengalensis Linn. (Moraceae), Gymnema sylvestre Retz. (Ascepidaceae), Holarrhena antidysentrica Wall. (Apocynaceae), Momordica charantia Linn. (Cucurbitaceae), Pistacia integerrima Stew. ex Brand (Anacardiaceae), Plumbago zeylanica Linn. (Plumbaginaceae), Pongamia glabra Linn. (Fabaceae), Pterocarpus marsupium Roxb. (Leguminosae), Santalum album Linn. (Santalaceae), Swertia chirata Buch Ham. (Gentinaceae), Terminalia chebula Retz. (Combretaceae), Tribulus terrestris Linn. (Zygophyllaceae) and Woodfordia fruticosa Kurz. (Lythraceae).