amylase activity by dns method protocol

Slight changes in enzyme concentration, time duration prior to and during the incubation, and time in the water bath resulted in significant changes in the inhibition percentage. Dilute to a final volume of 100 ml with reagent grade water. 7). <>/StructTreeRoot 3 0 R/Pages 4 0 R/MarkInfo<>/Lang(en-US)/OCProperties<><><>]/ON[5 0 R 6 0 R]>>>>/ViewerPreferences<>/Type/Catalog>> Can you see this HHS Vulnerability Disclosure, Help Amylase is an enzyme responsible for breaking down carbohydrates, it is found in saliva and pancreatic juice, and it helps the body digest food. Home - Protocols - Quantitative Analysis - Assay of Salivary Amylase enzyme activity. Spectrochim. The graph of inhibition percentage vs. inhibition concentration was plotted and checked for linearity (visually). endobj The .gov means its official. Prepare starch solution (10 mg/ml) 2. Because the determination of sugars is performed by reducing the aldehyde group of the sugar with 3,5-dinitrosalicylic acid (DNS) to form 3-amino-5-nitrosalicylic acid, the reaction involves high temperatures, making the determination on microplates difficult.99 Miller, G. L.; Anal. To achieve a good assay, there must be good agreement between the in vivo and in vitro results for the evaluated substance. Basic Med. The absorbance (A) of the samples is measured at =540 nm. Method. The reaction was stopped by adding 200 L of DNS (final concentration 19.2 mmol L-1) and boiling in a water bath for 15.6 min. The final aspect of the microplate, absorbance spectra, and illustrative calibration curve of different soluble starch standards (from left to right, respectively) of the starch-iodine quantitative assay used for the characterization of alpha-amylase and amyloglucosidase activities. These inhibitors delay the rate of absorption of glucose, thereby maintaining glucose levels in hyperglycemic individuals. MTCC 5214 when grown in solid-state fermentation. For a blood test, blood is Because acarbose is structurally similar to the substrate (starch), it competes for the active site on the enzyme. These bonds can be easily broken by water, so saliva is necessary for the enzyme to work. 4 0 obj The Km value is the substrate concentration needed to reach half the maximum velocity. [5]. Mina Karimi-Avargani kindly mail me saidurga48@gmail.com. I need your help for calculation of data. Place the vial in the block heater (or other alternative heating source) previously set at the target temperature of the study during the desired time of the assay (, Immediately stop the hydrolysis process by adding 750L of 1.0molL, Prepare the soluble starch standards as described in, Transfer 50L of the mixture (sample or standard), 150L of water, and 50L KI/I, Place the microplate in the plate reader, shake the plate (for 30s using the shaker function of the microplate reader) and read the absorbance at 580nm, at room temperature. Ten different experiments were performed. Plant extracts contain various phytochemicals, such as phenols, saponins, and alkaloids, that contribute to many observed pharmacologically relevant effects. The test is done in microtitre plates with a total volume of 260L and an assay time of 40min including the pre-incubation steps. A catalyst is a substance that hastens a chemical reaction but does not become part of the end product. The error percentage was calculated to be 5.46%, which indicates that the method is accurate. Although the small volume (100L) used in the original formulation of the assay [5] is an interesting option to minimize reagent consumption and waste disposal, the high temperatures that these enzymes can be submitted may pose problems of volume control caused by evaporation, which may have negative impact in the precision and accuracy of the method. Accessibility In this context, we herein describe an adaptation of a method proposed by Xiao et al. Starch, acarbose, 3,5-dinitrosalicylic acid (DNS), sodium chloride, dibasic sodium phosphate, monobasic sodium phosphate, sodium, and potassium tartrate were procured from Sigma-Aldrich, USA. This protocol helps you to understand the applications of amylase enzyme. strains showed the amylolytic activity. Sci. For the proposed method, the minimum detectable amount of starch (LOD) was 0.4mg. An example of the application of this assay is illustrated by Fig. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Most enzymes act specifically with only one reactant, called a substrate, to produce products. This section illustrates an example of the application of the method that includes the corresponding calculations for both methods herein described. Thus, it is important to optimize these two parameters to obtain a fixed value. Bethesda, MD 20894, Web Policies Next, for the assay experiment, 500L of enzyme solution 2 was mixed in a vial that contained 1500L of a soluble starch solution 2.00gL1 and 1000L of universal buffer. Amylase is an enzyme made by your pancreas and salivary glands that helps your body break down carbohydrates. This method is a redox reaction where DNS (yellow color) is reduced by reducing sugars to 3-amino-5-nitrosalicylic acid (red color) in an alkaline medium. Five plant extracts were used to determine -amylase inhibitory activity using the proposed method. The results were subjected to regression analysis to determine R2 and the regression equation for the plot. Required fields are marked *. <>>> Soluble starch (PN: S9765), starch from corn (PN: S4126), starch from potato (PN: S4251), starch from rice (PN: S7260), starch from wheat (PN: S5127, unmodified), I2 (PN: 207772, 99.8%), 85% (w/w) o-phosphoric acid (PN: 79620), and acetic acid (PN: 695092, 99.7%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hi, Laneesha Moozhikkal This link can be helpful for the calculation of alpha amylase activity. http://www.microbiology.biology.upatras.gr/en/proto A microscale in vitro method for the assessment of -amylase inhibition activity was optimized, validated and used for the analysis of enzyme inhibition using plant extracts in vitro. The samples are placed in a water bath (T=100C) for 5 min and then they are left to cool at room temperature. Keywords:optimization of enzyme method; validation of enzyme method; -amylase activity; design of experiments. Therefore, the objectives of this work were: (i) the consistent preparation of some reagents, especially the soluble starch and KI / I2 solution that generates I3, to improve batch to batch reproducibility and maximize the precision and accuracy of the of the starch-iodine method; (ii) the rescale of the activity assay, in order to minimize evaporation issues typical from microplate assays and to adapt it to different experimental requirements (in our particular case this was related with the US-assisted hydrolysis of starch); (iii) the adjustment of sensitivity by modifying the volumes of the starch-iodine microplate assay, to avoid precipitation and to minimize potential deviations to the Lamberts-Beer law of the absorbance measurements; and (iv) the measurement of glucose yield for amyloglucosidase through the glucose oxidase / peroxidase (GOPOD) method replicating the sample handling protocol used for measuring its activity, but using pure starches from different botanical sources instead of soluble starch. Determination of amylose/amylopectin ratio of starches. Positive controls are compounds with proven activity in humans, whereas negative controls are those without activity. In all cases, a sample blank was analyzed to demonstrate that there were no matrix interferences caused by the extract. Effect of type 2 diabetes mellitus caveolin-3 K15N mutation on glycometabolism. Therefore, this method should be considered as a valuable tool in clinical practice. Toxicol. Acarbose, a positive control, was used as an inhibitor for this experiment. Acarbose was used as an enzyme inhibitor. Experiments included different concentrations of the enzyme (0.1 and 0.2 U mL-1), preincubation time (5.2 and 9.2 min), incubation time (4.5 and 6.5 min), and time in a water bath (10.6 and 20.6 min). Standard deviation was calculated, and the relative standard deviation in terms of percentage was determined (Equation 2) to check the precision values on the experimentation day and other different days. Due to the presence of free carbonyl groups in sugars, they can reduce DNS and are oxidized to carboxyl groups. Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; de Sales, P. M.; de Souza, P. M.; Dartora, M.; Resck, I. S.; Simeoni, L. A.; Fonseca-Bazzo, Y. M.; de Oliveira-Magalhes P.; Silveira, D.; Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; Rahimzadeh, M.; Jahanshahi, S.; Moein, S.; Moein, M. R.; Valentina, P.; Ilango, K.; Chander, S.; Murugesan, S.; Paiva, R. V. N.; Chaves, A. C. T. A.; de Paula, V. F.; de Macedo, G. E. L.; Queiroz, R. F.; Yilmazer-Musa, M.; Griffith, A. M.; Michels, A. J.; Schneider, E.; Frei, B.; Szymaski, P.; Markowicz, M.; Mikiciuk-Olasik, E.; Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; Granados-Guzmn, G.; Castro-Ros, R.; de Torres, N. W.; Salazar-Aranda, R.; Satpathy, G.; Tyagi, Y. K.; Gupta, R. K.; Hou, W.; Li, Y.; Zhang, Q.; Wei, X.; Peng, A.; Chen, L.; Wei, Y.; Subramanian, R.; Asmawi, M. Z; Sadikun, A.; Wu, T.; Zhou, X.; Deng, Y.; Jing, Q.; Li, M.; Yuan, L.; Ha, K. S.; Jo, S. H.; Kang, B. H.; Apostolidis, E.; Lee, M. S.; Jang, H. D.; Kwon, Y. I.; Wang, Y.; Huang, S.; Shao, S.; Qian, L.; Xu, P.; Vadivel, V.; Nandety, A.; y Biesalski, H. K.; Tundis, R.; Loizzo, M. R.; Statti, G. A.; Menichini, F.; Iversen, P.; Beck, B.; Chen, Y.; Dere, W.; Devanarayan, V.; Eastwood, B. J.; Farmen, M. W.; Iturria, S. J.; Montrose, C.; Moore, R. A.; Weidner, J. R.; Sittampalam, G. S. In. Salivary amylase is essential for the digestion of carbohydrates. When the substrate concentration is increased, the inhibition by the competitive inhibitor is reversed. Before [Crossref]-1616 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980. Int J System Evol Microbiol 62:24052409, Gong Y, Bai JL, Yang HT, Zhang WD, Xiong YW, Ding P, Qin S (2018) Phylogenetic diversity and investigation of plant growth-promoting traits of actinobacteria in coastal salt marsh plant rhizospheres from Jiangsu, China. 2012, 37, 520. 8600 Rockville Pike ASSESSMENT OF -AMYLASE INHIBITION ACTIVITY BY AN OPTIMIZED AND VALIDATED IN VITRO MICROSCALE METHOD. Absorbance ( a ) of the end product 5.46 %, which indicates that the method accurate! At room temperature are oxidized to carboxyl groups Protocols - Quantitative Analysis - assay of salivary amylase an! Protocols - Quantitative Analysis - assay of salivary amylase enzyme activity an assay time of 40min including the steps! Helps you to understand the applications of amylase enzyme activity for linearity ( visually ) ( ). Assay time of 40min including the pre-incubation steps by the extract you understand... Checked for linearity ( visually ) is essential for the proposed method reaction does... 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