amylase activity by dns method protocol

Comparison of such results will lead to low reliability of these in vitro methods. In this method, starch by amylase is converted into maltose. The kinetic constant maximum velocity (Vmax) and the Michaelis-Menten constant (Km) were obtained from the Lineweaver-Burk and Michaelis-Menten plots, respectively. What are the functions of salivary amylase? Oliveira H.M., Correia V.S., Segundo M.A., Fonseca A.J.M., Cabrita A.R.J. The intra-day variation was below 1.6%. In vivo studies of species of Zanthoxylum revealed that the phenolic content of the plant had a potential hypoglycemic effect.3131 Quan, N. V.; Anh, H.; Lam, V. Q.; Takami, A.; Teschke, R.; Khanh, T. D.; Xuan, T. D.; Molecules 2022, 27, 774. Since the platescreening method is qualitative due to the poor correlation between enzyme activity and colony to clear zone ratio (Maki et al., 2009), all the four isolates were subjected to quantitative assays for cellulase, xylanase, and amylase activity to choose an efficient enzyme producer out of four screened. Amylase is an enzyme, or special protein, that helps you digest carbohydrates. An inhibition percentage near 50% was chosen as the best response, while the absorbance of the control at 0% inhibition (100% activity) resulted in the smallest photometric error. <>/Font<>/ExtGState<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI]>>/Contents[19 0 R 20 0 R]/Group<>/Type/Page/Tabs/S>> Rafael Resende Maldonado . amylase, any member of a class of enzymes that catalyze the hydrolysis (splitting of a compound by addition of a water molecule) of starch into smaller carbohydrate molecules such as maltose (a molecule composed of two glucose molecules). Relative standard deviation (expressed in percentage, RSD%) measured the precision of the assay. Pharmacol. {y)a?:(J F([[y#? Cool and dilute with 10ml of distilled water. 2011, 76, H188. Rev. Springer Nature is developing a new tool to find and evaluate Protocols. 7 0 obj It is an important enzyme and has wide range of functions like decomposing starch molecules in the human body. strains showed the amylolytic activity. Thanks for all of you, I increased the temperature to 37 C and decrease the volume of enzyme against the substrate and sample volumes but what I c Epub 2015 Feb 16. The structure of salivary amylase is a long, spiral-shaped molecule. [Crossref] Additionally, it is necessary to validate the assay method to demonstrate that it is acceptable for determining the pharmacological activity of a new chemical entity by a detectable signal that allows the biological process to be quantified. These findings demonstrated that our optimized and validated method is highly selective, enabling the identification of samples with or without potent activity. <> The relative standard deviation and Z value were found to be <4% and >0.93, respectively. Salivary amylase is essential for the digestion of carbohydrates. [Crossref], 10 White, R. E.; Annu. The optimum substrate concentration for the -amylase inhibition assay was found to be 0.38 mg mL-1 (1.11 mmol L-1). This protocol helps you to understand the applications of amylase enzyme. government site. Copyright 2016 Elsevier Ltd. All rights reserved. https://doi.org/10.1007/978-1-0716-1728-1_74, Shipping restrictions may apply, check to see if you are impacted, Tax calculation will be finalised during checkout. In this case, the control experiment was used to monitor any glucose formation unrelated to the enzyme activity. The solution for bypassing the precipitation of the starch-iodine complex was diluting the starch standard/sample with 150L of ultra-pure water followed by the addition of 50L of KI / I2 solution (triiodide) reagent (amylolytic enzyme activity assay, step 7). Glucose oxidase / peroxidase (GOPOD) (K-GLUC) assay kit was purchased from Megazyme (Wicklow, Ireland). For the starch-iodine method, absorbance measurements were conducted at 580nm, whereas for the GOPOD assay, the wavelength was set at 505nm. Achillea fragrantissima Forssk. This indicates that the plant extracts contain a strong inhibitor of the amylase enzyme. Would you like email updates of new search results? Selected plant products could provide better alternatives due to their low cost and safer use. Sci. Production of Actinobacteria Amylase by Fermentation in Solid State Using Residues of Licuri Palm (Syagrus coronata). Liu F, Zhu S, Ni L, Huang L, Wang K, Zhou Y. Endocrine. Amylase is the hydrolytic enzyme that breaks down many polysaccharides like Starch, Amylose, dextrins, and yields a disaccharide i.e., Maltose. 2021 May 1;343:128423. doi: 10.1016/j.foodchem.2020.128423. For measuring the amylase activity with DNS solution (Miller Method): 1. Prepare starch solution (10 mg/ml) 2. Prepare enzyme extract filtered with The error percentage was calculated to be 5.46%, which indicates that the method is accurate. The degree of inhibition was evaluated in terms of the concentration of the inhibitor that inhibited half of the enzyme activity (IC50). 1% Starch. Hence, it is absolutely necessary to optimize an assay method to achieve acceptable performance, with low variability and high signal with respect to noise.1313 Macarrn, R.; Hertzberg. [Crossref] In this study, the values of the Z factor were 0.93 and 0.96. Crops Prod. This work presents a protocol for the production of amylases by actinobacteria; amylases are important enzymes for different industrial sectors because they are able to perform in the hydrolysis of starch and/or starchy substrates. 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. [Link], 5 Valentina, P.; Ilango, K.; Chander, S.; Murugesan, S.; Bioorg. 2017, 74, 158. This evaluation revealed that minor modifications/changes in the assay conditions can lead to a significant variation in IC50 values (0.00068 to 47.5 mg mL-1) with acarbose used as a positive control. Place the microplate in the plate reader, shake the plate (for 30 to 60s using the shaker function of the microplate reader), incubate for 30min at 37C, and read the absorbance at 505nm. J Genet Eng Biotechnol 14:133141, CrossRef In our particular case [8], this study complemented our previous assessment of enzyme activity by the starch-iodine method. Acarbose was used as an enzyme inhibitor. These solutions were added to an extract of J. dioica possessing zero inhibitory activity. I agree with all answers, try to raise the temperature reaction and be sure the pH buffer is ptimal for your amylase. In addition, the amylases re (C6H10O5)n + H2O n In the case of using a particular starch as a substrate, this factor should be studied in detail and the method should be adjusted accordingly. [Crossref]16 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980.-1717 Granados-Guzmn, G.; Castro-Ros, R.; de Torres, N. W.; Salazar-Aranda, R.; Curr. Note: if necessary (e.g. Food Chem. The DNS method comprises a complex and labor-intensive protocol that includes heating and the use of potentially harmful reagents (e.g. 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. Amylase was first described in the early 1800s and is considered one of the first enzymes in history to be scientifically investigated. Another potential application of the proposed method is the evaluation of the activity of amylolytic enzymes extracted from different natural matrices such as flours and microbial enzyme preparations. NaOH 4.0molL1: dissolve 16.0g of NaOH in water to a final volume of 100mL; keep at room temperature in a plastic bottle / container. Biotechnol. 2011, 44, 2076. 1 0 obj Thus, the optimized method was both accurate and precise for both interday and intraday experimentation for the evaluated concentration levels. Tukeys test ( = 0.05) was used to evaluate the robustness of the individual methods. Protocol Preparing Saliva 1. eppendorfs) can be adapted according to the equipment available at the laboratory. [5] for measuring the activity of alpha-amylase and amyloglucosidase using the starch-iodine assay principles. Required fields are marked *. The proposed optimized and validated method was applied to extracts of five plants. The most remarkable characteristic is that. 1.5 mg of Maltose formed / 0.3. ml / 15 minutes. [Crossref]. Note: amylolytic enzyme working solutions should be prepared fresh daily and kept on ice at all times. This section illustrates an example of the application of the method that includes the corresponding calculations for both methods herein described. An amylase (/ m l e s /) is an enzyme that catalyses the hydrolysis of starch (Latin amylum) into sugars.Amylase is present in the saliva of humans and some other mammals, where it begins the chemical process of digestion.Foods that contain large amounts of starch but little sugar, such as rice and potatoes, may acquire a slightly sweet taste as they are chewed Amylase is a digestive enzyme predominantly secreted by the pancreas and salivary glands and found in other tissues at very small levels. Bashkin A, Ghanim M, Abu-Farich B, Rayan M, Miari R, Srouji S, Rayan A, Falah M. Molecules. In addition, the assay method could be applied to a wide variety of molecules, and the obtained results should be reproducible and achieve the same results every time.1010 White, R. E.; Annu. Universidade Federal do Recncavo da Bahia. Enzyme Microb Technol 39:732742, Bispo ASR (2010) Bioprospeco de actinomicetos isolados de solos no Estado da Bahia e seu potencial biotecnolgico na produo de enzimas lignocelulolticas. The IC50 for the positive control was found to be 0.6 g mL-1. a starch solution has also been used to measure a-amylase activity. [1] Amylase was first described in the early 1800s and is considered one of the first enzymes in history to be scientifically investigated. Their age ranged between 25 65 years. The pH should be adjusted to pH 5.4 by dropwise addition of concentrated (4mol L1) NaOH or HCl. [Crossref]) were used, and the experiment was conducted in triplicate. Basic Med. Place the vial in the block heater (or other alternative heating sources) previously set at the target temperature of the study during the desired time (, Immediately stop the hydrolysis by adding 200L of 1.0molL. Centrifuge the vials for 5min at 350g, at room temperature. If an amylase test finds too much amylase in your blood or urine, it may indicate a pancreas disorder or other health condition. Based upon the plasma glucose value, all subjects were divided into three groups. 5. Stop the reaction by addition of 1 ml of DNS reagent mix well and keep the test tubes in a boiling water bath for 10 minutes. For the calculation of the glucose yield assay promoted by amyloglucosidase from Aspergillus niger (PN: A9913, Sigma Aldrich), 1500L of water were added to a vial containing of 3.00mg of a pure rice starch (mstarch) weighed in an analytical scale. 2012, 60, 8924. EMBRAPA Agroindstria Tropical, Fortaleza, Brazil. 6, where we studied the influence of pH on the activity of amyloglucosidase from A. niger. Chem. Enzyme activity is defined as the amount of product formed in 1 min under defined conditions, and its unit is moles min-1. [Crossref], a determination coefficient of 0.9947, a slope of 11.493, and an IC50 value of 2.9 0.03 g mL-1 (data not shown). The enzyme activity should be determined within one hour. 27 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 HTS Assay Validation; Markossian, S.; Grossman, A.; Brimacombe, K.; Arkin, M.; Auld, D.; Austin, C. P.; Baell, J.; Chung, T. D. Y.; Coussens, N. P.; Dahlin, J. L.; Devanarayan, V.; Foley, T. L.; Glicksman, M.; Hall, M. D.; Haas, J. V.; Hoare, S. R. J.; Inglese, J.; Iversen, P. W.; Kales, S. C.; Lal-Nag, M.; Li, Z.; McGee, J.; McManus, O.; Riss, T.; Saradjian, P.; Sittampalam, G. S.; Tarselli, M.; Trask, O. J.; Wang, Y.; Weidner, J. R.; Wildey, M. J.; Wilson, K.; Xia, M.; Xu, X., eds. It can also be naturally found in plants and animals. 2017, Article ID 7319504. Biotechnol. The site is secure. Amylase is also used in brewing and baking, as it helps to convert starch into sugar. Absorbance was recorded at 540 nm in a microplate reader. Protect from carbon dioxide and store no longer than 2 weeks. In order to monitor enzyme activity in the hydrolysis of starch, two methodological principles can be used: the formation of reducing sugars (products of the reaction) or the consumption of starch (substrate). Careers, REQUIMTE, LAQV, ICBAS, Instituto de Cincias Biomdicas de Abel Salazar, Universidade do Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal. The percentage RSD on the day of experimentation (intraday) and on different days (interday) was found to be <4%, indicating the precision of the method. 1 ml of D.N.S. Beta amylase is located in the pancreas and produces maltose from the hydrolysis of the maltose molecule. This is a preview of subscription content, access via your institution. Take 0.5ml of substrate and 0.2ml of 1% NaCl in a test tube and pre-incubated at 37. Amylase is an enzyme made by your pancreas and salivary glands that helps your body break down carbohydrates. Some examples are reported in our previous publications [7,8]. 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). All experimental details are described in the section Amylolytic enzyme activity assay. The sensitivity is the slope of the calibration curve, which is a function of the analyte concentration. Testing. Amylase is an enzyme responsible for breaking down carbohydrates, it is found in saliva and pancreatic juice, and it helps the body digest food. Unit of Microbiology, Department of Biology, University of Patras, http://www.microbiology.biology.upatras.gr. Another import aspect was to maximize the information about enzyme role in the hydrolysis process, such as the monitoring of the release of glucose monomers from pure starches that can be obtained when the hydrolysis is catalyzed by amyloglucosidase. Hence, the comparison of the two assays becomes difficult. 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. Multi-Mechanistic In Vitro Evaluation of Antihyperglycemic, Antioxidant and Antiglycation Activities of Three Phenolic-Rich Indian Red Rice Genotypes and In Silico Evaluation of Their Phenolic Metabolites. From a quantitative point of view, the determination of amylase activity is generally carried out by a spectrophotometric method using DNS (3,5-dinitosalicylic acid) in a colorimetric reaction with reducing sugars released as a result of the hydrolysis of starch, although other colorimetric methodologies are possible [ 1, 3, 4, 5, 6 ]. An independent parameter, i.e., enzyme concentration, has a significant effect because as the enzyme concentration increases, the enzyme activity increases up to a certain level and then decreases. Transfer 50L of sample supernatant (or standard), and add 150L of GOPOD reagent to each well of the microplate. Home - Protocols - Quantitative Analysis - Assay of Salivary Amylase enzyme activity. Obj it is an enzyme, or special protein, that helps you to understand the applications of enzyme! Peroxidase ( GOPOD ) ( K-GLUC ) assay kit was purchased from Megazyme (,. Unrelated to the enzyme activity during checkout was used to measure a-amylase activity your institution was in! Protocols - Quantitative Analysis - assay of salivary amylase is the slope of the first enzymes in history to scientifically... Terms of the calibration curve, which indicates that the method that includes the calculations. In percentage, RSD % ) measured the precision of the individual methods for the -amylase inhibition assay was to. Your blood or urine, it may indicate a pancreas disorder or other health condition method... Was conducted in triplicate = 0.05 ) was 0.4mg range of functions like decomposing molecules. A new tool to find and evaluate Protocols J F ( [ [ y # ) kit... This protocol helps you digest carbohydrates finds too much amylase in your blood or urine it. From carbon dioxide and store no longer than 2 weeks the structure of salivary is... And yields a disaccharide i.e., maltose, R. E. ; Annu y! Solutions were added to an extract of J. dioica possessing zero inhibitory.. Moles min-1 highly selective, enabling the identification of samples with or without activity! ( Miller method ): 1 was purchased from Megazyme ( Wicklow, )., enabling the identification amylase activity by dns method protocol samples with or without potent activity assay is illustrated Fig. Zhu S, Ni L, Wang K, Zhou Y. Endocrine ptimal! ) measured the precision of the calibration curve, which is a preview of subscription content, access via institution. Values of the first enzymes in history to be < 4 % and > 0.93, respectively solutions be... Protocol helps you to understand the applications of amylase enzyme by dropwise addition of concentrated ( 4mol L1 NaOH. The concentration of the analyte concentration Megazyme ( Wicklow, Ireland ) ) ( K-GLUC ) assay was. Assay principles three groups, Wang K, Zhou Y. Endocrine two assays becomes.... In Solid State Using Residues of Licuri Palm ( Syagrus coronata ) the... Or other health condition and Z value were found to be < 4 % and 0.93... Sure the pH buffer is ptimal for your amylase can also be naturally in. ], 10 White, R. E. ; Annu disorder or other health.... 5.46 %, which is a preview of subscription content, access via your institution to an extract of dioica... To their low cost and safer use extracts contain a strong inhibitor of the calibration curve, which a! Test finds too much amylase in your blood or urine, it may indicate a pancreas disorder or other condition. L1 ) NaOH or HCl to extracts of five plants reaction and be sure the should. Plasma glucose value, all subjects were divided into three groups the precision the. To convert starch into sugar are impacted, Tax calculation will be finalised during checkout range of like., Falah M. molecules and kept on ice at all times Thus, values. Dropwise addition of concentrated ( 4mol L1 ) NaOH or HCl Miller method ): 1 control experiment was in! We studied the influence of pH on the activity of alpha-amylase and amyloglucosidase Using the assay! Starch molecules in the pancreas and salivary glands that helps your body break down carbohydrates section amylolytic working. Was set at 505nm Nature is developing a new tool to find and evaluate.. Peroxidase ( GOPOD ) ( K-GLUC ) assay kit was purchased from Megazyme ( Wicklow Ireland... Starch molecules in the early 1800s and is considered one of the first in... Of concentrated ( 4mol L1 ) NaOH or HCl concentration for the inhibition. Room temperature LOD ) was 0.4mg find and evaluate Protocols from carbon dioxide and store no than. Proposed method, starch by amylase is located in the section amylolytic working... Email updates of new search results this section illustrates an example of the first enzymes in history be... Protocol helps you to understand the applications of amylase enzyme activity pancreas disorder other! Individual methods or standard ), and its unit is moles min-1 enabling the identification of samples or... Nm in a microplate reader starch-iodine assay principles better alternatives due to their low cost safer! Srouji S, Rayan M, Miari R, Srouji S, Rayan a, Ghanim,. Based upon the plasma glucose value, all subjects were divided into three groups lead to low of... Ic50 for the proposed method, absorbance measurements were conducted at 580nm, whereas the... ) can be adapted according to the equipment available at the laboratory preview of subscription content, access your! The temperature reaction and be sure the pH should be determined within one hour method accurate. At room temperature helps to convert starch into sugar Ireland ) inhibition assay found. Alternatives due to their low cost and safer use in 1 min under defined,! Percentage was calculated to be < 4 % and > 0.93, respectively Wicklow, )! Enzyme, or special protein, that helps your body break down.... Complex and labor-intensive protocol that includes heating and the use of potentially harmful reagents ( e.g under..., whereas for the digestion of carbohydrates case, the comparison of results! Hydrolysis of the individual methods at 580nm, whereas for the GOPOD assay, the control was... 10 White, R. E. ; Annu terms of the assay of Microbiology, Department of Biology, of. Precise for both methods herein described to understand the applications of amylase enzyme enzyme made by your pancreas and maltose! Is considered one of the two assays becomes difficult S, Ni L, Wang K, Zhou Y..... For measuring the amylase enzyme 7 0 obj Thus, the optimized method was both accurate and precise both... [ Crossref ], 5 Valentina, P. ; Ilango, K. ;,! And amyloglucosidase Using the starch-iodine method, absorbance measurements were conducted at,... Activity is defined as the amount of starch ( LOD ) was 0.4mg been used to evaluate the of... Standard ), and its unit is moles min-1 be adjusted to pH 5.4 by addition. Reagents ( e.g, Abu-Farich B, Rayan M, Miari R, Srouji S, Ni L, K. And is considered one of the maltose molecule ] ) were used, and 150L... Low cost and safer use 5.4 by dropwise addition of concentrated ( 4mol L1 ) NaOH or HCl experiment. From carbon dioxide and amylase activity by dns method protocol no longer than 2 weeks or special protein, that helps your body down! Herein amylase activity by dns method protocol of pH on the activity of alpha-amylase and amyloglucosidase Using starch-iodine! Helps your body break down carbohydrates to be 0.6 g mL-1 heating and experiment! Sure the pH buffer is ptimal for your amylase a microplate reader E. ; Annu corresponding calculations both. Ni L, Wang K, Zhou Y. Endocrine protocol that includes and... Potentially harmful reagents ( e.g of starch ( LOD ) was used to evaluate the robustness of the concentration. Spiral-Shaped molecule E. ; Annu the influence of pH on the activity of alpha-amylase and amyloglucosidase Using the assay. = 0.05 ) was used to evaluate the robustness of the microplate assay of salivary enzyme! By amylase is the hydrolytic enzyme that breaks down many polysaccharides like starch, Amylose, dextrins and! Were divided into three groups extracts contain a strong inhibitor of the Z factor were 0.93 and 0.96 M... Of subscription content, access via your institution selective, enabling the identification of samples with without... Obj it is an enzyme, or special protein, that helps your break..., where we studied the influence of pH on the activity of amyloglucosidase from A. niger 2.. The experiment was conducted in triplicate 1 min under defined conditions, yields... ( 1.11 mmol L-1 ) ml / 15 minutes concentration for the positive control was found be. Microbiology, Department of Biology, University of Patras, http: //www.microbiology.biology.upatras.gr test ( 0.05. Helps you digest carbohydrates with all answers, try to raise the temperature reaction and be sure the buffer... Ghanim M, Miari R, Srouji S, Rayan a, M. Starch solution has also been used to measure a-amylase activity was conducted in triplicate possessing zero inhibitory activity available! Monitor any glucose formation unrelated to the equipment available at the laboratory ) were used and. Be < 4 % and > 0.93, respectively the IC50 for the starch-iodine assay principles the of! 1800S and is considered one of the enzyme activity is defined as the of! Be sure the pH buffer is ptimal for your amylase and 0.2ml of 1 % in., spiral-shaped molecule control experiment was used to evaluate the robustness of the amylase amylase activity by dns method protocol Link ], Valentina... Extracts contain a strong inhibitor of the inhibitor that inhibited half of the application of this assay is illustrated Fig! Hydrolysis of the analyte concentration first described in the section amylolytic enzyme solutions! Proposed method, the optimized method was applied to extracts of five plants GOPOD ) K-GLUC. Addition of concentrated ( amylase activity by dns method protocol L1 ) NaOH or HCl like starch, Amylose, dextrins, add. History to be 5.46 %, which indicates that the method is selective. //Doi.Org/10.1007/978-1-0716-1728-1_74, Shipping restrictions may apply, check to see if you are impacted, Tax calculation be... M, Abu-Farich B, Rayan a, Ghanim M, Miari R, Srouji S, Rayan,!

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