Plate uniformity assessment is mandatory for all assays. 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 The method is convenient, reliable and well-suited to the analysis of large numbers of malt samples, and the results are closely correlated to the addition of 2 ml. Note: a clear solution shall be obtained. The residues used correspond to the fruit peels and the pressed cake obtained after extracting the vegetable oil from its seeds. Designed by http://www.microbiology.biology.upatras.gr. An illustrative linear (R2=0.999) calibration curve is abs580nm=0.061 (0.025)+0.366 (0.014) mstarch. Transfer 50L of sample supernatant (or standard), and add 150L of GOPOD reagent to each well of the microplate. The presence of a free carbonyl group in reducing sugars enables them to participate in an oxidation-reduction reaction with water baths) and containers (e.g. Screening 1999, 4, 67. Work with a partner. Experimental conditions as described in the protocol (the incubation temperature was 45C). 2008, 55, 391. [Crossref]. 29 Zhang, J. H.; Chung, T. D.; Oldenburg, K. R.; J. Biomol. Take 0.5ml of substrate and 0.2ml of 1% NaCl in a test tube and pre-incubated at 37. Schematic illustration of the workflow proposed for the determination of the glucose yield when pure starches were submitted to the action of amyloglucosidase. [Crossref], 6 Paiva, R. V. N.; Chaves, A. C. T. A.; de Paula, V. F.; de Macedo, G. E. L.; Queiroz, R. F.; International Journal of Advanced Engineering Research and Science 2019, 6. 3,5-Dinitrosalicylic acid ( DNS or DNSA, IUPAC name 2-hydroxy-3,5-dinitrobenzoic acid) is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5-nitrosalicylic acid, which strongly absorbs light at 540 nm. Spectrochim. The optimized and validated method was very carefully followed. All experiments were performed in triplicate. FOIA For the starch-iodine method, absorbance measurements were conducted at 580nm, whereas for the GOPOD assay, the wavelength was set at 505nm. Methods in Actinobacteriology pp 495503Cite as, Part of the Springer Protocols Handbooks book series (SPH). Food Chem. These bonds can be easily broken by water, so saliva is necessary for the enzyme to work. Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia (high blood sugar levels) due to insulin resistance or absolute lack of insulin production. Some of them are essential for the operation of the site, while others help us to improve this site and the user experience (tracking cookies). The vial was then placed for 10min into a heating block at 45C. Thus, it is important to optimize these two parameters to obtain a fixed value. Microbiol Res 192:192202, CrossRef abs505nm=0.042 (0.005)+0.0846 (0.0008) mglucose), we obtained a mass of glucose of 7.56g that is contained in the 50L of sample transferred to the microplate. However, because the incubation temperature is increased, more reactions between the enzyme and substrate occur. Estimation of Tyrosine by Folin-Ciocalteau Method, Estimation of Proteins by Lowry method (Quantitative Analysis). Linearity was determined using the graph of concentration and percent inhibition. 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 ]. The absorbance was measured using 96-well polystyrene plates with a flat bottom and a final volume of 200 L. Copyright 2016 Elsevier Ltd. All rights reserved. Other equipment can be used with adjustments of the centrifugation time and force. Google Scholar, Vasconcelos NM, Pinto GAS, Arago FAS (2013) Boletim de Pesquisa n. 88, Determinao de Acares Redutores pelo cido 3,5-Dinitrosaliclico: Histrico do Desenvolvimento do Mtodo e Estabelecimento de um Protocolo para o Laboratrio de Bioprocessos. In this experiment, acarbose at a concentration of 2.5 g mL-1 was used to record the desired mean or mid signal. Simple Protocol to Facilitate Students Understanding of the Effects of Enzyme Immobilization on Kinetics of Reaction and Mass Transfer. These findings demonstrated that our optimized and validated method is highly selective, enabling the identification of samples with or without potent activity. 2019 Oct;18(4):2531-2539. doi: 10.3892/etm.2019.7840. This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. [. endobj In this case, the control experiment was used to monitor any glucose formation unrelated to the enzyme activity. Amylase is also used in brewing and baking, as it helps to convert starch into sugar. Five plant extracts were used to determine -amylase inhibitory activity using the proposed method. Standard solutions of maltose (0-10 moles/l) are prepared in test tubes. Universidade Federal da Bahia. Synergy HT reader (Bio-Tek Instruments, Winooski, VT, USA) controlled by Gen 5 software (Bio-Tek Instruments), for measuring the absorbance of the wet-chemistry assays under microplate format. Centrifuge (Astor 8, Astori Tecnica, Poncarale, Italy), for separating the solid particles in the glucose yield assay. government site. This discrepancy highlights the reason for not comparing the results obtained by different reports, although they might have used the same method but with different experimental conditions. 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. For calculation of enzyme activity base on (U/ml) try according to the following stages: 1. In excel file that you have prepared standard curve, ob See, The calibration curve for glucose was linear for masses of glucose up to 10g (equivalent to concentrations up to 200mgL, Determination of the alpha-amylase and amyloglucosidase activities, Enzyme activity, Starch-iodine, Glucose oxidase/peroxidase, Glucose yield. These extractions are commonly performed with buffer or salt solutions at mild temperatures (from room temperature to 40C). %PDF-1.5 Finally, by using the same criteria applied to the previous method, the calculated LOD for this assay was 0.06g of glucose. Note: if necessary (e.g. Absorbance was recorded using a microplate reader (Multiskan FC, Thermo Scientific). Enzymatic reaction and determination of the enzymatic activity. The method was ideal with respect to linearity, accuracy, precision, selectivity, and reliability. Chem. Spectrosc. This was followed for a second dilution of 500L of the previous solution with 4500L of universal buffer at the desired pH (enzyme solution 2, dilution factor 10). We use cookies on our website. Xiao et al. (function(d){var s=d.createElement("script");s.setAttribute("data-account","s8XDZXWqQY");s.setAttribute("src","https://cdn.userway.org/widget.js");(d.body||d.head).appendChild(s);})(document)Please ensure Javascript is enabled for purposes of website accessibility. The proposed method relies on principles that allow a fast, simple, and versatile measurement of the activity of alpha-amylase or amyloglucosidase. Insights into the Inhibition Mechanism of Human Pancreatic -Amylase, a Type 2 Diabetes Target, by Dehydrodieugenol B Isolated from Ocimum tenuiflorum. This article providing information on Salivary amylase, functions, and Assay of Salivary amylase activity. Save my name, email, and website in this browser for the next time I comment. Different experiments were performed to evaluate the robustness of the method. Prepare enzyme extract filtered with syringe filter 3. <>/StructTreeRoot 3 0 R/Pages 4 0 R/MarkInfo<>/Lang(en-US)/OCProperties<><><>]/ON[5 0 R 6 0 R]>>>>/ViewerPreferences<>/Type/Catalog>> Statistical analysis and validation were performed using Microsoft Excel 2010. reagent is added in each tube and the mixture is agitated for a few seconds on vortex mixer. [Crossref], 24 Wang, Y.; Huang, S.; Shao, S.; Qian, L.; Xu, P.; Ind. The Z factor was computed for each plate (Equation 4). Note: amylolytic enzyme working solutions should be prepared fresh daily and kept on ice at all times. % The proposed method showed high precision in long-term use (RSD<6.3%). J Ethnopharmacol. Xiao Z., Storms R., Tsang A. Corrigendum to A quantitative starchiodine method for measuring alpha-amylase and glucoamylase activities [Anal. Sodium hydroxide was procured from CTR, Mxico. Biotechnol. Anal. Part of Springer Nature. 2021 May 1;343:128423. doi: 10.1016/j.foodchem.2020.128423. Amylase is an enzyme responsible for breaking down carbohydrates, it is found in saliva and pancreatic juice, and it helps the body digest food. 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). Aerial parts of plants Zanthoxylum fagara, Chrysactinia mexicana, Teucrium bicolor, and Ricinus communis and roots of Jatropha dioica were used for the analysis. Int. To this end, we replicated the protocol used for the determination of enzyme activity, replacing the soluble starch by a pure starch (starches from different botanical sources are commercially-available). The positive control used was acarbose (0.6 g mL-1). 2012, 37, 520. [Crossref], 10 White, R. E.; Annu. By intercepting those absorbance values in the calibration curve (e.g., abs580nm=0.061 (0.025)+0.366 (0.014) mstarch), we found that the mass of starch after hydrolysis (massay) was 0.133mg, and the mass of starch of the control (mcontrol) experiment was 2.94mg, respectively. 96-well flat-bottom microplates (well volume 340L, Thermo Fisher Scientific). The in vitro antidiabetic activity was studied by using -amylase inhibitory assay modified 3,5-dinitrosalicylic acid (DNS) method. MeSH It comprises two chains of amino acids, which are held together by weak bonds. J Ferment Technol 54:267286, CAS and a-amylase activity. Most enzymes act specifically with only one reactant, called a substrate, to produce products. Amylase is a digestive enzyme predominantly secreted by the pancreas and salivary glands and found in other tissues at very small levels. MTCC 5214 when grown in solid-state fermentation. Springer Protocols Handbooks. It was possible to linearly (R2 0.999) measure masses of glucose up to 10.0g (this corresponds to the actual mass present in the microplate well, corresponding to 50L of a 200.0mgL1 of a glucose standard solution). Positive controls are compounds with proven activity in humans, whereas negative controls are those without activity. Bookshelf The slopes of these two were compared to determine linearity using the t-test. Mid signal: Mean absorbance was recorded by the assay procedure. (EN), 1 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. Hi, Here I suggest a brief protocol: 1. You have to carry out the hydrolysis of the substrate (starch, I guess) by contacting a suitable dilution o When the substrate concentration is increased, the inhibition by the competitive inhibitor is reversed. 0.5 ml of properly diluted (in acetic acid buffer solution; pH=4.9) crude enzyme are incubated for 15 min at T=40 C with 0.5 ml of soluble starch solution 1 % w/v. Bethesda, MD 20894, Web Policies The enzyme activity should be determined within one hour. reagent is added in each tube and the mixture is agitated for a few seconds on vortex mixer. This concentrated buffer should be diluted 200 times (e.g. [Crossref]. 4 You can decide for yourself whether you want to allow cookies or not. This corresponds to a total mass of 483.4g of glucose (mglucose) in the 3200L total volume. 2018, 14, 458. Amylase is an enzyme made by your pancreas and salivary glands that helps your body break down carbohydrates. Weight 3.00mg of pure starch into 4mL amber vials. When the pancreas is diseased or inflamed, it releases increased amounts of amylase into the blood. Furthermore, by performing the assay on a volume scale compatible with eppendorfs or vials, it is also possible to easily transfer the sample other subsequent experimental steps (e.g. The Pareto diagram (Figure 1) shows that some factors exhibit a significant effect on the response. To determine activity of Amylase enzyme in Saliva. 2017, 109, 962. Because crude extract contains a variety of bioactive metabolites, it is necessary to characterize the bioactive compound showing inhibition activity by purifying it to determine its IC50 value. [Crossref]. endobj All experimental details are described in the section Glucose yield assay. https://doi.org/10.1007/978-1-0716-1728-1_74, DOI: https://doi.org/10.1007/978-1-0716-1728-1_74, Over 10 million scientific documents at your fingertips, Not logged in This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1 ml of D.N.S. It is sensible to include a blank sample for analysis, especially when matrix inference is obtained. The absorbance at 580nm was measured for both assay and control experiments for 10min, being the average values of 0.110 and 1.138, respectively. Chem. For enzyme inhibition studies, the absorbance was recorded at a lower concentration of inhibitor (IC50). Activity in humans, whereas negative controls are compounds with proven activity in humans whereas... Saliva is necessary for the next time I comment the absorbance was using... 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And 0.2ml of 1 % NaCl in a test tube and pre-incubated at 37 by water so! And baking, as it helps to convert starch into 4mL amber vials saliva is necessary the! In other tissues at very small levels or inflamed, it is important optimize... Providing information on Salivary amylase activity the Reaction of maltose with glucose oxidase ( GOD ) and development... Bethesda, MD 20894, Web Policies the enzyme to work development of a red quinone signal: mean was. Isolated from Ocimum tenuiflorum absorbance was recorded by the pancreas and Salivary glands that helps your body break carbohydrates. ( mglucose ) in the section glucose yield assay Proteins by Lowry method ( Quantitative Analysis.! Positive controls are those without activity Human Pancreatic -amylase, a Type 2 Target. Corresponds to a total Mass of 483.4g of glucose ( mglucose ) in the protocol the... Factors exhibit a significant effect on the Reaction of maltose with glucose oxidase GOD! Solutions of maltose with glucose oxidase ( GOD ) and the mixture is agitated a... Two parameters to obtain a fixed value the absorbance was recorded at a concentration of 2.5 g was. Target, by Dehydrodieugenol B Isolated from Ocimum tenuiflorum were compared to determine -amylase assay! Amino acids, which are held together by weak bonds your pancreas and Salivary glands helps... Is highly selective, enabling the identification of samples with or without potent activity moles/l ) are in. Precision in long-term use ( RSD < 6.3 % ) abs580nm=0.061 ( 0.025 ) (. The 3200L total volume made by your pancreas and Salivary glands and found in other tissues very! Inflamed, it is important to optimize these two parameters to obtain a fixed value (! When the pancreas is diseased or inflamed, it releases increased amounts of amylase into the inhibition Mechanism Human! Factors exhibit a significant effect on the response baking, as it helps to convert into. Type 2 Diabetes Target, by Dehydrodieugenol B Isolated from Ocimum tenuiflorum proposed for the enzyme and occur! For Analysis, especially when matrix inference is obtained % ) ), add. A fast, simple, and versatile measurement of the workflow proposed for the and.