reagent is added in each tube and the mixture is agitated for a few seconds on vortex mixer. 8. In our particular case [8], this study complemented our previous assessment of enzyme activity by the starch-iodine method. It is responsible for breaking down carbohydrates into simpler sugars. [Link], 5 Valentina, P.; Ilango, K.; Chander, S.; Murugesan, S.; Bioorg. The inhibition percentage was calculated using Equation 1: where A is the absorbance of the negative control, and B is the absorbance of the solution containing an inhibitory agent. Absorbance was recorded using a microplate reader (Multiskan FC, Thermo Scientific). [Crossref], 2 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.; Food Chem. Analytical validation was performed based on different international guidelines. Epub 2015 Feb 16. Med 2011, Article ID 810207. 2014, 17, 465. For calculation of enzyme activity base on (U/ml) try according to the following stages: 1. J. Additionally, different solvents, such as ethanol (25%) and dimethyl sulfoxide (DMSO) (final concentrations of 0.15% and 0.25%), were included. We use cookies on our website. This is the first report of this plant showing -amylase inhibition activity. For each experiment, fresh and independent reagents were used, and the evaluation was performed on three different days. 2 0 obj 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. This assay was also very precise, with RSD values below 3% in all cases. This work addresses optimization, validation and application of an in vitro method for assessment of -amylase inhibition on a microscale level. The basic function of an enzyme is to increase the rate of a reaction. One milligram of each extract was weighed and dissolved in 1 mL of phosphate buffer (100 mmol L-1, pH 6.9); some solutions were sonicated to achieve complete dissolution. The use of a suitable volumetric scale for the assay was also an important concern for our development work. In general, they have a poor design of experiments and an inadequate statistical significance.1111 Finak, G.; Gottardo, R.; In Data Mining Techniques for the Life Sciences; Carugo, O., Eisenhaber, F., eds. MTCC 5214 when grown in solid-state fermentation. Federal government websites often end in .gov or .mil. Simultaneous, rapid, spectrophotometric determination of total starch, amylose and amylopectin. 96-well flat-bottom microplates (well volume 340L, Thermo Fisher Scientific). Five plant extracts were used to determine -amylase inhibitory activity using the proposed method. This process starts in the mouth and continues in the stomach and intestines. 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 ]. Without salivary amylase, we would not be able to digest carbohydrates properly and would not get the total nutritional value from these foods. 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 Pol. <>stream will also be available for a limited time. Would you like email updates of new search results? [Crossref], 19 Satpathy, G.; Tyagi, Y. K.; Gupta, R. K.; Food Res. 2014, 17, 465. This is not surprising, considering the importance of tertiary structure in enzyme function and non-covalent forces in determining the shape of enzymes. 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. Keywords:optimization of enzyme method; validation of enzyme method; -amylase activity; design of experiments. J Genet Eng Biotechnol 15:8794, Zhao Y, Zhao Y, Zhang Z, Wei Y, Wang H, Lu Q, Li Y, Wei Z (2017) Effect of thermo-tolerant actinomycetes inoculation on cellulose degradation and the formation of humic substances during composting. Thank you Marsha for your replay, actually I am trying to calculate IC50 or EC50 from my samples (plant extracts) to see if it can use as inhibitor To determine activity of Amylase enzyme in Saliva. If sodium azide is added, the reagent will be stable for more than one year. 1.5 X 4 mg of Maltose formed / 0.3 ml of Enzyme / 1 hour, 1.5 X 4 X 3.3 mg of Maltose formed / 1ml of Enzyme / 1 hour, 1.5 X 4 X 3.3 X 100 mg of Maltose formed / 100ml of Enzyme / 1 hour. McCleary B.V., McNally M., Monaghan D., Mugford D.C. 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. Note: this is a non-interchangeable rotor centrifuge (350g) where the vials used for the activity assay can be directly centrifuged (stacked in pairs). This section illustrates an example of the application of the method that includes the corresponding calculations for both methods herein described. Note: other heating sources (e.g. 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. 2020 Jan;67(1):87-94. doi: 10.1007/s12020-019-02118-1. Rev. Med 2011, Article ID 810207. To achieve a good assay, there must be good agreement between the in vivo and in vitro results for the evaluated substance. 2011, 47, 270. Dilute to a final volume of 100 ml with reagent grade water. An amylase test measures the amount of amylase in blood or urine (pee). ; Eli Lilly & Company and the National Center for Advancing Translational Sciences: Bethesda, 2004. Min signal: The minimum absorbance was recorded by the assay procedure. The sensitivity is the slope of the calibration curve, which is a function of the analyte concentration. and transmitted securely. 4. Chang YX, Ge AH, Donnapee S, Li J, Bai Y, Liu J, He J, Yang X, Song LJ, Zhang BL, Gao XM. The present method describes an improved experimental protocol that can be used to monitor the activity of two amylolytic enzymes: alpha-amylase and amyloglucosidase. For the GOPOD method under microplate format, we also performed a similar method characterization. Maltose 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. [Kinetic determination of alpha-amylase in serum and urine with an oligosaccharide as substrate--modification for a fully mechanized enzyme measuring device (author's transl)]. Mina Karimi-Avargani kindly mail me [email protected]. I need your help for calculation of data. Biotechnol. Toxicol. The vial was then placed for 10min into a heating block at 45C. Three categories of amylases, denoted alpha, beta, and gamma, differ in the way they attack the bonds of the starch molecules. 2012, 13, 427. Acarbose, a positive control, was used as an inhibitor for this experiment. More details and information about the extraction of other amylolytic enzymes from different natural matrices can be found in the method AOAC 2002.01, commonly named in the literature as the Ceralpha method [9]. In the present method of Millers reaction, the product to be analyzed was obtained by heating it in a boiling water bath. Before An enzymeisaprotein moleculethatisabiologicalcatalyst withthree characteristics. Beta amylase is located in the pancreas and produces maltose from the hydrolysis of the maltose molecule. [Crossref]. [. If an amylase test finds too much amylase in your blood or urine, it may indicate a pancreas disorder or other health condition. Spectrochim. Amber glass vials (15mm45mm8mm, e.g. Hence, the comparison of the two assays becomes difficult. Estimation of Tyrosine by Folin-Ciocalteau Method, Estimation of Proteins by Lowry method (Quantitative Analysis). 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. Please note that if you reject them, you may not be able to use all the functionalities of the site. Bethesda, MD 20894, Web Policies Among solvents, ethanol (up to 25%) and DMSO (up to 0.5%) at lower concentrations did not have a significant out-turn, indicating that the method is robust for the abovementioned conditions. The analytical protocol can also be conducted in mild conditions (room temperature), bypassing the heating steps and the protocol complexity that is present in the DNS method, as well in other alternative methods for the same analytes [6]. The experimental design and data handling were performed using Statgraphics Centurion XVI software (Statpoint Technologies, Inc., Warrenton, VA, USA). results of inhibition of alpha amylase enzyme by the selected plant extracts have been represented in Figure 1. After weighing 3.0g of flour into a 50mL flask, add 20.0mL malate buffer pH 5.4 (diluted from the concentrated solution). Selected plant products could provide better alternatives due to their low cost and safer use. Magnetic stirrer/heater plate (MAG-H, Gerhardt, Knigswinter, Germany), for preparing the soluble starch solution. According to the obtained results, the sensitivity of salivary amylase assay was found to be 95% and specificity was 100%. 7 0 obj Then, 100 L of starch was added (final concentration 0.38 mg mL-1) and incubated at 37 C for 5.5 min. PEst-C/EQB/LA0006/2011) and co-financed by FEDER (UID/QUI/50006/2013 NORTE-01-0145-FEDER-00011) under the Partnership Agreement PT2020. (1) the calculated activity for this enzyme was 1123UmL1. Different concentrations of acarbose (0.5 to 12 g mL-1) were added to the enzymatic solution, and an enzyme assay was performed as mentioned above. 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. Glucose yield (average values from two determinations) measured by the described glucose oxidase/peroxidase assay that resulted from the hydrolysis of four different pure starches mediated by an amyloglucosidase from Aspergillus niger (PN: A9913, Sigma Aldrich). Then the absorbance of each one of the unknown samples is measured and the concentration of the converting sugars is determined, based on the standard curve. In that case, the sensitivity of the determination will be modified. Abstract:NelsonSomogyi and 3,5-dinitrosalicylic acid (DNS) assays are the classical analytical methods for the determination of activity of starch-debranching enzymes, however, they have a narrow detection range and do not adapt to the quantitative measurement of linear polysaccharides. The error percentage was calculated to be 5.46%, which indicates that the method is accurate. 6, where we studied the influence of pH on the activity of amyloglucosidase from A. niger. An amylase test measures the amount of amylase in your blood or urine (pee). A comparative study towards a tailor-made enzymatic hydrolysis of starch. From these samples, salivary amylose enzyme assays were carried out. Other equipment can be used with adjustments of the centrifugation time and force. This extract was the closest to the acarbose positive control. Note: a clear solution shall be obtained. 19 0 obj Learn more Hence, the disparity between the slopes of the standard and sample highlights the presence of a matrix effect, which affects sensitivity. Method. 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. Optimization of the -amylase inhibition assay was performed using fractionalized factorial design (2^6-1). 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. The test is done in microtitre plates with a total volume of 260L and an assay time of 40min including the pre-incubation steps. In this experiment, acarbose at a concentration of 2.5 g mL-1 was used to record the desired mean or mid signal. phenol). [Crossref], Stay informed of issues for this journal through your RSS reader, Text Unit of Microbiology, Department of Biology, University of Patras, http://www.microbiology.biology.upatras.gr. This is a preview of subscription content, access via your institution. The https:// ensures that you are connecting to the 2023 Springer Nature Switzerland AG. If no amylase activity is present in the saliva, how many mg of starch will be detected in the first time point of the assay? By applying Eq. The variability test is conducted at three response levels with varied signal intensities.1515 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. The site is secure. Salivary amylase is present in high concentrations in the saliva of young children and decreases as we age. Keywords: Absorbance was recorded at 540 nm using a microplate reader. Hence, a reliable protocol for preparing the starch solution is an important aspect regarding the need for a batch to batch reproducibility. It comprises two chains of amino acids, which are held together by weak bonds. 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. Food Sci. Based upon the plasma glucose value, all subjects were divided into three groups. For enzymes, denaturation can be defined as the loss of enough structure, rendering the enzyme inactive. Furthermore, this improved method is very flexible and allows a broadband application, being able to be easily adjusted according to the requirements of each study or the equipment and instrumentation available at each laboratory. All experimental details are described in the section Amylolytic enzyme activity assay. 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These foods ; Gupta, R. K. ; Chander, S. ; Murugesan, S. ; Murugesan, ;! Be able to digest carbohydrates properly and would not get the total nutritional value from samples! Total volume of 100 ml with reagent grade water be able to digest carbohydrates properly would... According to the obtained results, the comparison of the determination will be stable for more one. In vivo and in vitro method for assessment of -amylase inhibition amylase activity by dns method protocol microscale... The following stages: 1 the need for a batch to batch reproducibility starch amylose. ):87-94. doi: 10.1007/s12020-019-02118-1 for more than one year also be available for a few on! By heating it in a boiling water bath the comparison of the analyte concentration volume of 100 ml with grade! Total nutritional value from these foods importance of tertiary structure in enzyme function non-covalent. Two amylolytic enzymes: alpha-amylase and amyloglucosidase assessment of -amylase inhibition activity different international guidelines in vivo in. Be stable for more than one year assay time of 40min including the steps! Glucose value, all subjects were divided into three groups ], 19 Satpathy, G. Tyagi! All subjects were divided into three groups slope of the method that includes the corresponding calculations for both herein... Of subscription content, access via your institution the obtained results, the product be!, we would not be able to digest carbohydrates properly and would get. 50Ml flask, add 20.0mL malate buffer pH 5.4 ( diluted from the concentrated ). Enzyme inactive estimation of Tyrosine by Folin-Ciocalteau amylase activity by dns method protocol, estimation of Proteins by method!
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