Plate uniformity assessment is mandatory for all assays. Amylase was first described in the early 1800s and is considered one of the first enzymes in history to be scientifically investigated. Boric acid (PN: A79-212, 99.5%) and NaOH (PN: S/4920/60) were purchased from Thermo-Fisher Scientific (Waltham, MA, USA). Factors showing a significant effect on the inhibition percentage were selected and optimized using a basic sequential simplex method. Gamma amylase is found in the small intestine and completes the hydrolysis of maltose into glucose. Glucose oxidase / peroxidase (GOPOD) (K-GLUC) assay kit was purchased from Megazyme (Wicklow, Ireland). Enhanced consistence of reagent preparation. The DNS method comprises a complex and labor-intensive protocol that includes heating and the use of potentially harmful reagents (e.g. 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). Statistical analysis and validation were performed using Microsoft Excel 2010. High intra-day and inter-day reproducibility. For enzyme inhibition studies, the absorbance was recorded at a concentration with 50% inhibition (IC60). As a result of the intensive demand for this plant in folk medicinal uses, its scarcity has become problematic. Transfer 50L of sample supernatant (or standard), and add 150L of GOPOD reagent to each well of the microplate. 7). For calculation of enzyme activity base on (U/ml) try according to the following stages: 1. Some examples of amylolytic enzymes assessed by this method were: amyloglucosidase from Aspergillus niger (PN: A9913) (illustrative results with this enzyme will be shown in the next sections), and alpha-amylase from Bacillus licheniformis (PN: A3306, heat-stable), purchased from Sigma-Aldrich, amyloglucosidase Spirizyme Achieve and alpha-amylase from B. licheniformis Liquozyme SC DS (AA-2), supplied by Novozymes (Bagsvaerd, Denmark). Gangadharan D., Sivaramakrishnan S. Amylolytic enzymes. From these samples, salivary amylose enzyme assays were carried out. FOIA J. The reduced 3-amino-5-nitrosalicylic acid is red-brown in color and can be quantified spectrophotometrically with an absorption maximum at 540 nm. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). As an example, we summarize in this section the extraction protocol for the measurement of alpha-amylase activity in wheat flour [9]. Int J Biol Macromol 159:763772, Antony R, Sanyal A, Kapse N, Dhakephalkar PK, Thamban M, Nair S (2016) Microbial communities associated with Antarctic snow pack and their biogeochemical implications. Appl Microbiol Biotechnol 54:764771, Khandeparkar RDS, Bhosle NB (2006) Isolation, purification and characterization of the xylanase produced by Arthrobacter sp. Previous studies citing the assays of in vitro antihyperglycemic activity measurements, including detailed procedures and the use of positive controls, were collected and analyzed (Table 1). Amylase level testing can be done with a blood or urine test. 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. (Family: Asteraceae) has been used as a natural remedy in the Arabian region for its antihyperglycemic activity. 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 ]. Among the amylolytic enzymes, which act on starchs polymeric chain as well in its related oligo- and polysaccharides, alpha-amylase, and amyloglucosidase are the most widespread biocatalysts used in the hydrolytic process [2]. 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 absorbance of the well was monitored at 505nm, and the value recorded was 0.681. The samples are placed in a water bath (T=100C) for 5 min and then they are left to cool at room temperature. 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]. 3. This extract was the closest to the acarbose positive control. Acarbose was used as an enzyme inhibitor. In addition, the effect of substrate concentration on enzyme activity was analyzed. 2012, 60, 8924. After weighing 3.0g of flour into a 50mL flask, add 20.0mL malate buffer pH 5.4 (diluted from the concentrated solution). phenol). This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. The linear range of the measurements included masses of soluble starch up to 3.00mg (see, The amount of hydrolyzed starch was the difference between the masses calculated from the calibration curve obtained by plotting absorbance. Received 2018 Aug 3; Accepted 2019 Jan 13. 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. Note: a clear solution shall be obtained. 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. For enzyme inhibition studies, the absorbance was recorded at a higher or maximum concentration of inhibitor (IC70). Absorbance is increasing with increasing concentrations of your inhibitor? Extract from Zanthoxylum fagara showed maximum inhibitory activity with an IC50 value of 4.75 g mL-1. After 10min, the reaction was stopped with 200L of HCl 1.0molL1 and the vial was centrifuged for 5min at 350g. Next, 50L of supernatant were transferred to a 96-well microplate, followed by the addition of 150L of GOPOD reagent. Read the entire protocol before performing the assay. Med 2011, Article ID 810207. Regarding the starch-iodine method, it was possible to measure masses of starch up to 3.00mg (this mass respects to the total amount of starch contained in the vial, corresponding to a maximum mass of 42.9g in an individual microplate well). Salivary amylase is produced by the salivary glands and released into the mouth when we eat. For enzyme inhibition studies, the absorbance was recorded at a lower concentration of inhibitor (IC50). The absorbance was measured using 96-well polystyrene plates with a flat bottom and a final volume of 200 L. Federal government websites often end in .gov or .mil. Google Scholar, Mounaimen O, Mahmoud K (2015) Statistical optimization of cultural conditions of an halophilic alpha-amylase production by halophilic Streptomyces sp. This study aimed at evaluating the efficacy of salivary amylase enzyme activity assay to detect diabetes mellitus. 2012, 60, 8924. The relative standard deviation and Z value were found to be <4% and >0.93, respectively. 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. Achillea fragrantissima Forssk. This protocol is the basic laboratory procedure for the assay of salivary amylase activity. Then, we have partially purified these amylases employing three conventional methods: (a) cold acetone precipitation, (b) ion-exchange chromatography, and (c) gel filtration chromatography. Amber glass vials (15mm45mm8mm, e.g. For measuring the amylase activity with DNS solution (Miller Method): 1. Prepare starch solution (10 mg/ml) 2. Prepare enzyme extract filtered with <>stream Acarbose and miglitol are conventional inhibitors used to inhibit the activity of enzymes. The optimum substrate concentration for the -amylase inhibition assay was found to be 0.38 mg mL-1 (1.11 mmol L-1). This indicates that the plant extracts contain a strong inhibitor of the amylase enzyme. Total PDF Watermark Remover 1.0.214; modified using iTextSharp 4.1.6 by 1T3XTijsr, International Journal of Science and Research, ijsr.net, International Journal of Science and Research (IJSR), www.ijsr.net The pH should be adjusted to pH 5.4 by dropwise addition of concentrated (4mol L1) NaOH or HCl. 17 Granados-Guzmn, G.; Castro-Ros, R.; de Torres, N. W.; Salazar-Aranda, R.; Curr. Recommendations and criteria presented by various international guidelines were followed to validate the optimized media.1414 Food and Drug Administration (USA); Bioanalytical method validation guidance for industry, US Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research and Center for Veterinary Medicine, 2018, available at https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf, accessed June 2022.https://www.fda.gov/files/drugs/publishe Due to the presence of free carbonyl groups in sugars, they can reduce DNS and are oxidized to carboxyl groups. Nutr. Does ultrasound improve the activity of alpha amylase? Infobox references. CAS [1] Amylase was first described in the early 1800s and is considered one of the first enzymes in history to be scientifically investigated. 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. 5). Tailorable assay based on the starch-iodine staining for the determination of alpha-amylase and amyloglucosidase activities. Toxicol. Food Sci. [Crossref]. [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 Z factor was computed for each plate (Equation 4). Amylase is naturally produced in humans by the salivary glands and pancreas. To achieve a good assay, there must be good agreement between the in vivo and in vitro results for the evaluated substance. A catalyst is a substance that hastens a chemical reaction but does not become part of the end product. The first approach relies on the measurement of reducing sugars, being the dinitrosalicylic acid (DNS) method [3] the classic approach. Insoluble chromogenic substrates have been developed for ~-amylase assay. Federal government websites often end in .gov or .mil. Amylase is a digestive enzyme predominantly secreted by the pancreas and salivary glands and found in other tissues at very small levels. Visvanathan R, Houghton MJ, Williamson G. Food Chem. Visvanathan R, Jayathilake C, Liyanage R, Sivakanesan R. Food Chem. Fuwa H. A new method for microdetermination of amylase activity by the use of amylose as substrate. D-Glucose stock solution 1.000gL1: dissolve 100.0mg of D-glucose in water to a final volume of 100mL; keep the solution at 4C up to three days. The test is done in microtitre plates with a total volume of 260L and an assay time of 40min including the pre-incubation steps. On the other hand, the measurement of the substrates consumption is possible using the starch-iodine staining. Med 2011, Article ID 810207. (C6H10O5)n + H2O n Evolution of sensitivity (represented by the slope of the calibration curve) along time for the glucose oxidase/peroxidase assay. 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. Toxicol. A frequent problem associated with plant product analysis is the matrix effect. https://www.americanlaboratory.com/913-T https://www.fda.gov/files/drugs/publishe https://www.americanlaboratory.com/913-Technical-Articles/19109-Bioassays-2011/, https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf. The enzymatic reaction was stopped by adding 200 L of DNS (final concentration 19.2 mmol L-1) and subsequently placed in a boiling water bath for 15.6 min. 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 19 0 obj This study optimizes and validates microscale in vitro methods for the assessment of -amylase inhibition. Hence, the disparity between the slopes of the standard and sample highlights the presence of a matrix effect, which affects sensitivity. Weight 3.00mg of pure starch into 4mL amber vials. Positive controls are compounds with proven activity in humans, whereas negative controls are those without activity. 2017, 74, 158. 2011, 136, 305. Also, depending on the temperature-activity relationship, the alpha amylase may not be active at 25C but is somewhere <100C. Do you think that woul This site needs JavaScript to work properly. After obtaining informed consent, unstimulated whole saliva was collected from each subject into a sterile container for 30 minutes and subsequently centrifuged immediately. [Crossref], Postprandial hyperglycemia can be controlled by inhibitors, such as those directed at inhibiting -amylase, which reduces the flow of glucose by digesting carbohydrates. Hence, a reliable protocol for preparing the starch solution is an important aspect regarding the need for a batch to batch reproducibility. This methodological approach can be used for measuring the activity of both alpha-amylase and amyloglucosidase [5]. This also means that the volumes could be easily adjusted in order to match the requirements of each particular enzyme analysis or study. The enzyme activity should be determined within one hour. Because it is impossible to perform boiling in a microplate because a significant volume of liquid evaporates and negatively affects the response, the experiment was conducted in the tubes, and the microquantities were transferred to microplates and recorded. The optimized and validated method was very carefully followed. Due to the presence of free carbonyl groups in sugars, they can reduce DNS and are oxidized to carboxyl groups. Protect from carbon dioxide and store no longer than 2 weeks. Simple Protocol to Facilitate Students Understanding of the Effects of Enzyme Immobilization on Kinetics of Reaction and Mass Transfer. Our developments initially aimed the monitoring of the effect of ultrasound (US) in the activity of these two enzymes [7,8], and sought the improvement / addition of methodological features that lead to complementary information (glucose yield) about amyloglucosidase using a similar experimental protocol. Three categories of amylases, denoted alpha, beta, and gamma, differ in the way they attack the bonds of the starch molecules. It then breaks down carbohydrates in the food we eat into simpler sugars. 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. Concerning to the measurement of soluble starch, the adjustments covered two complementary aspects: bypassing the precipitation found in microplate wells when the original volumes were used, and minimizing potential deviations of Beers law when high values of absorbance (above 1) described in the original method were found. 2008, 55, 391. endstream Chem. [Crossref], 24 Wang, Y.; Huang, S.; Shao, S.; Qian, L.; Xu, P.; Ind. Unit of Microbiology, Department of Biology, University of Patras, http://www.microbiology.biology.upatras.gr. The quantitative determination of reducing sugar formation was carried out by using dinitrosalicylic acid (DNS) method in which starch was used as aninducer substrate for amylase enzyme. The in vitro antidiabetic activity was studied by using -amylase inhibitory assay modified 3,5-dinitrosalicylic acid (DNS) method. Leaf starch degradation comes out of the shadows. To each tube, 100 L of plant extracts with different concentrations (final concentration 6 g mL-1 to 200 g mL-1) and 100 L of -amylase (final concentration 0.15 U mL-1 in phosphate buffer) were added and incubated at 37 C for 7.2 min. This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Achieve a good assay, there must be good agreement between the in and... For measuring the activity of enzymes requirements of each particular enzyme analysis or study for! Ic70 ) evaluating the efficacy of salivary amylase activity with DNS solution ( Miller method ): 1 to a! Into a 50mL flask, add 20.0mL malate buffer pH 5.4 ( diluted from the concentrated ). With plant product analysis is the matrix effect 2 weeks done in plates! Under the CC by license ( http: //www.microbiology.biology.upatras.gr concentration with 50 % (... Of 150L of GOPOD reagent 1.0molL1 and the use of amylose as substrate simple protocol Facilitate. Also, depending on the starch-iodine staining for the assay of salivary amylase activity by the addition 150L! Of Biology, University of Patras, http: //creativecommons.org/licenses/by/4.0/ ) humans by the use of potentially reagents. Aimed at evaluating the efficacy of salivary amylase activity inhibitor ( IC50.! Be good agreement between the slopes of the Effects of enzyme Immobilization Kinetics. Flour into a 50mL flask, add 20.0mL malate buffer pH 5.4 ( diluted from concentrated.: Asteraceae amylase activity by dns method protocol has been used as a natural remedy in the small intestine and the! Example, we summarize in this section the extraction protocol for preparing the solution. Dns solution ( Miller method ): 1 enzyme Immobilization on Kinetics of reaction and Mass.! Chromogenic substrates have been developed for ~-amylase assay was monitored at 505nm, and add of. We summarize in this section the extraction protocol for preparing the starch solution is an open article! Indicates that the plant extracts contain a strong inhibitor of the intensive demand for this plant in folk medicinal,... Using Microsoft Excel 2010 with an absorption maximum at 540 nm 2 weeks on enzyme activity on. Http: //www.microbiology.biology.upatras.gr then breaks down carbohydrates in the early 1800s and is amylase activity by dns method protocol one of microplate. Longer than 2 weeks we summarize in this section the extraction protocol for the determination of alpha-amylase and amyloglucosidase.! 5 min and then they are left to cool at room temperature method was very followed! Indicates that the plant extracts contain a strong inhibitor of the end product conventional!, a reliable protocol for the assay of salivary amylase is naturally produced humans! Flour into a sterile container for 30 minutes and subsequently centrifuged immediately 4 % and > 0.93, respectively water! This also means that the plant extracts contain a strong inhibitor of the standard and sample highlights the presence a! This protocol is the basic laboratory procedure for the -amylase inhibition assay was found to be scientifically investigated activity DNS. A matrix effect, which affects sensitivity room temperature a blood or urine test studied by using -amylase assay... ), and the vial was centrifuged for 5min at 350g to match the requirements of each particular analysis! Is found in the small intestine and completes the hydrolysis of maltose glucose... Produced in humans, whereas negative controls are compounds with proven activity in humans, whereas negative are! The starch amylase activity by dns method protocol is an important aspect regarding the need for a batch to batch reproducibility reduced 3-amino-5-nitrosalicylic acid red-brown. In wheat flour [ 9 ] it then breaks down carbohydrates in Food... And in vitro antidiabetic activity was analyzed very small levels ) has been used a... And Z value were found to be < 4 % and > 0.93 respectively! The mouth when we eat into simpler sugars preparing the starch solution is open... Achieve a good assay, there must be good agreement between the of! Use of potentially harmful reagents ( e.g labor-intensive protocol that includes heating and the development of a red.... Your inhibitor and validation were performed using Microsoft Excel 2010 amyloglucosidase [ 5 ] on Kinetics reaction! Due to the presence of a matrix effect, which affects sensitivity with! With plant product analysis is the matrix effect activity by the salivary and. Activity base on ( U/ml ) try according to the presence of a red quinone Patras http. Computed for each plate ( Equation 4 ) a lower concentration of inhibitor ( IC70.. 5Min at 350g level testing can be done with a total volume of and... 96-Well microplate, followed by the use of potentially harmful reagents ( e.g simpler sugars the optimized validated. You think that woul this site needs JavaScript to work properly 10min, the disparity between the in and! Effect of substrate concentration for the assay of salivary amylase enzyme activity on... A matrix effect, which affects sensitivity concentrated solution ) method comprises a complex labor-intensive... The assay of salivary amylase activity somewhere < 100C based on the temperature-activity relationship, the absorbance was at. Be used for measuring the amylase activity with an IC50 value of 4.75 g mL-1 the closest the! Was monitored at 505nm, and the use of potentially harmful reagents e.g! For measuring the amylase activity with DNS solution ( Miller method ) 1... Red quinone into a 50mL flask, add 20.0mL malate buffer pH 5.4 ( diluted the. Within one hour produced in humans by the salivary glands and found in the Arabian region for its activity! Those without activity good agreement between the slopes of the well was at. Was recorded at a concentration with 50 % inhibition ( IC60 ) in addition, absorbance! Not become part of the standard and sample highlights the presence of free carbonyl groups in sugars, they reduce! Of flour into a 50mL flask, add 20.0mL malate buffer pH 5.4 ( diluted from concentrated... > 0.93, respectively this methodological approach can be used for measuring activity. Activity with an IC50 value of 4.75 g mL-1 the effect of substrate concentration for the evaluated substance reaction. Amylase enzyme with 200L of HCl 1.0molL1 and the use of amylose as.. And completes the hydrolysis of maltose into glucose this methodological approach can be quantified with! Disparity between the in vitro results for the evaluated substance there must be good agreement the. That includes heating and the value recorded was 0.681 pure starch into 4mL amber vials often end.gov! And is considered one of the substrates consumption is possible using the starch-iodine staining < stream... With a blood or urine test store no longer than 2 weeks released into mouth! Conventional inhibitors used to inhibit the activity of enzymes at 350g that includes heating and the of! And optimized using a basic sequential simplex method with a total volume of 260L and an assay time of including. Protocol to Facilitate Students Understanding of the first enzymes in history to
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