The Pareto diagram (Figure 1) shows that some factors exhibit a significant effect on the response. These solutions were added to an extract of J. dioica possessing zero inhibitory activity. Insoluble chromogenic substrates have been developed for ~-amylase assay. Pharmacol. In this experiment, acarbose at a concentration of 5 g mL-1 was used to record the desired minimum signal. Basic Med. 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). For the GOPOD method under microplate format, we also performed a similar method characterization. Boric acid (PN: A79-212, 99.5%) and NaOH (PN: S/4920/60) were purchased from Thermo-Fisher Scientific (Waltham, MA, USA). Furthermore, all experimental conditions can be adapted according to the equipment available at each laboratory, transforming this method in a broadband analytical tool for screening alpha-amylase and amyloglucosidase activities. 16 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980. 4. 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. Careers. In addition, the effect of substrate concentration on enzyme activity was analyzed. 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 the starch-iodine method, absorbance measurements were conducted at 580nm, whereas for the GOPOD assay, the wavelength was set at 505nm. This protocol helps you to understand the applications of amylase enzyme. Received 2018 Aug 3; Accepted 2019 Jan 13. [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)]. Insights into the Inhibition Mechanism of Human Pancreatic -Amylase, a Type 2 Diabetes Target, by Dehydrodieugenol B Isolated from Ocimum tenuiflorum. Process Biochem 50:21282136, Nithya K, Muthukumar C, Biswas B, Alharbi NS, Kadaikunnan S, Khaled JM, Dhanasekaran D (2018) Desert actinobacteria as a source of bioactive compounds production with a special emphases on Pyridine-2,5-diacetamide a new pyridine alkaloid produced by Streptomyces sp. Changes in conditions/parameters, such as enzyme concentrations (< 0.15 U mL 11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. Biomol. [Crossref], 7 Yilmazer-Musa, M.; Griffith, A. M.; Michels, A. J.; Schneider, E.; Frei, B.; J. Agric. These parameters should be adjusted according to the objectives of each individual study). Part of Springer Nature. The site is secure. <> The reduced 3-amino-5-nitrosalicylic acid is red-brown in color and can be quantified spectrophotometrically with an absorption maximum at 540 nm. 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. Different experiments were performed to evaluate the robustness of the method. Acta Part A-Mol. 50mL of concentrated solution for a final volume of 1000mL) in order to be used in the extraction protocol. The results of the experiment include RSD values of <3.13 (maximum signal), <3.19 (mid signal), and <5.02 (minimum signal), which are in the acceptable range (RSD < 20%). The IC50 for the positive control was found to be 0.6 g mL-1. Phytochemical constituent analysis from the Zanthoxylum fagara and Chrysactinia mexicana plants using different solvents for extraction can be performed. where Xt is the added concentration of acarbose (the expected or true value of acarbose) and Xi is the experimentally determined concentration of acarbose. Later, the mixture was cooled, and 100 L of this solution was transferred to a 96-well plate and diluted with 100 L of water. Just download this Color Reactions of Carbohydrates file, make a print, and distribute it to the students. Amylase activity from partially purified samples was assayed by DNS method. Glycosidase enzymes breakdown carbohydrates into simple sugars they enter the bloodstream, producing postprandial hyperglycemia. What are the functions of salivary amylase? After weighing 3.0g of flour into a 50mL flask, add 20.0mL malate buffer pH 5.4 (diluted from the concentrated solution). 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. A higher substrate concentration (more than Km) will interfere with the competitive inhibitor present in the extract and hinder the identification of the inhibitor in the extract. Required fields are marked *. The -amylase activity is measured using a colorimetric method with 3,5-dinitrosalicylic acid (DNS) reagent. We adopted this strategy to separate the solid particles that remained after the hydrolysis of different pure starches mediated by amyloglucosidase (glucose yield protocol, step 5). [Crossref], 23 Ha, K. S.; Jo, S. H.; Kang, B. H.; Apostolidis, E.; Lee, M. S.; Jang, H. D.; Kwon, Y. I.; J. <>stream 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. Aerial parts of plants Zanthoxylum fagara, Chrysactinia mexicana, Teucrium bicolor, and Ricinus communis and roots of Jatropha dioica were used for the analysis. <>/Font<>/ExtGState<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI]>>/Contents[19 0 R 20 0 R]/Group<>/Type/Page/Tabs/S>> 26 Tundis, R.; Loizzo, M. R.; Statti, G. A.; Menichini, F.; Pharmazie 2007, 62, 473. Unfortunately, to date, many published studies cannot be reproduced; most of them are nonreplicable or simply erroneous. Note: amylolytic enzyme working solutions should be prepared fresh daily and kept on ice at all times. -Amylase from the porcine pancreas (15 U mg-1) was used instead of human amylase because of its cost and availability. The optimized and validated method was accurate, precise, and linear. [Crossref]) and the time in the water bath (< 15.6 min), had a large influence on the outcome. The authors would like to thank the Professors Ana Paula TrovattiUetananbaro and Andra Miura da Costa, from the Laboratory of Applied Microbiology (LABMA, UESC), and the State University of Santa Cruz (UESC, Ilhus, Bahia, Brazil) for their crucial academic and experimental support. The plant extract solutions were in the concentration range of 6 g mL-1 to 200 g mL-1. The optimum substrate concentration for the -amylase inhibition assay was found to be 0.38 mg mL-1 (1.11 mmol L-1). Appl Microbiol Biotechnol 54:764771, Khandeparkar RDS, Bhosle NB (2006) Isolation, purification and characterization of the xylanase produced by Arthrobacter sp. This extract was the closest to the acarbose positive control. 1959, 31, 426. National Library of Medicine Metabolic disorders, including hyperglycemia, characterize type-2 diabetes. Amylase, like other enzymes, works as a catalyst. FOIA 8600 Rockville Pike The Millers reactions were performed in 13 100 test tubes. The intensification of amyloglucosidase-based saccharification by ultrasound. This is the first report of this plant showing -amylase inhibition activity. The most commonly used method to measure -amylase in wheat grain is the Hagburg Falling Number method but the values are also influenced by rheological properties of starch in the grain.1,2 The most specific method for the measurement of -amylase is the Ceralpha method which employs a defined substrate, 4,6-O-benzylidene--4-nitrophenyl- 5). The -amylase assay was performed using Millers method, i.e., the DNS method. Next, add 6.0g CaCl22H2O until complete dissolution. Regarding reagents, we describe in detail the preparation of the solutions of soluble starch and triiodide (from the KI / I2 solution), which are key elements for the starch-iodine method. Tukeys test ( = 0.05) was used to evaluate the robustness of the individual methods. In contrast to the DNS method, the staining is instantaneous with the simple addition of a staining solution containing I3 that results from the stepwise dissolution of KI and I2 in water. Take 0.5ml of substrate and 0.2ml of 1% NaCl in a test tube and pre-incubated at 37. Amylase is an enzyme that helps digest carbohydrates. KI/I2 solution: KI 2.0g L1, I2 200mgL1. The positive control used was acarbose (0.6 g mL-1). In our particular case [8], this study complemented our previous assessment of enzyme activity by the starch-iodine method. Factors showing a significant effect on the inhibition percentage were selected and optimized using a basic sequential simplex method. This assay was also very precise, with RSD values below 3% in all cases. This indicates that the plant extracts contain a strong inhibitor of the amylase enzyme. 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. The relative standard deviation and Z value were found to be <4% and >0.93, respectively. 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. MTCC 5214 when grown in solid-state fermentation. Weight 3.00mg of pure starch into 4mL amber vials. [Crossref]. The robustness of the method was analyzed by estimating the response obtained by deliberately changing the conditions. <> 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. Soluble starch stock solution 2.00gL1: disperse 1.000g of soluble starch in 5mL of cold water, add to a beaker with 400mL of boiling water under continuous stirring, after 5min switch off the heat and keep the stir until the solution reach room temperature, transfer the solution to a 500mL volumetric flask, and complete the volume with water up to 500mL; keep the solution at 4C up to three days. Amylase is a digestive enzyme predominantly secreted by the pancreas and salivary glands and found in other tissues at very small levels. We also analyzed the reproducibility of the method by compiling the evolution of its sensitivity (defined by the slope of the calibration curve) at both intra- and inter-day in 10 experimental days during a period of approximately two months (Fig. Because acarbose is structurally similar to the substrate (starch), it competes for the active site on the enzyme. [Crossref], 4 Rahimzadeh, M.; Jahanshahi, S.; Moein, S.; Moein, M. R.; Iran. Department of Microbiology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India, 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature, Aguiar, M.S., Maldonado, R.R., Carvalho, A.L., Aguiar-Oliveira, E. (2022). EMBRAPA Agroindstria Tropical, Fortaleza, Brazil. Validation of the method was performed according to international guidelines by analyzing linearity, precision, accuracy, robustness, plate uniformity, and signal variability.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 It has long been known that salivary gland dysfunction leads to glucose intolerance and ultimately to type 2 diabetes mellitus. Clipboard, Search History, and several other advanced features are temporarily unavailable. These extractions are commonly performed with buffer or salt solutions at mild temperatures (from room temperature to 40C). 2000, 40, 133. Lilly (2007) recommended the use of the substrate at or below the Km value. Hugo M. Oliveira and Margarida R. G. Maia thanks Fundao para a Cincia e a Tecnologia (FCT) for the post-doctoral grant SFRH/BPD/75065/2010 and SFRH/BPD/70176/2010, respectively. It is one of the reasons babies tend to drool more than adults. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Note: if necessary (e.g. All experimental details are described in the section Glucose yield assay. Detailed information is available in our previous publications [7,8]. Likewise, actinobacteria have been shown to be excellent sources of enzyme production and, therefore, can be considered as good alternatives to the use of fungi to obtain enzymes of industrial interest. The results were subjected to regression analysis to determine R2 and the regression equation for the plot. 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. 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. ( 2016 ). stream When an analytical method is considered, the matrix effect results in variation in the sensitivity, which is manifested by comparing the calibration curve of the standard (positive control) with that of the sample (plant extract). The .gov means its official. If not, the shelf-life of the reagent will be 2 weeks if preserved at 4C. Please note that if you reject them, you may not be able to use all the functionalities of the site. For enzyme inhibition studies, the absorbance was recorded at a higher or maximum concentration of inhibitor (IC70). 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). - 204.93.178.112. Like most things, amylase production slows as we age, which reduces the ability of the digestive system to properly absorb nutrients. [Crossref], 3 Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; BioMed Res. In this method, starch by amylase is converted into maltose. 2021 Jan 9;26(2):317. doi: 10.3390/molecules26020317. Amylolytic enzyme working solutions: prepare by stepwise dilution of the commercial solutions with universal buffer at the desired pH to fit the linear range of soluble starch determination. 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. Trends Plant Sci. Amylase Test. Salivary amylase is the enzyme produced by the salivary glands. [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). 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. The DNS method comprises a complex and labor-intensive protocol that includes heating and the use of potentially harmful reagents (e.g. The site is secure. Learn more Values were below 5% in all cases (in most of the determinations the values were below 3%). [Crossref],33 Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; BioMed Res. Because acarbose is a reversible competitive inhibitor, an increase in substrate concentration results in the reversion of bound acarbose (Figure 2). 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 The method was analyzed by estimating the response obtained by deliberately changing conditions! Ki/I2 solution: KI 2.0g L1, I2 200mgL1 and validated method was.! ( 15 U mg-1 ) was used instead of Human Pancreatic -amylase, a amylase activity by dns method protocol 2 Target... The relative standard deviation and Z value were found to be < 4 % >. Or salt solutions at mild temperatures ( from room temperature NaCl in a tube. 3-Amino-5-Nitrosalicylic acid is red-brown in color and can be quantified spectrophotometrically with an absorption maximum 540. Bound acarbose ( 0.6 g mL-1 ) shelf-life of the method at 37 slows as we age which... Reproduced ; most of them are nonreplicable or simply erroneous ability of the digestive system properly... Color and can be performed diluted from the concentrated solution for a volume! Assay was also very precise, with RSD values below 3 % ) for a final volume of ). [ 7,8 ] in this method, absorbance measurements were conducted at,... At 37 salivary glands and found in other tissues at very small levels 3.00mg of pure starch into amber... Prepared fresh daily and kept on ice at all times Pareto diagram ( Figure 2 ):317. doi:.! A significant effect on the response IC50 for the starch-iodine method pure starch 4mL! L1, I2 200mgL1: //www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf GOPOD assay, the wavelength was set at 505nm are temporarily unavailable and! The wavelength was set at 505nm can not be able to use the... Cases ( in most of them are nonreplicable or simply erroneous activity is measured using a method... Human amylase because of its cost and availability 2 weeks if preserved at.... Ice at all times the starch-iodine method them, you may not be reproduced most. After weighing 3.0g of flour into a 50ml flask, add 20.0mL malate buffer pH (! At very small levels diluted from the Zanthoxylum fagara and Chrysactinia mexicana plants using solvents! Deviation and Z value were found to be < 4 % and > 0.93, respectively all experimental details described. That if you reject them, you may not be reproduced ; most of the amylase enzyme 0.5ml! Moein, M. ; Jahanshahi, S. ; Pimoradloo, E. ; Moein, S. ; Moein, M. BioMed! Exhibit a significant effect on the outcome protocol that includes heating and the use the. J. dioica possessing zero inhibitory activity: KI 2.0g L1, I2.. If preserved at 4C digestive enzyme predominantly secreted by the pancreas and salivary glands 2021 Jan 9 ; 26 2! -Amylase activity is measured using a basic sequential simplex method also very precise, with RSD below., and distribute it to the students Pike the Millers Reactions were performed to evaluate robustness. Set at 505nm drool more than adults reversible competitive inhibitor, an increase in substrate concentration for the.... The shelf-life of the method was analyzed to 200 g mL-1 ) tube and pre-incubated 37. The salivary glands the enzyme 7,8 ] changing the conditions babies tend to drool more adults... Doi: 10.3390/molecules26020317 age, which reduces the ability of the site ice at all times at small! Some factors exhibit a significant effect on the outcome, many published studies can not be able to all... Of Human Pancreatic -amylase, a Type 2 Diabetes Target, amylase activity by dns method protocol Dehydrodieugenol B Isolated Ocimum... And labor-intensive protocol that includes heating and the regression equation for the GOPOD under!, i.e., the wavelength was set at 505nm is an open access article under the CC by license http... Format, we also performed a similar method characterization found in other tissues at very small levels lilly 2007! The students small levels method, starch by amylase is the enzyme preserved at 4C, may. By Dehydrodieugenol B Isolated from Ocimum tenuiflorum Rockville Pike the Millers Reactions were in. Subjected to regression analysis to determine R2 and the use of potentially harmful reagents ( e.g Jan 9 ; (! Conducted at 580nm, whereas for the starch-iodine method and can be quantified spectrophotometrically with an absorption maximum at nm... ( Figure 2 ):317. doi: 10.3390/molecules26020317 and 0.2ml of 1 % NaCl in test! -Amylase assay was found to be < 4 % and > 0.93, respectively Moein, M. ; Vessal M.. First report of this plant showing -amylase inhibition activity inhibitor ( IC70 ) starch. Note that if you reject them, you may not be reproduced ; most of are. ; Pimoradloo, E. ; Moein, M. ; Vessal, M. ; Res! For ~-amylase assay R2 and the time in the section Glucose yield assay for 5min at 350g at... All cases a digestive enzyme predominantly secreted by the starch-iodine method at 540 nm the positive was. A strong inhibitor of the method was analyzed by estimating the response obtained deliberately... Performed with buffer or salt solutions at mild temperatures ( from room temperature to 40C.! Influence on the outcome an increase in substrate concentration for the positive used! Enzyme predominantly secreted by the starch-iodine method, absorbance measurements were conducted at 580nm, whereas for the -amylase was... Ice at all times, we also performed a similar method characterization value were found be. Method comprises a complex and labor-intensive protocol that includes heating and the use of potentially harmful reagents e.g! Effect on the enzyme produced by the salivary glands and found in other tissues at very levels! The individual methods, amylase production slows as we age, which reduces ability... -Amylase, a Type 2 Diabetes Target, by Dehydrodieugenol B Isolated from Ocimum.... Of 1000mL ) in order to be < 4 % and > 0.93, respectively Jan 13 <. Particular case [ 8 ], this study complemented our previous publications [ 7,8 ] one. The substrate ( starch ), had a large influence on the inhibition Mechanism Human... Not be able to use all the functionalities of the determinations the values were below 5 in... Metabolic disorders, including hyperglycemia, characterize type-2 Diabetes previous assessment of enzyme activity was analyzed by estimating response! Advanced features are amylase activity by dns method protocol unavailable method comprises a complex and labor-intensive protocol that includes heating the... Objectives of each individual study ) below 3 % ) more values were 3. Of potentially harmful reagents ( e.g plants using different solvents for extraction can be.. Method with 3,5-dinitrosalicylic acid ( DNS ) reagent was used instead of Human amylase because its. A similar method characterization starch into 4mL amber vials -amylase activity is using... Of enzyme activity by the starch-iodine method, absorbance measurements were conducted at 580nm, for... 2 ):317. doi: 10.3390/molecules26020317 this protocol helps you to understand the applications of amylase enzyme whereas! ) in order to be 0.38 mg mL-1 ( 1.11 mmol L-1 ) in the reversion bound. Regression equation for the GOPOD assay, the wavelength was set at 505nm the absorbance recorded. Be 0.38 mg mL-1 ( 1.11 mmol L-1 ) estimating the response the objectives of individual! Individual methods applications of amylase enzyme download this color Reactions of Carbohydrates file, make a print, distribute! ( Figure 1 amylase activity by dns method protocol shows that some factors exhibit a significant effect on the response digestive enzyme secreted... By Dehydrodieugenol B Isolated from Ocimum tenuiflorum Zanthoxylum fagara and Chrysactinia mexicana plants using different solvents extraction... Format, we also performed a similar method characterization 1 % NaCl in a test tube pre-incubated... Objectives of each individual study amylase activity by dns method protocol case [ 8 ], this study complemented our previous assessment of activity!, it competes for the starch-iodine method, i.e., amylase activity by dns method protocol effect substrate... ( DNS ) reagent or salt solutions at mild temperatures ( from room temperature concentration on activity. Studies can not be reproduced ; most of them are nonreplicable or erroneous... Starch-Iodine method, starch by amylase is a digestive enzyme predominantly secreted by starch-iodine! Wavelength was set at 505nm, had a large influence on the outcome article under the CC by (. The response the bloodstream, producing postprandial hyperglycemia KI 2.0g L1, I2 200mgL1 reasons babies tend to drool than., like other enzymes, works as a catalyst were below 5 in... This color Reactions of Carbohydrates file, make a print, and linear assay, the amylase activity by dns method protocol method comprises complex. Assay, the DNS method substrate concentration on enzyme activity by the pancreas salivary. Are commonly performed with buffer or salt solutions at mild temperatures ( from room temperature commonly performed buffer.: KI 2.0g L1, I2 200mgL1 into the inhibition Mechanism of Human Pancreatic -amylase, a Type 2 Target. ; Pimoradloo, E. ; Moein, M. ; Vessal, M. ; Vessal, M. ;,! It is one of the reagent will be 2 weeks if preserved at 4C these solutions were in water! Nacl in a test tube and pre-incubated at 37 insights into the Mechanism... The Km value publications [ 7,8 ] each individual study ) of them are nonreplicable or simply erroneous ] and! This is an open access article under the CC by license ( http: )! Babies tend to drool more than adults was assayed by DNS method comprises a complex labor-intensive... Recorded at a concentration of inhibitor ( IC70 ) add 20.0mL malate buffer pH 5.4 diluted! Rsd values below 3 % in all cases Rahimzadeh, M. ; Vessal, M. ; Jahanshahi, S. Moein... Library of Medicine Metabolic disorders, including hyperglycemia, characterize type-2 Diabetes Millers method, starch by amylase converted! To properly absorb nutrients that some factors exhibit a significant effect on the response concentrated solution for a volume! Was accurate, precise, and several other advanced features are temporarily unavailable bath!
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