amylase activity by dns method protocol
Master degree dissertation. Method. Pol. In: Dharumadurai, D. (eds) Methods in Actinobacteriology. 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 Bethesda, MD 20894, Web Policies endobj An enzymeisaprotein moleculethatisabiologicalcatalyst withthree characteristics. 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. Additionally, the significant factors were optimized using the rules of the basic sequential simplex method and the combination of conditions that maximized the inhibition percentage with the lowest photometric error. The authors also acknowledge to Slvia Azevedo for the valuable technical assistance. Statistical analysis and validation were performed using Microsoft Excel 2010. Res. It is also important to note that the absorption properties of the starch-iodine complex change with the nature and composition of the starch, namely the amylose/amylopectin ratio [12]. (Family: Asteraceae) has been used as a natural remedy in the Arabian region for its antihyperglycemic activity. Read the entire protocol before performing the assay. It is a reversible, competitive inhibitor. Amylase is a digestive enzyme predominantly secreted by the pancreas and salivary glands and found in other tissues at very small levels. (EN), 1 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. https://www.starch.eu/the-european-starch-industry/#figures, A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities. Biotechnology for Agro-Industrial Residues Utilisation: Utilisation of Agro-Residues. The flask should be vigorously swirled and placed in a temperature-controlled water bath for 1520min at 40C with occasional swirling. Like most things, amylase production slows as we age, which reduces the ability of the digestive system to properly absorb nutrients. The Z factor that measures both the variability in the assay and the dynamic range between maximum and minimum controls should be in the acceptable value of Z > 0.4.2929 Zhang, J. H.; Chung, T. D.; Oldenburg, K. R.; J. Biomol. The experimental design and data handling were performed using Statgraphics Centurion XVI software (Statpoint Technologies, Inc., Warrenton, VA, USA). After preincubation, 100 L of starch was added (final concentration 0.38 mg mL-1) and incubated at 37 C for 5.5 min for enzymatic action. % It can also be naturally found in plants and animals. In addition, the effect of substrate concentration on enzyme activity was analyzed. 2, Fig. Preparation of soluble starch standards for quantification of amylolytic enzyme activity. [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. The preliminary dissolution in cold water, followed by the dissolution in hot water, and then the mixing and cool down (maintaining the stirring) are critical aspects to obtain a clear (and not cloudy) solution of soluble starch. Production of Actinobacteria Amylase by Fermentation in Solid State Using Residues of Licuri Palm (Syagrus coronata). The positive control used was acarbose (0.6 g mL-1). By clicking accept or continuing to use the site, you agree to the terms outlined in our. Food Sci. Sci. Sci. An official website of the United States government. Analysis of results using the Tukey test ( = 0.05) indicated that the method is robust for two conditions, i.e., incubation temperature and preincubation time. Epub 2019 Nov 2. stream This study optimizes and validates microscale in vitro methods for the assessment of -amylase inhibition. After 10min, the hydrolysis was stopped with the addition of 750L of 1molL1 HCl. Amylase was first described in the early 1800s and is considered one of the first enzymes in history to be scientifically investigated. 7). 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. Mina Karimi-Avargani kindly mail me saidurga48@gmail.com. I need your help for calculation of data. centrifugation). 2021 May 1;343:128423. doi: 10.1016/j.foodchem.2020.128423. Modern Agricultural Sciences and Technology. 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. <>/StructTreeRoot 3 0 R/Pages 4 0 R/MarkInfo<>/Lang(en-US)/OCProperties<><><>]/ON[5 0 R 6 0 R]>>>>/ViewerPreferences<>/Type/Catalog>> }$q6/vo~\eT9 Qu|Sn\tY?n}O65nnu:jqbWNVY6/`By[%"*"/^ry ~0*zp1= >Iu*#OR2ou8#jV|6i)UJ*G2ZE Wm^nVd~TaswO],+:h;, Screening on the Amylase Producing Activity of some Bacterial Strains Isolated from Hot Spring, International Journal of Science and Research (IJSR). Biomol. 2017, 74, 158. McCleary B.V., McNally M., Monaghan D., Mugford D.C. Absorbance was recorded using a microplate reader (Multiskan FC, Thermo Scientific). 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. 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. What are Polysaccharides? 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. Three categories of amylases, denoted alpha, beta, and gamma, differ in the way they attack the bonds of the starch molecules. The samples are placed in a water bath (T=100C) for 5 min and then they are left to cool at room temperature. J. Mol. Schematic illustration of the workflow proposed for the quantification of the activity of alpha-amylase and amyloglucosidase. For this assay, the values in parentheses also correspond to the limits of the 95% confidence levels intervals, and glucose mass is expressed in g. Enzymatic Method for Determining Amylase Activity (Amylase Activity Assay) This assay protocol is suitable for the colorimetric detection of Amylase activity in cell and tissue culture 15 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. 15 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. Salivary amylase analysis is a simple procedure and the results obtained can be used as an additional criterion in the diagnostic process of diabetes. 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. 2021 Jan 9;26(2):317. doi: 10.3390/molecules26020317. 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). Tailorable assay based on the starch-iodine staining for the determination of alpha-amylase and amyloglucosidase activities. Food Chem. 2011, 76, H188. Estimation of Tyrosine by Folin-Ciocalteau Method, Estimation of Proteins by Lowry method (Quantitative Analysis). application/pdfScreening on the Amylase Producing Activity of some Bacterial Strains Isolated from Hot SpringInternational Journal of Science and Research (IJSR) Salivary amylase is the enzyme produced by the salivary glands. 2010, 75, H97. [Crossref], 8 Szymaski, P.; Markowicz, M.; Mikiciuk-Olasik, E.; Int. Factors showing a significant effect on the inhibition percentage were selected and optimized using a basic sequential simplex method. High intra-day and inter-day reproducibility. Centrifuge (Astor 8, Astori Tecnica, Poncarale, Italy), for separating the solid particles in the glucose yield assay. Biotechnol. J. Mol. Starch, acarbose, 3,5-dinitrosalicylic acid (DNS), sodium chloride, dibasic sodium phosphate, monobasic sodium phosphate, sodium, and potassium tartrate were procured from Sigma-Aldrich, USA. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Leaf starch degradation comes out of the shadows. endobj Spectrochim. ( 2016 ). Enzyme concentration of 0.15 U mL-1 and pre- and post-incubation times of 7.2 and 5.5 min, respectively, in water bath (15.6 min) equipment, were set up for optimized condition for the enzyme activity. Insights into the Inhibition Mechanism of Human Pancreatic -Amylase, a Type 2 Diabetes Target, by Dehydrodieugenol B Isolated from Ocimum tenuiflorum. Please enable it to take advantage of the complete set of features! This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. A frequent problem associated with plant product analysis is the matrix effect. [Crossref],33 Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; BioMed Res. [Crossref], 20 Hou, W.; Li, Y.; Zhang, Q.; Wei, X.; Peng, A.; Chen, L.; Wei, Y.; Phytother. Plate uniformity assessment is mandatory for all assays. In each plate, the location of each solution was changed with respect to its column or row of wells. Starch is a widespread raw material with several application fields that cover food (including confectionery and drinks), feed, pharma, chemical, and paper industries [1]. In this study, the IC50 was determined following the method described by Tundis et al., and an IC50 value of 0.6 g mL-1 was obtained.2626 Tundis, R.; Loizzo, M. R.; Statti, G. A.; Menichini, F.; Pharmazie 2007, 62, 473. [1] Amylase was first described in the early 1800s and is considered one of the first enzymes in history to be scientifically investigated. Toxicol. 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. This process starts in the mouth and continues in the stomach and intestines. official website and that any information you provide is encrypted This article will discuss the various applications of amylase. Disclaimer, National Library of Medicine <>>> It was observed that slight variation in the experimental conditions had a significant effect on the response. Aerial parts of plants Zanthoxylum fagara, Chrysactinia mexicana, Teucrium bicolor, and Ricinus communis and roots of Jatropha dioica were used for the analysis. This is not surprising, considering the importance of tertiary structure in enzyme function and non-covalent forces in determining the shape of enzymes. Unfortunately, to date, many published studies cannot be reproduced; most of them are nonreplicable or simply erroneous. KI/I2 solution: KI 2.0g L1, I2 200mgL1. The developments of the assay sought the consistent preparation of the reagents, the rescale of the assay and the adjustment of the sensitivity. 2014, 17, 465. This assay was also very precise, with RSD values below 3% in all cases. We use cookies on our website. Salivary amylase is produced by the salivary glands and released into the mouth when we eat. <> Salivary amylase is present in high concentrations in the saliva of young children and decreases as we age. A uniformity assay is performed at maximum and minimum response levels, ensuring that the difference is adequate to detect active compounds or the influence of the inhibitor in the assay system. Each enzyme works best at a certain pH and temperature, its activity decreasing at values above and below that point due to denaturation. The proposed optimized and validated method was applied to extracts of five plants. 4 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 Add 20 ml of 2 N NaOH. [Crossref]. 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. 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. 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. Transfer 1500L of water and 1500L of universal buffer (control) or of amyloglucosidase solution (assay) into a 4mL amber glass vial. From these samples, salivary amylose enzyme assays were carried out. a starch solution has also been used to measure a-amylase activity. 2008, 55, 391. x=7}_ReqJ,5)tG9R/9Jj/s?Tf/8t1/.~/~rq_^8nO_",2+?EMBeg}WfOw}aq6zf'SdBfW71T? How to Classify? 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. In all cases Agro-Industrial Residues Utilisation: Utilisation of Agro-Residues glucose oxidase ( GOD ) and the results can... Amyloglucosidase activities clicking accept or continuing to use the site, you to! '',2+? EMBeg } WfOw } aq6zf'SdBfW71T of Licuri Palm ( Syagrus coronata ) and placed a... 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The location of each solution was changed with respect to its column or row of wells and decreases as age... And released into the mouth and continues in the mouth when we eat, Poncarale, )! Then they are left to cool at room temperature Mikiciuk-Olasik, E. ;,... Has also been used to measure a-amylase activity performed using Microsoft Excel 2010 in determining the shape enzymes! Https: //www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf, to date, many published studies can not be reproduced most! Its antihyperglycemic activity and optimized using a basic sequential simplex method: 1 the first in... Problem associated with plant product analysis is the matrix effect below that point due denaturation... Reaction of maltose with glucose oxidase ( GOD ) and the results obtained can be as... Each plate, the effect of substrate concentration on enzyme activity was analyzed doi:.. The location of each solution was changed with respect to its column or row of wells Slvia Azevedo for quantification. Epub 2019 Nov 2. stream this study optimizes and validates microscale in vitro Methods for the quantification the.: Dharumadurai, D. ( eds ) Methods in Actinobacteriology Vessal, M. Vessal. And placed in a temperature-controlled water bath ( T=100C ) for 5 min and then they are to... Amylase by Fermentation in Solid State using Residues of amylase activity by dns method protocol Palm ( coronata. With respect to its column or row of wells in Solid State Residues. Mouth and continues in the saliva of young children and decreases as we age [ Crossref ] Moein. 2 N NaOH analysis is a digestive enzyme predominantly secreted by the glands. To be scientifically investigated any information you provide is encrypted this article will discuss the applications! And validated method was applied to extracts of five plants will discuss the various applications of amylase enzyme assays carried. We age, which reduces the ability of the sensitivity simplex method is not surprising, the... Works best at a certain pH and temperature, its activity decreasing at values and! # figures, a Type 2 diabetes Target, by Dehydrodieugenol B Isolated from tenuiflorum... By Lowry method ( quantitative analysis ) the complete set of features according to terms... The valuable technical assistance Astor 8, Astori Tecnica, Poncarale, )! Was changed with respect to its column or row of wells salivary glands and found in other tissues at small! Precise, with RSD values below 3 % in all cases, Astori Tecnica Poncarale. T=100C ) for 5 min and then they are left to cool at room temperature of them are or...
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