amylase activity by dns method protocol

In: Singh-Nee Nigam P., Pandey A., editors. 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]. The higher is the intensity of color, the higher is the concentration of reducing sugars in the solution. %PDF-1.5 Chem. 2012, 60, 8924. 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. The linear range of amylase inhibition by acarbose was 1.5-5 g mL-1, with a regression equation of y = 7.5784x + 37.80, a determination coefficient of 0.9906, and an IC50 of 1.61 0.06 g mL-1 (Figure 3). Transfer 1500L of water and 1500L of universal buffer (control) or of amyloglucosidase solution (assay) into a 4mL amber glass vial. EMBRAPA Agroindstria Tropical, Fortaleza, Brazil. 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 2017, 74, 158. 37% (w/w) HCl (PN: 131020) was purchased from Panreac (Barcelona, Spain). 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. Briefly centrifuge vials before opening. Cool and dilute with 10ml of distilled water. [Crossref]. ( 2016 ). where SD is the standard deviation of each measurement, and AVG is the average of such measurements. This site needs JavaScript to work properly. Acarbose was used as an enzyme inhibitor. 1 ml of D.N.S. Epub 2018 Sep 19. The kinetic constant maximum velocity (Vmax) and the Michaelis-Menten constant (Km) were obtained from the Lineweaver-Burk and Michaelis-Menten plots, respectively. 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. Amylase Test. 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 Different parameters, such as enzyme concentration, substrate concentration (starch), preincubation time, incubation time, incubation temperature, and time in the water bath, were evaluated. 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]. 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 The .gov means its official. The reagent has a dark orange colour and is stable for several days in room temperature. 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. This protocol is the basic laboratory procedure for the assay of salivary amylase activity. One negative control (absence of inhibitor) was set up to obtain 100% enzyme activity. J Genet Eng Biotechnol 14:133141, CrossRef Basic Med. 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. Humana, New York, NY. The new method is also compared with the most commonly used 3,5-dinitrosalicylic acid (DNSA) method for determining -amylase activity. Two plant extracts (Zanthoxylum fagara and Chrysactinia mexicana) inhibited -amylase activity. In this experiment, acarbose at a concentration of 2.5 g mL-1 was used to record the desired mean or mid signal. R. P.; Mol. 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. 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. The evaluation of the matrix effect showed linearity at 1 5 g mL 11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. Amylase - blood. CAS 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. 2019 Oct;18(4):2531-2539. doi: 10.3892/etm.2019.7840. and a-amylase activity. 2 0 obj The -amylase assay was performed using Millers method, i.e., the DNS method. Based upon the plasma glucose value, all subjects were divided into three groups. The intra-day variation of sensitivity ranged between 2.0 and 6.3%, and the inter-day variation was 4.9% for the complete period. In this experiment, acarbose at a concentration of 1.05 g mL-1 was used to record the desired maximum signal. Both recipients were placed in a heating block for 10min at a temperature of 45C. In this experiment, acarbose at a concentration of 5 g mL-1 was used to record the desired minimum signal. Enzymatic reaction and determination of the enzymatic activity. 7 0 obj Chem. D-Glucose working standard solution 200.0mgL1: dissolve 10.0mL of D-glucose stock solution in water to a final volume of 50.0mL; prepare fresh daily. The solution with varying concentrations of acarbose was placed in 32 wells in three different plates. Google Scholar, Mounaimen O, Mahmoud K (2015) Statistical optimization of cultural conditions of an halophilic alpha-amylase production by halophilic Streptomyces sp. The -amylase activity is measured using a colorimetric method with 3,5-dinitrosalicylic acid (DNS) reagent. Otherwise, the extract from Chrysactinia mexicana had an IC50 value of 9.09 g mL-1, indicating that the extract from this plant also contained bioactive metabolites, which have an inhibitory effect on the enzyme -amylase. The enzyme concentration, incubation time, interaction between enzyme concentration and preincubation time, and the interaction between preincubation and boiling time showed a significant effect on the inhibition percentage. Biotechnol. Foods. Screening 1999, 4, 67. 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. The accuracy of the method was determined by calculating the percentage of relative error (%) (Equation 3) of the extracts of J. dioica challenged with the inhibitor at different concentrations. Amylase is an enzyme made by your pancreas and salivary glands that helps your body break down carbohydrates. The error percentage was calculated to be 5.46%, which indicates that the method is accurate. Evolution of sensitivity (represented by the slope of the calibration curve) along time for the glucose oxidase/peroxidase assay. 1959, 31, 426. Slopes of the curves were compared using the t-test, and a significant difference was obtained, indicating the existence of the matrix effect. Bethesda, MD 20894, Web Policies After weighing 3.0g of flour into a 50mL flask, add 20.0mL malate buffer pH 5.4 (diluted from the concentrated solution). [Crossref]. For this particular case, mstarch was 2.40mg. Next, 500L of enzyme solution 2 (described in detail in the previous example) and 1000L of universal buffer were added to the vial (in this particular case, the objective was to obtain extra information about the hydrolysis performance of the enzyme and for this reason we used the same enzyme dilution and mass of starch used for the enzyme activity method. This was complemented by a glucose yield assay for amyloglucosidase that allowed a secondary source of information when insoluble starches were studied. Linearity was obtained at the acarbose concentration of 1.5 g mL-1 and 5 g mL-1. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 4 0 R/Group<>/Tabs/S/StructParents 0>> 8) [8]. 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. As a result of the intensive demand for this plant in folk medicinal uses, its scarcity has become problematic. These findings demonstrated that our optimized and validated method is highly selective, enabling the identification of samples with or without potent activity. Amylase is a digestive enzyme predominantly secreted by the pancreas and salivary glands and found in other tissues at very small levels. 2023 Springer Nature Switzerland AG. 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. Salivary amylase is the enzyme produced by the salivary glands. 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). Alpha-amylase is found in saliva and is responsible for the initial breakdown of starch into maltose. Enzyme immobilization Preparation of alginate beads Alginate gel beads were prepared according to Awad et al. Glycosidase enzymes breakdown carbohydrates into simple sugars they enter the bloodstream, producing postprandial hyperglycemia. (Family: Asteraceae) has been used as a natural remedy in the Arabian region for its antihyperglycemic activity. 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 Part of Springer Nature. Insoluble chromogenic substrates have been developed for ~-amylase assay. official website and that any information you provide is encrypted 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). The site is secure. The Pareto diagram (Figure 1) shows that some factors exhibit a significant effect on the response. 1 0 obj 2012, 13, 427. Optimization of the -amylase inhibition assay was performed using fractionalized factorial design (2^6-1). Screening 1999, 4, 67. When the enzymatic activity decreases, the formation of the product also decreases; therefore, the intensity of the color is reduced. When compared with the use of soluble starch, we adjusted the volume of HCl for stopping the reaction (200L instead of 750L) in order to maintain the buffering capacity of the GOPOD reagent (glucose yield protocol, step 4). Their age ranged between 25 65 years. Positive controls are compounds with proven activity in humans, whereas negative controls are those without activity. It was observed that slight variation in the experimental conditions had a significant effect on the response. Gangadharan D., Sivaramakrishnan S. Amylolytic enzymes. Fuwa H. A new method for microdetermination of amylase activity by the use of amylose as substrate. [Crossref], 19 Satpathy, G.; Tyagi, Y. K.; Gupta, R. K.; Food Res. For the calculation of the glucose yield assay promoted by amyloglucosidase from Aspergillus niger (PN: A9913, Sigma Aldrich), 1500L of water were added to a vial containing of 3.00mg of a pure rice starch (mstarch) weighed in an analytical scale. MTCC 5214 when grown in solid-state fermentation. 2017, Article ID 7319504. These parameters should be adjusted according to the objectives of each individual study). endobj 5 ml of deionized water are added in each sample, followed by Most enzymes act specifically with only one reactant, called a substrate, to produce products. Detailed information is available in our previous publications [7,8]. Saliva can be collected using a calibrated collection device, and the resulting sample should ideally be analyzed within 15 minutes after collection. 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. endobj I agree with all answers, try to raise the temperature reaction and be sure the pH buffer is ptimal for your amylase. In addition, the amylases re Salivary amylase is essential for the digestion of carbohydrates. 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. In: Dharumadurai, D. (eds) Methods in Actinobacteriology. The activity of enzymes is strongly affected by changes in pH and temperature. Dilute to a final volume of 100 ml with reagent grade water. Microbiol Res 207:116133, Srinivas A, Rahul K, Sasikala C, Subhash Y, Ramaprasad EVV, Ramana CV (2012) Georgenia satyanarayanai sp. Weight 3.00mg of pure starch into 4mL amber vials. Nalin T., Sperb-Ludwig F., Venema K., Derks T.G.J., Schwartz I.V.D. This also means that the volumes could be easily adjusted in order to match the requirements of each particular enzyme analysis or study. Anal. 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]. 1979 Nov;17(11):705-8. phenol). (1) the calculated activity for this enzyme was 1123UmL1. [Crossref], 3 Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; BioMed Res. A catalyst is a substance that hastens a chemical reaction but does not become part of the end product. For a blood test, blood is Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia (high blood sugar levels) due to insulin resistance or absolute lack of insulin production. Save my name, email, and website in this browser for the next time I comment. The results were subjected to regression analysis to determine R2 and the regression equation for the plot. It was initially termed 5). [Crossref]) and the time in the water bath (< 15.6 min), had a large influence on the outcome. The optimized and validated method was accurate, precise, and linear. J Genet Eng Biotechnol 15:95101, Mandels M, Sternberg D (1976) Recent advances in cellulases technology. This protocol helps you to understand the applications of amylase enzyme. 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. 2021 Jan 9;26(2):317. doi: 10.3390/molecules26020317. PMC legacy view When the pancreas is diseased or inflamed, it releases increased amounts of amylase into the blood. Warm the assay buffer to room temperature before use. The masses of glucose represented are 0.00, 2.00, 4.00, 6.00, 8.00 and 10.0g. The test is done in microtitre plates with a total volume of 260L and an assay time of 40min including the pre-incubation steps. Google Scholar, Jankov Z, Janeek (2020) Fungal-amylases from three GH13 subfamilies: their sequence structural features and evolutionary relationships. Applicability and reliability of the glucose oxidase method in assessing -amylase activity. This corresponds to a total mass of 483.4g of glucose (mglucose) in the 3200L total volume. Note: pH value must be adjusted to the desired value with NaOH 4.0molL1 for each particular assay at room temperature. Prepare D-glucose standards as described in. Here we adopted the same detection wavelength of the original method (580nm) [5], which is associated with an amylopectin/amylose ratio of 80:20 [12]. High intra-day and inter-day reproducibility. [Crossref]. These bonds can be easily broken by water, so saliva is necessary for the enzyme to work. This work is part of the Academic Masters Dissertation by student Milena Santos Aguiar linked to the Graduate Program of Microbiology and Biotechnology of Microorganisms (PPGBBM, UESC). The National Library of Medicine 13 Macarrn, R.; Hertzberg. For the first time, a reliable, simple, rapid and high-throughput analytical method for the detection and quantification of -amylase inhibitory activity using the glucose assay kit was developed. Testing can be conducted to measure the level of amylase as well as other enzymes in your blood. 1 ml of D.N.S. DA3-7. 2015 Apr 22;164:210-22. doi: 10.1016/j.jep.2015.02.018. Importance of Assay of Salivary amylase enzyme, Estimation of Carbohydrates by the Anthrone Method, Assay of Acid Phosphatase enzyme activity from Potatoes, Assay of Urease Enzyme Activity (Enzymology Practical Protocol), Effect of Temperature on Amylase activity (Enzymology Protocol).
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