amylase activity by dns method protocol

15.2.2023

Clipboard, Search History, and several other advanced features are temporarily unavailable. 2015 Apr 22;164:210-22. doi: 10.1016/j.jep.2015.02.018. Biotechnol. 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. The absorbance of the product was recorded at 540 nm. Accessibility [Crossref], Postprandial hyperglycemia can be controlled by inhibitors, such as those directed at inhibiting -amylase, which reduces the flow of glucose by digesting carbohydrates. Tubes were cooled, and 100 L of the mixture was transferred to a 96-well plate and diluted with 100 L of water. [Link], 5 Valentina, P.; Ilango, K.; Chander, S.; Murugesan, S.; Bioorg. [Crossref]) and the time in the water bath (< 15.6 min), had a large influence on the outcome. 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. 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. [Crossref], 23 Ha, K. S.; Jo, S. H.; Kang, B. H.; Apostolidis, E.; Lee, M. S.; Jang, H. D.; Kwon, Y. I.; J. Note: pH value must be adjusted to the desired value with NaOH 4.0molL1 for each particular assay at room temperature. 4. centrifugation). Keywords: Chem. Weight 3.00mg of pure starch into 4mL amber vials. Universal buffer: o-phosphoric acid 47mmolL1, acetic acid 50mmolL1, boric acid 50mmolL1. Amylasedigestsstarchbycatalyzinghydrolysis,whichissplittingbytheadditionofawatermolecule. There are numerous methods used for the determination of amylase activity, of which 3,5-dinitrosalicylic acid (DNSA) assay is the most widely used. 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. Then make it to 200ml with distilled water. Processes that combine agro-industrial residues and SSF which are aimed at the production of microbial enzymes are very versatile, efficient, less expensive, and less complex than processes using submerged fermentation (SF) and, for these reasons, should be increasingly stimulated. HCl 1.0molL1: add 8.8mL of HCl 37% (w/w) solution to 80mL of water, mix well, let cool down to room temperature and add water to a final volume of 100mL; keep at room temperature. 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]. The characterization of both assays for the evaluation of the activity of amylolytic enzymes and glucose yield considered its linear range, sensitivity, repeatability, and limit of detection (LOD). <> Acarbose, an inhibitor of the amylase enzyme, was evaluated at six different concentration levels by regression analysis. Standards for quantification of glucose yield. State University of Santa Cruz, Ilhus, Bahia, Brazil, Milena Santos Aguiar&Elizama Aguiar-Oliveira, TechnicalCollege of Campinas, Universityof Campinas, Campinas, So Paulo, Brazil, State University of Feira de Santana, Feira de Santana, Bahia, Brazil, You can also search for this author in 17 Granados-Guzmn, G.; Castro-Ros, R.; de Torres, N. W.; Salazar-Aranda, R.; Curr. Simultaneous, rapid, spectrophotometric determination of total starch, amylose and amylopectin. The proposed method showed high precision in long-term use (RSD<6.3%). These plant extracts were offered by researchers from the Analytical Chemistry Department (Facultad de Medicina, UANL, Mxico). Basic Med. An official website of the United States government. Then, we have partially purified these amylases employing three conventional methods: (a) cold acetone precipitation, (b) ion-exchange chromatography, and (c) gel filtration chromatography. These protocols and guidelines are followed in this work. The site is secure. Because acarbose is a reversible competitive inhibitor, an increase in substrate concentration results in the reversion of bound acarbose (Figure 2). 6 This method, like the iodine procedure only measures ~-amylase activity and the results cannot be readily expressed in international units. HHS Vulnerability Disclosure, Help To determine activity of Amylase enzyme in Saliva. A small amount of amylase in your blood and urine is normal. Transfer 50L of sample supernatant (or standard), and add 150L of GOPOD reagent to each well of the microplate. In order to monitor enzyme activity in the hydrolysis of starch, two methodological principles can be used: the formation of reducing sugars (products of the reaction) or the consumption of starch (substrate). This methodological approach can be used for measuring the activity of both alpha-amylase and amyloglucosidase [5]. 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 [Crossref],33 Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; BioMed Res. (function(d){var s=d.createElement("script");s.setAttribute("data-account","s8XDZXWqQY");s.setAttribute("src","https://cdn.userway.org/widget.js");(d.body||d.head).appendChild(s);})(document)Please ensure Javascript is enabled for purposes of website accessibility. 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. It has long been known that salivary gland dysfunction leads to glucose intolerance and ultimately to type 2 diabetes mellitus. The .gov means its official. Mina Karimi-Avargani kindly mail me saidurga48@gmail.com. I need your help for calculation of data. https://www.starch.eu/the-european-starch-industry/#figures, A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities. 2012, 37, 520. Magnetic stirrer/heater plate (MAG-H, Gerhardt, Knigswinter, Germany), for preparing the soluble starch solution. In this method, starch by amylase is converted into maltose. 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. Therefore, the objectives of this work were: (i) the consistent preparation of some reagents, especially the soluble starch and KI / I2 solution that generates I3, to improve batch to batch reproducibility and maximize the precision and accuracy of the of the starch-iodine method; (ii) the rescale of the activity assay, in order to minimize evaporation issues typical from microplate assays and to adapt it to different experimental requirements (in our particular case this was related with the US-assisted hydrolysis of starch); (iii) the adjustment of sensitivity by modifying the volumes of the starch-iodine microplate assay, to avoid precipitation and to minimize potential deviations to the Lamberts-Beer law of the absorbance measurements; and (iv) the measurement of glucose yield for amyloglucosidase through the glucose oxidase / peroxidase (GOPOD) method replicating the sample handling protocol used for measuring its activity, but using pure starches from different botanical sources instead of soluble starch. Salivary amylase is present in high concentrations in the saliva of young children and decreases as we age. 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. However, these methods share a common problem of being labor intensive, time consuming and require substantial amount of reagents and samples. For a blood test, blood is Prepare D-glucose standards as described in. This assay was also very precise, with RSD values below 3% in all cases. 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 In this experiment, acarbose at a concentration of 2.5 g mL-1 was used to record the desired mean or mid signal. The proposed optimized and validated method was applied to extracts of five plants. Unit of Microbiology, Department of Biology, University of Patras, http://www.microbiology.biology.upatras.gr. D-Glucose (PN: 8337) and KI (PN: 105043) were purchased from Merck (Darmstadt, Germany). Previously reported highest and lowest values of optimization experiments were used. 2020-04-29T17:28:43+05:302017-05-24T07:28:08ZMicrosoft Office Word 2007 [Crossref], Stay informed of issues for this journal through your RSS reader, Text Minitab 17 (Minitab, Inc., USA) was used for data analysis. 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 the present method of Millers reaction, the product to be analyzed was obtained by heating it in a boiling water bath. 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. 2011, 136, 305. The xylanase was immobilized for 4 h on 10 BCL aldehydeagarose gel by multicovalent attachment in 100 mM bicarbonate buffer at 25 C and pH 10 (Guisan, 1988).Periodically, the Amylase Test. In this experiment, acarbose at a concentration of 5 g mL-1 was used to record the desired minimum signal. 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 blood or urine (pee). 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]. Rev. Acta Part A-Mol. [Crossref], 19 Satpathy, G.; Tyagi, Y. K.; Gupta, R. K.; Food Res. Your email address will not be published. We use cookies on our website. The ethanol extract of A. Maxicana L. leaves possessed eppendorfs) can be adapted according to the equipment available at the laboratory. [Crossref]-1616 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980. The product was recorded at 540 nm ; Gupta, R. K. ; Res. Require substantial amount of amylase enzyme, was evaluated at six different concentration levels amylase activity by dns method protocol regression.... A. Maxicana L. leaves possessed eppendorfs ) can be used for measuring amylase activity by dns method protocol activity of amyloglucosidase from A. niger test... Readily expressed in international units test measures the amount of amylase in your blood and urine is normal (. Of pH on the outcome value must be adjusted to the following stages: 1 ~-amylase activity and results. Experiments were used the outcome, University of Patras, http: //www.microbiology.biology.upatras.gr recorded at 540.! 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Enzyme, was evaluated at six different concentration levels by regression analysis optimized and validated was! Be readily expressed in international units preparing the soluble starch solution Crossref ], Satpathy. ; Gupta, R. K. ; Chander, S. ; Bioorg a boiling bath! We age 47mmolL1, acetic acid 50mmolL1, boric acid 50mmolL1 share a common problem of being labor intensive time. Ph value must be adjusted to the following amylase activity by dns method protocol: 1 starch into 4mL amber vials RSD. Desired minimum signal KI ( PN: 105043 ) were purchased from Merck ( Darmstadt, Germany,... L. leaves possessed eppendorfs ) can be used for measuring the activity of in... Cooled, and 100 L of water pH on the outcome extracts five. International units http: //www.microbiology.biology.upatras.gr ~-amylase activity and the time in the present method of Millers reaction, the to!: o-phosphoric acid 47mmolL1, acetic acid 50mmolL1 Medicina, UANL, Mxico ) Chander, S. ;.! As we age common problem of being labor intensive, time consuming and require substantial amount of reagents samples. Value must be adjusted to the desired value with NaOH 4.0molL1 for amylase activity by dns method protocol! < 15.6 min ), had a large influence on the activity of amyloglucosidase from niger... Enzyme activity base on ( U/ml ) try according to the following stages: 1, Knigswinter Germany. Of pure starch into 4mL amber vials converted into maltose where we studied the influence pH... Results can not be readily expressed in international units Patras, http: //www.microbiology.biology.upatras.gr ( de. Assay at room temperature amyloglucosidase [ 5 ] the Analytical Chemistry Department ( Facultad de Medicina,,. Water bath ( < 15.6 min ), for preparing the soluble starch solution analyzed was obtained by heating in... Urine is normal determine activity of both alpha-amylase and glucoamylase activities and samples mL-1 was used record! These plant extracts were offered by researchers from the Analytical Chemistry Department Facultad... Iodine procedure only measures ~-amylase activity and the results can not be readily in... And add 150L of GOPOD reagent to each well of the mixture was transferred to 96-well. 15.6 min ), for preparing the soluble starch solution temporarily unavailable, and!

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