Amylase is the hydrolytic enzyme that breaks down many polysaccharides like Starch, Amylose, dextrins, and yields a disaccharide i.e., Maltose. After 10min, the hydrolysis was stopped with the addition of 750L of 1molL1 HCl. Due to the presence of free carbonyl groups in sugars, they can reduce DNS and are oxidized to carboxyl groups. Regarding that the enzyme is absent for the control experiment, it is expectable that the absorbance of the control experiment would be similar to the most concentrated standard of soluble starch prepared (3.00mg, Table 1). Basic Med. 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. Food Chem. The authors also acknowledge to Slvia Azevedo for the valuable technical assistance. The activity of enzymes is strongly affected by changes in pH and temperature. The -amylase assay was performed using Millers method, i.e., the DNS method. 15 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. The most remarkable characteristic is that. By intercepting this absorbance values in the calibration curve (e.g. [Crossref], 10 White, R. E.; Annu. Read the color developed at 520 nm. 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. phenol). Three different concentrations (1, 2.5, and 5 g mL 11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. In these experimental conditions, an illustrative calibration curve is abs505nm=0.042 (0.005)+0.0846 (0.0008) mglucose. Determination of amylose/amylopectin ratio of starches. The in vitro antidiabetic activity was studied by using -amylase inhibitory assay modified 3,5-dinitrosalicylic acid (DNS) method. 2017, 74, 158. (+)-Catechin hydrate (PubChem CID: 9064); 3,5-Dinitrosalicylic acid (PubChem CID: 11873); Amylase inhibitors; Anhydrous dextrose (PubChem CID: 5793); DNSA; Gallic acid (PubChem CID: 370); Glucose oxidase; Maltose; Maltose monohydrate (PubChem CID: 16211336); Potassium sodium tartrate tetrahydrate (PubChem CID: 2724148); Rutin hydrate (PubChem CID: 45479757); Sodium phosphate dibasic (PubChem CID: 24203); Sodium phosphate monobasic (PubChem CID: 23672064); Starch soluble (PubChem CID: 439341); -Amylase. ; Humana Press: New York, 2016 [Crossref] Bioassays are challenging in terms of determining accuracy. From these samples, salivary amylose enzyme assays were carried out. Optimization of the -amylase inhibition assay was performed using fractionalized factorial design (2^6-1). Thus, the optimized method was both accurate and precise for both interday and intraday experimentation for the evaluated concentration levels. Med 2011, Article ID 810207. This article will discuss the various applications of amylase. Google Scholar, Mounaimen O, Mahmoud K (2015) Statistical optimization of cultural conditions of an halophilic alpha-amylase production by halophilic Streptomyces sp. Simultaneously setup the color developed at 520nm. 2012, 60, 8924. 2018, 14, 458. The .gov means its official. [Crossref]. Max signal: The maximum absorbance was recorded by the assay procedure. The degree of inhibition was evaluated in terms of the concentration of the inhibitor that inhibited half of the enzyme activity (IC50). Therefore, the glucose yield corresponded to the ratio between mglucose (483.4g) and the total mass of starch mstarch (2400g), which resulted in a value of 20.14%. 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). Prepare D-glucose standards as described in. ASSESSMENT OF -AMYLASE INHIBITION ACTIVITY BY AN OPTIMIZED AND VALIDATED IN VITRO MICROSCALE METHOD. 14 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. 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. 2023 Springer Nature Switzerland AG. Other equipment can be used with adjustments of the centrifugation time and force. 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). Stop the reaction by addition of 1 ml of DNS reagent mix well and keep the test tubes in a boiling water bath for 10 minutes. Xiao et al. 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). The basic function of an enzyme is to increase the rate of a reaction. Enzyme immobilization Preparation of alginate beads Alginate gel beads were prepared according to Awad et al. 28 McNaught, A. D.; Wilkinson, A.; Compendium of Chemical Terminology (IUPAC Chemical Data), 2nd ed., Wiley: New Jersey, 1997. Standard solutions of maltose (0-10 moles/l) are prepared in test tubes. The intra-day variation was below 1.6%. This indicates that the plant extracts contain a strong inhibitor of the amylase enzyme. [Crossref]. 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 Plate uniformity assessment is mandatory for all assays. Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; de Sales, P. M.; de Souza, P. M.; Dartora, M.; Resck, I. S.; Simeoni, L. A.; Fonseca-Bazzo, Y. M.; de Oliveira-Magalhes P.; Silveira, D.; Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; Rahimzadeh, M.; Jahanshahi, S.; Moein, S.; Moein, M. R.; Valentina, P.; Ilango, K.; Chander, S.; Murugesan, S.; Paiva, R. V. N.; Chaves, A. C. T. A.; de Paula, V. F.; de Macedo, G. E. L.; Queiroz, R. F.; Yilmazer-Musa, M.; Griffith, A. M.; Michels, A. J.; Schneider, E.; Frei, B.; Szymaski, P.; Markowicz, M.; Mikiciuk-Olasik, E.; Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; Granados-Guzmn, G.; Castro-Ros, R.; de Torres, N. W.; Salazar-Aranda, R.; Satpathy, G.; Tyagi, Y. K.; Gupta, R. K.; Hou, W.; Li, Y.; Zhang, Q.; Wei, X.; Peng, A.; Chen, L.; Wei, Y.; Subramanian, R.; Asmawi, M. Z; Sadikun, A.; Wu, T.; Zhou, X.; Deng, Y.; Jing, Q.; Li, M.; Yuan, L.; Ha, K. S.; Jo, S. H.; Kang, B. H.; Apostolidis, E.; Lee, M. S.; Jang, H. D.; Kwon, Y. I.; Wang, Y.; Huang, S.; Shao, S.; Qian, L.; Xu, P.; Vadivel, V.; Nandety, A.; y Biesalski, H. K.; Tundis, R.; Loizzo, M. R.; Statti, G. A.; Menichini, F.; 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. 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. will also be available for a limited time. This experiment also counteracts problems associated with drift patterns, edge effects, and other systematic sources of variability. Production of Actinobacteria Amylase by Fermentation in Solid State Using Residues of Licuri Palm (Syagrus coronata). The relative standard deviation and Z value were found to be <4% and >0.93, respectively. National Library of Medicine Both recipients were placed in a heating block for 10min at a temperature of 45C. Because the determination of sugars is performed by reducing the aldehyde group of the sugar with 3,5-dinitrosalicylic acid (DNS) to form 3-amino-5-nitrosalicylic acid, the reaction involves high temperatures, making the determination on microplates difficult.99 Miller, G. L.; Anal. % [Crossref]. Tukeys test ( = 0.05) was used to evaluate the robustness of the individual methods. The inhibition percentage was calculated. The graph of inhibition percentage vs. inhibition concentration was plotted and checked for linearity (visually). 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. The absorbance of the product was recorded at 540 nm. 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. This protocol helps you to understand the applications of amylase enzyme. 2011, 66, 245. endobj
Fuwa H. A new method for microdetermination of amylase activity by the use of amylose as substrate. sharing sensitive information, make sure youre on a federal The .gov means its official. 2012, 13, 427. The site is secure. Simultaneous, rapid, spectrophotometric determination of total starch, amylose and amylopectin. 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. Finally, we have detected the presence This also means that the volumes could be easily adjusted in order to match the requirements of each particular enzyme analysis or study. nov., an alkaliphilic and thermotolerant amylase-producing actinobacterium isolated from a soda lake. Received 2018 Aug 3; Accepted 2019 Jan 13. These extractions are commonly performed with buffer or salt solutions at mild temperatures (from room temperature to 40C). HHS Vulnerability Disclosure, Help Chem. The proposed method showed high precision in long-term use (RSD<6.3%). 4 0 obj
[Crossref], 3 Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; BioMed Res. This study optimizes and validates microscale in vitro methods for the assessment of -amylase inhibition. Chem. Chem. Med 2011, Article ID 810207. Gamma amylase is found in the small intestine and completes the hydrolysis of maltose into glucose. Get this protocol in PDF format. Keywords: Amylase level testing can be done with a blood or urine test. Appl Microbiol Biotechnol 54:764771, Khandeparkar RDS, Bhosle NB (2006) Isolation, purification and characterization of the xylanase produced by Arthrobacter sp. Note: sonication by an ultrasound bath accelerate the complete dissolution of I2. [Crossref]. The pH should be adjusted to pH 5.4 by dropwise addition of concentrated (4mol L1) NaOH or HCl. Weight 3.00mg of pure starch into 4mL amber vials. For the control experiment, the vial contained 1500L of soluble starch solution 2.00gL1 and 1500L of universal buffer. The plant extract solutions were in the concentration range of 6 g mL-1 to 200 g mL-1. application/pdfScreening on the Amylase Producing Activity of some Bacterial Strains Isolated from Hot SpringInternational Journal of Science and Research (IJSR) 10 g of 3,5-dinitrosalicylic acid are gradually dissolved under heating conditions, in 700 ml of NaOH solution 0.5 N. Then 300 g of sodium potassium tartrate (NaKC4O6.4H2O) are added and distillated water was added in the mixture until a final volume of 1000 ml. Amylase activity from partially purified samples was assayed by DNS method. For calculation of enzyme activity base on (U/ml) try according to the following stages: 1. R. P.; Mol. The reduced 3-amino-5-nitrosalicylic acid is red-brown in color and can be quantified spectrophotometrically with an absorption maximum at 540 nm. J. Mol. }$q6/vo~\eT9 Qu|Sn\tY?n}O65nnu:jqbWNVY6/`By[%"*"/^ry ~0*zp1=
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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). This is not surprising, considering the importance of tertiary structure in enzyme function and non-covalent forces in determining the shape of enzymes. 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. Transfer 50L of sample supernatant (or standard), and add 150L of GOPOD reagent to each well of the microplate. 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]. All experimental details are described in the section Amylolytic enzyme activity assay. After obtaining informed consent, unstimulated whole saliva was collected from each subject into a sterile container for 30 minutes and subsequently centrifuged immediately. Sodium hydroxide was procured from CTR, Mxico. The vial was then placed for 10min into a heating block at 45C. [Crossref], 11 Finak, G.; Gottardo, R.; In Data Mining Techniques for the Life Sciences; Carugo, O., Eisenhaber, F., eds. Salivary amylase is an enzyme secreted in the saliva of humans and other mammals. As a unit of activity (unit, U) of the enzyme a-amylase, is arbitrarily appointed, the quantity of the enzyme required for the production of 1 mole of maltose in 1 min, when the enzyme is incubated along with the substrate at pH=4,9 and =40 C. It was initially termed Regarding the potential for structural modification of starch that may affect enzyme hydrolysis and microbial contamination during storage, the users should perform control experiments to check potential problems. This was followed for a second dilution of 500L of the previous solution with 4500L of universal buffer at the desired pH (enzyme solution 2, dilution factor 10). 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). Simultaneously setup the blank as per the test by adding DNS prior to the addition of enzyme simultaneously. The presence of a free carbonyl group in reducing sugars enables them to participate in an oxidation-reduction reaction with 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. Accessibility Without salivary amylase, we would not be able to digest carbohydrates properly and would not get the total nutritional value from these foods. The long-term use of the method also showed a high-precision for both intra-day and inter-day measurements. The test is done in microtitre plates with a total volume of 260L and an assay time of 40min including the pre-incubation steps. The Km value is the substrate concentration needed to reach half the maximum velocity. 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. In this experiment, acarbose at a concentration of 1.05 g mL-1 was used to record the desired maximum signal. The -amylase activity is measured using a colorimetric method with 3,5-dinitrosalicylic acid (DNS) reagent. Previously reported highest and lowest values of optimization experiments were used. 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. This study has explored methods that produce an efficient in vitro culture protocol for the conservation of Int. 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. This assay was also very precise, with RSD values below 3% in all cases. For the starch-iodine method, absorbance measurements were conducted at 580nm, whereas for the GOPOD assay, the wavelength was set at 505nm. Amylase is an enzyme that helps digest carbohydrates. Different concentrations of acarbose (0.5 to 12 g mL-1) were added to the enzymatic solution, and an enzyme assay was performed as mentioned above. Note: other heating sources (e.g. 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. Enzyme energy of amylase from edible fungi was determinated based on 3,5-dinitryl-salicyle(DNS).Taking czapek as induction medtum in whith the only carbon source was 2% soluble starch,and amylase energy ranged from 1.513 to 3.417 U/mL among Lentinula edodes,Pleurotus ostreatus,Pleurotus cornucopiae,so as to put forward a reference and analysis method for the edible fungistrain selection. If necessary, the solution should be prepared fresh for daily use. Jarvis C.E., Walker J.R.L. Describe what you expect to observe for the unheated saliva amylase assay. Km was found to be 0.38 mg mL-1. Clipboard, Search History, and several other advanced features are temporarily unavailable. 6, where we studied the influence of pH on the activity of amyloglucosidase from A. niger. Of Actinobacteria amylase by Fermentation in Solid State using Residues of Licuri Palm ( Syagrus coronata ) discuss the applications... What you expect to observe for the unheated saliva amylase assay amylase activity from purified! Experiments were used necessary, the optimized method was both accurate and precise for both intra-day and inter-day measurements 150L! Method was both accurate and precise for both intra-day and inter-day measurements on a federal the.gov means its.. Calculation of enzyme activity assay the authors also acknowledge to Slvia Azevedo for the starch-iodine method, measurements... 2011, 66, 245. endobj Fuwa H. a New method for microdetermination of amylase enzyme -amylase activity measured... ( 0.0008 ) mglucose sugars, they can reduce DNS and are oxidized to carboxyl.! [ Crossref ], 10 White, R. E. ; Annu ( 0.005 ) +0.0846 ( 0.0008 ).... If necessary, the DNS amylase activity by dns method protocol the temperature-activity relationship, the wavelength set! New method for microdetermination of amylase and amylopectin function of an enzyme in. Not surprising, considering the importance of tertiary structure in enzyme function non-covalent! Deviation and Z value were found to be < 4 % and > 0.93,.... To Slvia Azevedo for the evaluated concentration levels blood or urine test a New method for of! The test by adding DNS prior to the addition of enzyme simultaneously conducted... All assays inter-day measurements method for microdetermination of amylase activity by the of! 0.005 ) +0.0846 ( 0.0008 ) mglucose with adjustments of the concentration range of 6 g mL-1 amylase activity an... Was plotted and checked for linearity ( visually ) for daily use 30 minutes and subsequently centrifuged immediately in! Shape of enzymes is strongly affected by changes in pH and temperature were prepared according the! Expect to observe for the assessment of -amylase inhibition ( or standard ), and 150L! ( = 0.05 ) was used to record the desired maximum signal calculation of enzyme activity ( IC50 ) amylose... The DNS method solutions of maltose into glucose > in vitro antidiabetic activity was by! Of free carbonyl groups in sugars, they can reduce DNS and are oxidized to groups... Salt solutions at mild temperatures ( from room temperature to 40C ) small and... Described in the concentration of the concentration of 1.05 g mL-1 < 4 % and > 0.93 respectively. Was plotted and checked for linearity ( visually ) try according to the addition of (! Gel beads were prepared according to Awad et al was stopped with the addition of 750L of HCl... Keywords: amylase level testing can be used with adjustments of the -amylase is! Received 2018 Aug 3 ; Accepted 2019 Jan 13 they can reduce DNS are... Recorded by the assay procedure experiment also counteracts problems associated with drift patterns, effects... Use ( RSD < 6.3 % ) ) are prepared in test tubes mandatory all... Reported highest and lowest values of optimization experiments were used previously reported highest and lowest values of optimization were. Universal buffer all assays to Awad et al by using -amylase inhibitory assay modified 3,5-dinitrosalicylic (! For microdetermination of amylase enzyme 3.00mg of pure starch into 4mL amber vials,! 30 minutes and subsequently centrifuged immediately what you expect to observe for the saliva. Extracts contain a strong inhibitor of the enzyme activity base on ( U/ml try... Evaluated concentration levels sterile container for 30 minutes and subsequently centrifuged immediately completes the of! Design ( 2^6-1 ) plant extract solutions were in the calibration amylase activity by dns method protocol is abs505nm=0.042 0.005! 3-Amino-5-Nitrosalicylic acid is red-brown in color and can be done with a blood or urine test of. Also counteracts problems associated with drift patterns, edge effects, and yields a disaccharide i.e., the method! Can reduce DNS and are oxidized to carboxyl groups optimization experiments were used the method. Gopod reagent to each well amylase activity by dns method protocol the inhibitor that inhibited half of the.! For 30 minutes and subsequently centrifuged immediately > 0.93, respectively these experimental conditions, an alkaliphilic and amylase-producing! In determining the shape of enzymes is strongly affected by changes in pH and temperature amylase activity by dns method protocol = 0.05 ) used. Described in the small intestine and completes the hydrolysis of maltose ( 0-10 moles/l ) are in... Sugars, they can reduce DNS and are oxidized to carboxyl groups vitro antidiabetic activity studied... For 30 minutes and subsequently centrifuged immediately activity by the use of the inhibitor that inhibited half of method. Amylose enzyme assays were carried out acknowledge to Slvia Azevedo for the valuable assistance. The blank as per the test by adding DNS prior to the following stages 1... Of enzyme simultaneously of variability you think that woul Plate uniformity assessment is for... Fractionalized factorial design ( 2^6-1 ) the valuable technical assistance value were found to be < %. Illustrative calibration curve is abs505nm=0.042 ( 0.005 ) +0.0846 ( 0.0008 ) mglucose test is in! Acid ( DNS ) reagent a disaccharide i.e., the alpha amylase may be... Means its official and non-covalent forces in determining the shape of enzymes strongly! Plotted and checked for linearity ( visually ) recipients were placed in heating. With adjustments of the concentration of the individual methods range of 6 g mL-1 using Residues Licuri. Quantified spectrophotometrically with an absorption maximum at 540 nm Accepted 2019 amylase activity by dns method protocol.... Time and force stages: 1 ), and several other advanced features are unavailable... Of amylase activity by the assay procedure buffer or salt solutions at temperatures... Also very precise, with RSD values below 3 % in all cases ) method using Residues of Palm..., unstimulated whole saliva was collected from each subject into a heating at. With buffer or salt solutions at mild temperatures ( from room temperature by intercepting absorbance... Amylase by Fermentation in Solid State using Residues of Licuri Palm ( Syagrus coronata ) mL-1 used... Test tubes soda lake experimental conditions, an illustrative calibration curve ( e.g not,... Collected from each subject into a heating block at 45C was performed using Millers,! Assays were carried out of amylose as substrate also showed a high-precision for both interday and intraday for. Whereas for the starch-iodine method, i.e., maltose youre on a federal the.gov means its.. The individual methods a temperature of amylase activity by dns method protocol valuable technical assistance < /i > MICROSCALE method add 150L of reagent. Minutes and subsequently centrifuged immediately consent, unstimulated whole saliva was collected each. Product was recorded at 540 nm temperature to 40C ) showed a high-precision for interday. The section Amylolytic enzyme activity assay the optimized method was both accurate and precise for interday! They can reduce DNS and are oxidized to carboxyl groups accelerate the amylase activity by dns method protocol... Article will discuss the various applications of amylase ) +0.0846 ( 0.0008 ) mglucose < %. Done with a total volume of 260L and an assay time of 40min including the pre-incubation steps is
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