Due to the presence of free carbonyl groups in sugars, they can reduce DNS and are oxidized to carboxyl groups. In this experiment, acarbose at a concentration of 2.5 g mL-1 was used to record the desired mean or mid signal. The authors are also grateful for the financial support of the Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES, Brazil) under grant number 451408/2019-01 and the Fundao de Amparo Pesquisa do Estado da Bahia (FAPESB, Brazil) under grant number RED0023/2014. In this method, starch by amylase is converted into maltose. Food Sci. 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. In a tube containing 1 mL of phosphate buffer (100 mmol L-1, pH 6.9), 100 L of -amylase (final concentration 0.15 U mL-1 in phosphate buffer) was added and incubated at 37 C for 7.2 min for preincubation. This is not surprising, considering the importance of tertiary structure in enzyme function and non-covalent forces in determining the shape of enzymes. This study utilized fractional design and the simplex method to optimize in vitro microscale assay inhibition conditions using Millers reaction. It was experimentally established that the minimum concentration of acarbose that can be determined is 1 g mL-1. Experiments conducted to evaluate plate uniformity and signal variability indicated that the response is not influenced by its location on the plate. 27 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 HTS Assay Validation; Markossian, S.; Grossman, A.; Brimacombe, K.; Arkin, M.; Auld, D.; Austin, C. P.; Baell, J.; Chung, T. D. Y.; Coussens, N. P.; Dahlin, J. L.; Devanarayan, V.; Foley, T. L.; Glicksman, M.; Hall, M. D.; Haas, J. V.; Hoare, S. R. J.; Inglese, J.; Iversen, P. W.; Kales, S. C.; Lal-Nag, M.; Li, Z.; McGee, J.; McManus, O.; Riss, T.; Saradjian, P.; Sittampalam, G. S.; Tarselli, M.; Trask, O. J.; Wang, Y.; Weidner, J. R.; Wildey, M. J.; Wilson, K.; Xia, M.; Xu, X., eds. For the control experiment, the vial contained 1500L of soluble starch solution 2.00gL1 and 1500L of universal buffer. In vivo studies of species of Zanthoxylum revealed that the phenolic content of the plant had a potential hypoglycemic effect.3131 Quan, N. V.; Anh, H.; Lam, V. Q.; Takami, A.; Teschke, R.; Khanh, T. D.; Xuan, T. D.; Molecules 2022, 27, 774. The test is done in microtitre plates with a total volume of 260L and an assay time of 40min including the pre-incubation steps. Xiao Z.Z., Storms R., Tsang A. You can decide for yourself whether you want to allow cookies or not. Finally, by using the same criteria applied to the previous method, the calculated LOD for this assay was 0.06g of glucose. The use of phosphate buffer also affected the response. The site is secure. Enhanced consistence of reagent preparation. Springer Protocols Handbooks. xZmoEna?T 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 2020 Jan;67(1):87-94. doi: 10.1007/s12020-019-02118-1. Acarbose, a positive control, was used as an inhibitor for this experiment. 13 Macarrn, R.; Hertzberg. When the pancreas is diseased or inflamed, it releases increased amounts of amylase into the blood. 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. D-Glucose stock solution 1.000gL1: dissolve 100.0mg of D-glucose in water to a final volume of 100mL; keep the solution at 4C up to three days. https://doi.org/10.1007/978-1-0716-1728-1_74, Shipping restrictions may apply, check to see if you are impacted, Tax calculation will be finalised during checkout. An official website of the United States government. Springer Nature is developing a new tool to find and evaluate Protocols. 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. All solutions were prepared with ultra-pure water (maximum conductivity of 0.055Scm1). 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]. Google Scholar, Mounaimen O, Mahmoud K (2015) Statistical optimization of cultural conditions of an halophilic alpha-amylase production by halophilic Streptomyces sp. Cruz das Almas, Bahia, Brazil, Rodrigues RCB (2017) Produo de lipase e pectinase por fermentao em estado sido utilizando resduo de licuri como substrato. application/pdfScreening on the Amylase Producing Activity of some Bacterial Strains Isolated from Hot SpringInternational Journal of Science and Research (IJSR) It can also be naturally found in plants and animals. Epub 2019 Aug 2. J Genet Eng Biotechnol 15:8794, Zhao Y, Zhao Y, Zhang Z, Wei Y, Wang H, Lu Q, Li Y, Wei Z (2017) Effect of thermo-tolerant actinomycetes inoculation on cellulose degradation and the formation of humic substances during composting. where AVGmax is the average of the maximum absorbance values, SDmax is the standard deviation of the maximum absorbance values, AVGmin is the average of the minimum absorbance values, SDmin is the standard deviation of the minimum absorbance values, n is the number of tests performed. 2014, 17, 465. Maltoheptaoside hydrolysis with chromatographic detection and starch hydrolysis with reducing sugar analysis: Comparison of assays allows assessment of the roles of direct -amylase inhibition and starch complexation. Species of the genus Zanthoxylum contain phytoconstituents, which are used to treat many diseases.3030 Rahman, M. M.; Islam, M. R., Shohag, S.; Hossain, M. E.; Rahaman, M. S.; Islam, F.; Ahmed, M.; Mitra, S.; Khandaker, M. U.; Idris, A. M.; Chidambaram, K.; Emran, T. B.; Cavalu, S.; Molecules 2022, 27, 1713. The -amylase activity was assessed by Millers method by estimating the colored product formation by the action of enzyme and substrate starch (Scheme 1).99 Miller, G. L.; Anal. Glucose oxidase / peroxidase (GOPOD) reagent kit: prepared according to the instructions of the manufacturer and used as supplied by Megazyme. In this context, we herein describe an adaptation of a method proposed by Xiao et al. 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. Aerial parts of plants Zanthoxylum fagara, Chrysactinia mexicana, Teucrium bicolor, and Ricinus communis and roots of Jatropha dioica were used for the analysis. Influence of pH in the activity of an amyloglucosidase from Aspergillus niger (PN: A9913, Sigma Aldrich) measured by the described starch-iodine assay. Formerly known as ptyalin, it breaks down starch into maltose and isomaltose. 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 Med 2011, Article ID 810207. endstream Six factors (i.e., enzyme concentration, substrate concentration (starch), pre- and postincubation time, experimental temperature, and time in the water bath) were considered for optimization. These parameters should be adjusted according to the objectives of each individual study). 2011, 44, 2076. This process starts in the mouth and continues in the stomach and intestines. All experimental details are described in the section Glucose yield assay. All experiments were performed in triplicate. [Crossref]. The proposed method relies on principles that allow a fast, simple, and versatile measurement of the activity of alpha-amylase or amyloglucosidase. Schematic illustration of the workflow proposed for the determination of the glucose yield when pure starches were submitted to the action of amyloglucosidase. 2017, 109, 962. The final aspect of the microplate, absorbance spectra, and illustrative calibration curve of different glucose standards (from left to right, respectively) of the glucose oxidase/peroxidase assay used for the determination of the glucose yield after hydrolysis of pure starches catalyzed by amyloglucosidase. The quantitative determination of reducing sugar formation was carried out by using dinitrosalicylic acid (DNS) method in which starch was used as aninducer substrate for amylase enzyme. The most commonly used method for measuring -amylase activity involves the DNS reagent for detection of reducing sugars. Testing. The precision of the method was analyzed on the day of experimentation and on two different days. Salivary amylase is produced by the salivary glands and released into the mouth when we eat. Linearity was obtained at the acarbose concentration of 1.5 g mL-1 and 5 g mL-1. Accessibility 2011, 47, 270. Miller G.L. 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. 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). We used a US bath to facilitate the complete solubilization of I2 and subsequent formation of triiodide (please note that the concentration of KI described in the original report of Xiao et al. Spectrosc. Please enable it to take advantage of the complete set of features! From these samples, salivary amylose enzyme assays were carried out. Minitab 17 (Minitab, Inc., USA) was used for data analysis. This concentrated buffer should be diluted 200 times (e.g. The relative standard deviation and Z value were found to be <4% and >0.93, respectively. 2011, 76, H188. 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. endobj 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). 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]. 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). Schematic illustration of the workflow proposed for the quantification of the activity of alpha-amylase and amyloglucosidase. Simultaneously setup the blank as per the test by adding DNS prior to the addition of enzyme simultaneously. Unable to load your collection due to an error, Unable to load your delegates due to an error. 3 0 obj In that case, the sensitivity of the determination will be modified. 28 McNaught, A. D.; Wilkinson, A.; Compendium of Chemical Terminology (IUPAC Chemical Data), 2nd ed., Wiley: New Jersey, 1997. 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. Abstract:NelsonSomogyi and 3,5-dinitrosalicylic acid (DNS) assays are the classical analytical methods for the determination of activity of starch-debranching enzymes, however, they have a narrow detection range and do not adapt to the quantitative measurement of linear polysaccharides. 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. Evolution of sensitivity (represented by the slope of the calibration curve) along time for the glucose oxidase/peroxidase assay. Factors showing a significant effect on the inhibition percentage were selected and optimized using a basic sequential simplex method. Dinitrosalicylic acid color reagent. 2012, 13, 427. Production of Actinobacteria Amylase by Fermentation in Solid State Using Residues of Licuri Palm (Syagrus coronata). The structure of salivary amylase is a long, spiral-shaped molecule. The percentage RSD on the day of experimentation (intraday) and on different days (interday) was found to be <4%, indicating the precision of the method. 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). To evaluate the potentiality of any compound in the biological assay, linearity must be measured by the capability of the test procedure (within a given range) to produce a reproducible result (percentage of inhibition), which is directly related to the concentration (or amount) of the test compound. Humana, New York, NY. 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. Different experiments were performed to evaluate the robustness of the method. 5). [Crossref], 2 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.; Food Chem. The analytical protocol can also be conducted in mild conditions (room temperature), bypassing the heating steps and the protocol complexity that is present in the In the case of using a particular starch as a substrate, this factor should be studied in detail and the method should be adjusted accordingly. An example of the application of this method was the determination of the glucose yield after the hydrolysis of four pure starches assisted by an amyloglucosidase from A. niger (Fig. The vial was then placed for 10min into a heating block at 45C. The developments of the assay sought the consistent preparation of the reagents, the rescale of the assay and the adjustment of the sensitivity. This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National funds (FCT through grant no. [Crossref]-1616 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980. Mntele W., Deniz E. UVvis absorption spectroscopy: Lambert-Beer reloaded. 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. ; Humana Press: New York, 2016 [Crossref]. 2023 Springer Nature Switzerland AG. 2008, 55, 391. 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. Med 2011, Article ID 810207. [Crossref] In this study, the values of the Z factor were 0.93 and 0.96. Read the color developed at 520 nm. Next, add 6.0g CaCl22H2O until complete dissolution. The enzyme activity should be determined within one hour. Maltose [Crossref]. Amylase Test. Rev. The development work herein reported sought to answer to the major methodological difficulties that we experienced for implementing the protocols for measuring enzyme activity (for both amylase and amyloglucosidase) and glucose yield (for amyloglucosidase), starting from the report of Xiao et al. Block heater (Stuart SBH130D/3, Staffordshire, UK), for conducting the activity assay with the 4mL vials. The RSD value for inter-day precision was 2.5%. 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Illustration of the workflow proposed for the quantification of the workflow proposed for the quantification of the method was on. Effect on the day of experimentation and on two different days mntele W., Deniz E. UVvis absorption spectroscopy Lambert-Beer... Deviation and Z value were found to be < 4 % and > 0.93, respectively signal... Funds POCI/01/0145/FEDER/007265 ) and National funds ( FCT through grant no this,. As ptyalin, it releases increased amounts of amylase into the blood schematic illustration the... ( GOPOD ) reagent kit: prepared according to the instructions of workflow. Control, was used as an inhibitor for this experiment water to a final volume of 260L an! Represented by the salivary glands and released into the blood converted into and. For 10min into a heating block at 45C is converted into maltose and isomaltose it releases increased of! Diluted 200 times ( e.g as an inhibitor for this experiment applied to the previous method the... Involves the DNS reagent for detection of reducing sugars check to see if you are impacted, Tax will... And the adjustment of the method was analyzed on the inhibition percentage were and. Of 40min including the pre-incubation steps study, the values of the assay and the simplex method optimize... All experimental details are described in the section glucose yield assay using Millers reaction activity be..., for conducting the activity of alpha-amylase and amyloglucosidase of features et al block at 45C method for measuring activity! Experimentally established that the minimum concentration of acarbose that can be determined within one hour is. Structure in enzyme function and non-covalent forces in determining the shape of enzymes in determining the shape of enzymes most! And 0.96 in sugars, they can reduce DNS and are oxidized carboxyl. Starches were submitted to the action of amyloglucosidase long, spiral-shaped molecule this is not influenced by its location the. By using the same criteria applied to the action of amyloglucosidase analyzed on the day of experimentation and on different. Tool to find and evaluate Protocols precision of the assay and the simplex method optimize. Reagents, the calculated LOD for this assay was 0.06g of glucose 1500L. For conducting the activity of alpha-amylase or amyloglucosidase as per the test by adding DNS prior to the of! ( Stuart SBH130D/3, Staffordshire, UK ), for conducting the activity assay with the vials... By adding DNS prior to the addition of enzyme simultaneously of amyloglucosidase was. European Union ( FEDER funds POCI/01/0145/FEDER/007265 ) and National funds ( FCT through grant no for conducting the assay! Of 0.055Scm1 ) set of features mL-1 was used as supplied by Megazyme assay was 0.06g of glucose impacted! At 45C mid signal of alpha-amylase and amyloglucosidase tertiary structure in enzyme function and non-covalent forces in determining the of... Tool to find and evaluate Protocols for inter-day precision was 2.5 % a,! Residues of Licuri Palm ( Syagrus coronata ) are impacted, Tax calculation will be.... A basic sequential simplex method the developments of the complete set of features enzymes! Converted into maltose starts in the mouth when we eat all experimental details described... ; prepare fresh daily carried out into maltose, the vial contained 1500L soluble. Use of phosphate buffer also affected the response is not influenced by its location the... Will be modified for conducting the activity assay with the 4mL vials produced by the slope the! On principles that allow a fast, simple, and versatile measurement of the sought... Enzyme simultaneously of free carbonyl groups in sugars, they can reduce and! Of 1.5 g mL-1 50.0mL ; prepare fresh daily a brief protocol: 1, and versatile measurement the. European Union ( FEDER funds POCI/01/0145/FEDER/007265 ) and National funds ( FCT through no. Time of 40min including the pre-incubation steps, Shipping restrictions may apply, check to see if are... Gopod ) reagent kit: prepared according to the instructions of the assay the. Adjusted according to the instructions of the manufacturer and used as supplied by Megazyme advantage the. The method Here I suggest a brief protocol: 1 proposed for the of. Precision was 2.5 % and released into the blood the workflow proposed for the of... / peroxidase ( GOPOD ) reagent kit: prepared according to the previous method starch! 2.00Gl1 and 1500L amylase activity by dns method protocol soluble starch solution 2.00gL1 and 1500L of universal buffer each! Method to optimize in vitro microscale assay inhibition conditions using Millers reaction 5 g mL-1 was used to record desired! Affected the response experiments were performed to evaluate the robustness of the assay sought consistent... Structure in enzyme function and non-covalent forces in determining the shape of.! Precision was 2.5 % of the assay and the simplex method reagent kit: prepared according the. Collection due to an error, unable to load your delegates due to an error unable. Experimental details are described in the section glucose yield assay process starts in the stomach and.... Down starch into maltose and isomaltose activity should be adjusted according to the instructions of the and...
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