19 0 obj 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]. 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. 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. Amylase is the hydrolytic enzyme that breaks down many polysaccharides like Starch, Amylose, dextrins, and yields a disaccharide i.e., Maltose. Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia (high blood sugar levels) due to insulin resistance or absolute lack of insulin production. The linear range of the measurements included masses of soluble starch up to 3.00mg (see, The amount of hydrolyzed starch was the difference between the masses calculated from the calibration curve obtained by plotting absorbance. grown on orange waste powder. 2018, 14, 458. [Crossref]) were used, and the experiment was conducted in triplicate. Appl Microbiol Biotechnol 54:764771, Khandeparkar RDS, Bhosle NB (2006) Isolation, purification and characterization of the xylanase produced by Arthrobacter sp. https://www.starch.eu/the-european-starch-industry/#figures, A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities. For enzyme inhibition studies, the absorbance was recorded at a concentration with 50% inhibition (IC60). 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. J. Before 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. Amylase level testing can be done with a blood or urine test. [Crossref], 4 Rahimzadeh, M.; Jahanshahi, S.; Moein, S.; Moein, M. R.; Iran. Simultaneously setup the color developed at 520nm. Salivary amylase is essential for the digestion of carbohydrates. 0.5 ml of properly diluted (in acetic acid buffer solution; pH=4.9) crude enzyme are incubated for 15 min at T=40 C with 0.5 ml of soluble starch solution 1 % w/v. Insoluble chromogenic substrates have been developed for ~-amylase assay. [Crossref] Although other protocols are based either on the glucose oxidase reaction or on the determination by fluorescence, they are commonly used in microplates. 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 Accessibility 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. 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. 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. Amylase is the hydrolytic enzyme that breaks down many polysaccharides like Starch, Amylose, dextrins, and yields a disaccharide i.e., Maltose. Then, 50L of each vial were transferred to the 96-well microplate, followed by the addition of 150L of water and 50L KI/I2 reagent. Thank you Marsha for your replay, actually I am trying to calculate IC50 or EC50 from my samples (plant extracts) to see if it can use as inhibitor By intercepting this absorbance values in the calibration curve (e.g. An official website of the United States government. Most enzymes act specifically with only one reactant, called a substrate, to produce products. amylase, any member of a class of enzymes that catalyze the hydrolysis (splitting of a compound by addition of a water molecule) of starch into smaller carbohydrate molecules such as maltose (a molecule composed of two glucose molecules). Concerning to the measurement of soluble starch, the adjustments covered two complementary aspects: bypassing the precipitation found in microplate wells when the original volumes were used, and minimizing potential deviations of Beers law when high values of absorbance (above 1) described in the original method were found. Chem. This study utilized fractional design and the simplex method to optimize in vitro microscale assay inhibition conditions using Millers reaction. Med 2011, Article ID 810207. [Crossref]16 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980.-1717 Granados-Guzmn, G.; Castro-Ros, R.; de Torres, N. W.; Salazar-Aranda, R.; Curr. 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. Place the microplate in the plate reader, shake the plate (for 30 to 60s using the shaker function of the microplate reader), incubate for 30min at 37C, and read the absorbance at 505nm. E)CQb_w)33;3:'jOjd_nx~xln%x=,syv:'{xq70r,ugN'@5TzFPD:1$K$d Food Sci. Both recipients were placed in a heating block for 10min at a temperature of 45C. This article will discuss the various applications of amylase. Amylase is naturally produced in humans by the salivary glands and pancreas. The most remarkable characteristic is that. [Crossref], 8 Szymaski, P.; Markowicz, M.; Mikiciuk-Olasik, E.; Int. PMC legacy view The abovementioned protocol was followed with different concentrations of substrates. Simultaneous, rapid, spectrophotometric determination of total starch, amylose and amylopectin. All solutions were prepared with ultra-pure water (maximum conductivity of 0.055Scm1). Salivary amylase is present in high concentrations in the saliva of young children and decreases as we age. These parameters should be adjusted according to the objectives of each individual study). It is responsible for breaking down carbohydrates into simpler sugars. The RSD value for inter-day precision was 2.5%. 2, Fig. }$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). 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. Furthermore, this improved method is very flexible and allows a broadband application, being able to be easily adjusted according to the requirements of each study or the equipment and instrumentation available at each laboratory. The -amylase assay was performed using Millers method, i.e., the DNS method. [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. document.getElementById( "ak_js_1" ).setAttribute( "value", ( new Date() ).getTime() ); Assay of Salivary Amylase enzyme activity. An official website of the United States government. Universal buffer: o-phosphoric acid 47mmolL1, acetic acid 50mmolL1, boric acid 50mmolL1. Chem. D-Glucose (PN: 8337) and KI (PN: 105043) were purchased from Merck (Darmstadt, Germany). It is made in the pancreas and the glands that make saliva. sharing sensitive information, make sure youre on a federal 4 Relative standard deviation (expressed in percentage, RSD%) measured the precision of the assay. Zhang, J. H.; Chung, T. D.; Oldenburg, K. R.; 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.; Quan, N. V.; Anh, H.; Lam, V. Q.; Takami, A.; Teschke, R.; Khanh, T. D.; Xuan, T. D.; Analytical Chemistry Department, School of Medicine, Autonomous University of Nuevo Len, Madero y Aguirre Pequeo, Mexico, Text [Crossref],33 Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; BioMed Res. Sci. Experimental conditions as described in the protocol (the incubation temperature was 45C). 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. Due to the presence of free carbonyl groups in sugars, they can reduce DNS and are oxidized to carboxyl groups. Save my name, email, and website in this browser for the next time I comment. Metabolic disorders, including hyperglycemia, characterize type-2 diabetes. The optimum substrate concentration for the -amylase inhibition assay was found to be 0.38 mg mL-1 (1.11 mmol L-1). [Crossref]. Unable to load your collection due to an error, Unable to load your delegates due to an error. McCleary B.V., McNally M., Monaghan D., Mugford D.C. The https:// ensures that you are connecting to the Hugo M. Oliveira and Margarida R. G. Maia thanks Fundao para a Cincia e a Tecnologia (FCT) for the post-doctoral grant SFRH/BPD/75065/2010 and SFRH/BPD/70176/2010, respectively. For a blood test, blood is The use of soluble starch also allowed the standardization of the working conditions, avoiding any influence of the solubility of the starch in the final result. Km was found to be 0.38 mg mL-1. It was initially termed diastaste but was later renamed amylase in the early 20th 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. Epub 2015 Feb 16. 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. [Crossref], 18 Apostolidis, E.; Lee, C. M.; J. 6 This method, like the iodine procedure only measures ~-amylase activity and the results cannot be readily expressed in international units. Beta amylase is located in the pancreas and produces maltose from the hydrolysis of the maltose molecule. 50mL of concentrated solution for a final volume of 1000mL) in order to be used in the extraction protocol. 2 0 obj Anal. MTCC 5214 when grown in solid-state fermentation. Weight 3.00mg of pure starch into 4mL amber vials. Amylase activity from partially purified samples was assayed by DNS method. Biochem. The graph of inhibition percentage vs. inhibition concentration was plotted and checked for linearity (visually). The amount of Maltose in the given unknown sample is _________ grams of Maltose formed per 100ml of enzyme per one hour. Kolmogorov-Smirnov and Tukey tests were performed, and P < 0.05 was considered significant. 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. Evolution of sensitivity (represented by the slope of the calibration curve) along time for the starch-iodine assay. Spectrosc. The new method is tested for linearity, sensitivity, precision, reproducibility and applicability. Learn more, Banerjee S, Maiti TK, Roy RN (2016) Identification and product optimization of amylolytic Rhodococcus opacus GAA 31.1 isolated from gut of Gryllotalpa africana. Oliveira H.M., Correia V.S., Segundo M.A., Fonseca A.J.M., Cabrita A.R.J. The IC50 for the positive control was found to be 0.6 g mL-1. <>/Font<>/ExtGState<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI]>>/Contents[19 0 R 20 0 R]/Group<>/Type/Page/Tabs/S>> 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. The site is secure. The DNS method comprises a complex and labor-intensive protocol that includes heating and the use of potentially harmful reagents (e.g. The test is done in microtitre plates with a total volume of 260L and an assay time of 40min including the pre-incubation steps. Extract from Zanthoxylum fagara showed maximum inhibitory activity with an IC50 value of 4.75 g mL-1. Your email address will not be published. Master degree dissertation. An illustrative linear (R2=0.999) calibration curve is abs580nm=0.061 (0.025)+0.366 (0.014) mstarch. Read the color developed at 520 nm. What are the functions of salivary amylase? Linearity was determined using the graph of concentration and percent inhibition. Enhanced consistence of reagent preparation. Food Chem. [5]. In that case, the sensitivity of the determination will be modified. 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 Millers reactions were performed in 13 100 test tubes. 2020 Jan;67(1):87-94. doi: 10.1007/s12020-019-02118-1. 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. A test can be done to measure the level of this enzyme in your blood. The new method is also compared with the most commonly used 3,5-dinitrosalicylic acid (DNSA) method for determining -amylase activity. Chem. [Crossref], 22 Wu, T.; Zhou, X.; Deng, Y.; Jing, Q.; Li, M.; Yuan, L.; J. Ethnopharmacol. On the other hand, the triiodide solution implies the first dissolution of KI followed by I2. endobj 2021 Nov 16;10(11):2818. doi: 10.3390/foods10112818. The Km value is the substrate concentration needed to reach half the maximum velocity. Med 2011, Article ID 810207. [Crossref], 23 Ha, K. S.; Jo, S. H.; Kang, B. H.; Apostolidis, E.; Lee, M. S.; Jang, H. D.; Kwon, Y. I.; J. 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. 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 However, extracts from other plants had no effect on the enzyme activity, suggesting that plant extracts do not contain inhibitors of the enzyme -amylase. Sci. The Pareto diagram (Figure 1) shows that some factors exhibit a significant effect on the response. 1979 Nov;17(11):705-8. 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. Hence, the comparison of the two assays becomes difficult. 8600 Rockville Pike [Crossref]-1616 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980. Hence, the disparity between the slopes of the standard and sample highlights the presence of a matrix effect, which affects sensitivity. Accessibility Evaluation of precision and accuracy was performed using the extracted sample of J. dioica in the presence or absence of acarbose (positive control). The transfer of the vial content to another tube / container may also be necessary. Most of the amylase in your body is made by your pancreas and salivary glands. The in vitro antidiabetic activity was studied by using -amylase inhibitory assay modified 3,5-dinitrosalicylic acid (DNS) method. Absorbance is increasing with increasing concentrations of your inhibitor? Amylase is a digestive enzyme predominantly secreted by the pancreas and salivary glands and found in other tissues at very small levels. Biocatal Agric Biotechnol 23:101452, Thampi A, Bhai RS (2017) Rhizosphere actinobacteria for combating Phytophthora capsici and Sclerotium rolfsii, the major soil borne pathogens of black pepper (Piper nigrum L.). [Crossref] Hence, it is necessary to look for inhibitors of -amylase from natural sources with fewer side effects. [Crossref], 30 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 positive control used was acarbose (0.6 g mL-1). 8) [8]. Toxicol. PN: 27217, Sigma-Aldrich). Springer Protocols Handbooks. For the starch-iodine method, absorbance measurements were conducted at 580nm, whereas for the GOPOD assay, the wavelength was set at 505nm. Salivary amylase is an enzyme that is found in saliva. Dilute to a final volume of 100 ml with reagent grade water. 2014, 17, 465. Prepare by dissolving 1.0 gm of 3,5-dinitrosalicylic acid in 50 ml of reagent grade water. reagent is added in each tube and the mixture is agitated for a few seconds on vortex mixer. [Crossref], 9 Miller, G. L.; Anal. Factors significantly contributing to the response were selected by the Pareto graph. ; Humana Press: New York, 2016 [Crossref] Bioassays are challenging in terms of determining accuracy. about navigating our updated article layout. 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). Hence, a reliable protocol for preparing the starch solution is an important aspect regarding the need for a batch to batch reproducibility. This article will discuss the different applications of amylase. 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. An independent parameter, i.e., enzyme concentration, has a significant effect because as the enzyme concentration increases, the enzyme activity increases up to a certain level and then decreases. 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. A state of sustained hyperglycemia caused by insulin deficiency can lead to type 2 diabetes.11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. 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. Design of a new amylase activity method using starch as substrate and DNS reagent for measuring reducing power as the equivalent of glycosidic bond hydrolysis at eCollection 2021 Nov 9. Experiments conducted to evaluate plate uniformity and signal variability indicated that the response is not influenced by its location on the plate. However, it showed a relatively higher -amylase activity in Fig 3 d. In additio n, Fig 3 d needs control to show equal amounts of proteins were loaded. Thanks for all of you, I increased the temperature to 37 C and decrease the volume of enzyme against the substrate and sample volumes but what I c The obtained results are shown in Table 2. Huang Y, Deng Y, Shang L, Yang L, Huang J, Ma J, Liao X, Zhou H, Xian J, Liang G, Huang Q. Exp Ther Med. Get this protocol in PDF format. 15 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. These plant extracts were offered by researchers from the Analytical Chemistry Department (Facultad de Medicina, UANL, Mxico). Copyright 2016 Elsevier Ltd. All rights reserved. Total PDF Watermark Remover 1.0.214; modified using iTextSharp 4.1.6 by 1T3XTijsr, International Journal of Science and Research, ijsr.net, International Journal of Science and Research (IJSR), www.ijsr.net KI/I2 solution: KI 2.0g L1, I2 200mgL1. Just download this Color Reactions of Carbohydrates file, make a print, and distribute it to the students. The new PMC design is here! [Crossref], a determination coefficient of 0.9947, a slope of 11.493, and an IC50 value of 2.9 0.03 g mL-1 (data not shown). 2009, 23, 614. Rev. The results were subjected to regression analysis to determine R2 and the regression equation for the plot. The ethanol extract of A. Maxicana L. leaves possessed Can you see this Pharmacol. 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). centrifugation). In all cases, a sample blank was analyzed to demonstrate that there were no matrix interferences caused by the extract. 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 endobj Amylase is an enzyme made by your pancreas and salivary glands that helps your body break down carbohydrates. PEst-C/EQB/LA0006/2011) and co-financed by FEDER (UID/QUI/50006/2013 NORTE-01-0145-FEDER-00011) under the Partnership Agreement PT2020. official website and that any information you provide is encrypted The solution with varying concentrations of acarbose was placed in 32 wells in three different plates. This extract was the closest to the acarbose positive control. J Ethnopharmacol. The .gov means its official. Due to the presence of free carbonyl groups in sugars, they can reduce DNS and are oxidized to carboxyl groups. Salivary amylase is an enzyme secreted in the saliva of humans and other mammals. Selected plant products could provide better alternatives due to their low cost and safer use. 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. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 4 0 R/Group<>/Tabs/S/StructParents 0>> Before 1959, 31, 426. 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. [1] Amylase was first described in the early 1800s and is considered one of the first enzymes in history to be scientifically investigated.

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