2020 Jan;67(1):87-94. doi: 10.1007/s12020-019-02118-1. Visvanathan R, Houghton MJ, Williamson G. Food Chem. 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. Accessibility 2017, Article ID 7319504. 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. The extract of A. lebbeck at a concentration of 1000g/ Most of the amylase in your body is made by your pancreas and salivary glands. The intensification of amyloglucosidase-based saccharification by ultrasound. These bonds can be easily broken by water, so saliva is necessary for the enzyme to work. ; Humana Press: New York, 2016 [Crossref]. The optimized and validated method was accurate, precise, and linear. [Crossref]. Most enzymes act specifically with only one reactant, called a substrate, to produce products. Federal government websites often end in .gov or .mil. Absorbance was recorded at 540 nm using a microplate reader. To search for new compounds, it is necessary to carry out pharmacological screening for a large number of potential -amylase inhibitors in a short period, for which high-throughput screening can be successfully used.88 Szymaski, P.; Markowicz, M.; Mikiciuk-Olasik, E.; Int. The robustness of the method was analyzed by estimating the response obtained by deliberately changing the conditions. Changes in these parameters do not have any effect on the inhibition percentage. 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. [Crossref], 10 White, R. E.; Annu. A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities. 6, where we studied the influence of pH on the activity of amyloglucosidase from A. niger. official website and that any information you provide is encrypted % In this experiment, acarbose at a concentration of 5 g mL-1 was used to record the desired minimum signal. and transmitted securely. It was initially termed Analysis of results using the Tukey test ( = 0.05) indicated that the method is robust for two conditions, i.e., incubation temperature and preincubation time. In addition, the effect of substrate concentration on enzyme activity was analyzed. [Crossref], 19 Satpathy, G.; Tyagi, Y. K.; Gupta, R. K.; Food Res. Glucose oxidase / peroxidase (GOPOD) (K-GLUC) assay kit was purchased from Megazyme (Wicklow, Ireland). 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. Experiments included different concentrations of the enzyme (0.1 and 0.2 U mL-1), preincubation time (5.2 and 9.2 min), incubation time (4.5 and 6.5 min), and time in a water bath (10.6 and 20.6 min). The -amylase assay was performed using Millers method, i.e., the DNS method. KI/I2 solution: KI 2.0g L1, I2 200mgL1. 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. 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 Detailed information is available in our previous publications [7,8]. Soluble starch (PN: S9765), starch from corn (PN: S4126), starch from potato (PN: S4251), starch from rice (PN: S7260), starch from wheat (PN: S5127, unmodified), I2 (PN: 207772, 99.8%), 85% (w/w) o-phosphoric acid (PN: 79620), and acetic acid (PN: 695092, 99.7%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 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 Keywords: 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. endobj Based upon the plasma glucose value, all subjects were divided into three groups. The proposed method showed high precision in long-term use (RSD<6.3%). 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. Universal buffer: o-phosphoric acid 47mmolL1, acetic acid 50mmolL1, boric acid 50mmolL1. Your email address will not be published. [Crossref] Additionally, it is necessary to validate the assay method to demonstrate that it is acceptable for determining the pharmacological activity of a new chemical entity by a detectable signal that allows the biological process to be quantified. For the enzyme amyloglucosidase from Aspergillus niger (PN: A9913, Sigma Aldrich), 10L of the commercial enzyme solution were mixed with 1990L of universal buffer at the desired pH (enzyme solution 1, dilution factor 200). [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). Med 2011, Article ID 810207. Unit of Microbiology, Department of Biology, University of Patras, http://www.microbiology.biology.upatras.gr. 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. This discrepancy highlights the reason for not comparing the results obtained by different reports, although they might have used the same method but with different experimental conditions. [Crossref],33 Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; BioMed Res. 2020-04-29T17:28:43+05:302017-05-24T07:28:08ZMicrosoft Office Word 2007 Learn more A test can be done to measure the level of this enzyme in your blood. It helps you protect your students from spelling mistakes and volumetric errors. R. P.; Mol. Screening 1999, 4, 67. After 10min, the reaction was stopped with 200L of HCl 1.0molL1 and the vial was centrifuged for 5min at 350g. Next, 50L of supernatant were transferred to a 96-well microplate, followed by the addition of 150L of GOPOD reagent. The use of a suitable volumetric scale for the assay was also an important concern for our development work. Prepare There are numerous methods used for the determination of amylase activity, of which 3,5-dinitrosalicylic acid (DNSA) assay is the most widely used. This method intends to facilitate the implementation of a new routine determination of alpha-amylase and amyloglucosidase activities in any laboratory that could deal with amylolytic enzymes by providing all the necessary experimental details and conditions that could maximize the performance of the method. 351 (2006) 146148]. Constructing a standard curve / graph for maltose helps us to estimate concentration of reducing sugars present in an unknown sample and for determining the activity of amylase enzyme in water baths) and containers (e.g. Insoluble chromogenic substrates have been developed for ~-amylase assay. <> [Crossref],77 Yilmazer-Musa, M.; Griffith, A. M.; Michels, A. J.; Schneider, E.; Frei, B.; J. Agric. Dinitrosalicylic acid color reagent. From these samples, salivary amylose enzyme assays were carried out. Trends Plant Sci. The samples are placed in a water bath (T=100C) for 5 min and then they are left to cool at room temperature. Tukeys test ( = 0.05) was used to evaluate the robustness of the individual methods. The diagnosis of saliva amylase enzyme activity deficiency requires an oral test with a standard protocol. 2023 Springer Nature Switzerland AG. Dilute to a final volume of 100 ml with reagent grade water. National Library of Medicine 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. This can be regarded as a standard approach since this is a common composition ratio found in different starches [13]. This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. 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]. The kinetic constants for the enzyme were determined by varying the substrate concentration from 0.03 mg mL-1 to 0.6 mg mL-1. When the substrate concentration is increased, the inhibition by the competitive inhibitor is reversed. LOD was calculated as the starch mass equivalent to the absorbance that resulted from the addition of the blank signal (standard solution without soluble starch) to 3 times the corresponding standard deviation [14]. Oliveira H.M., Correia V.S., Segundo M.A., Fonseca A.J.M., Cabrita A.R.J. J. Mol. 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. 12 Stevenson, R. A.; American Laboratory, 2011, available at https://www.americanlaboratory.com/913-Technical-Articles/19109-Bioassays-2011/, accessed June 2022. This method is based on the binding of iodine to terminals of the starchs polymeric chain [4] that results in a blue colored complex that can be also quantitatively monitored by UVvis spectrophotometry [5]. This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National funds (FCT through grant no. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). DA3-7. The experimental design and data handling were performed using Statgraphics Centurion XVI software (Statpoint Technologies, Inc., Warrenton, VA, USA). It then breaks down carbohydrates in the food we eat into simpler sugars. Factors showing a significant effect on the inhibition percentage were selected and optimized using a basic sequential simplex method. 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. Enhanced consistence of reagent preparation. See, The calibration curve for glucose was linear for masses of glucose up to 10g (equivalent to concentrations up to 200mgL, Determination of the alpha-amylase and amyloglucosidase activities, Enzyme activity, Starch-iodine, Glucose oxidase/peroxidase, Glucose yield. Note: amylolytic enzyme working solutions should be prepared fresh daily and kept on ice at all times. 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). (EN), 1 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. -Amylase from the porcine pancreas (15 U mg-1) was used instead of human amylase because of its cost and availability. McCleary B.V., McNally M., Monaghan D., Mugford D.C. Received 2018 Aug 3; Accepted 2019 Jan 13. Before The intra-day variation was below 1.6%. The new PMC design is here! The use of a microscale method offers several advantages such as 1) fewer chemicals needed, 2) reduction in material cost, 3) ease of disposal of the reaction mixture after experimentation, 4) reduction of the safety risk, and 5) multiple experiments can be performed at the same time. What are Polysaccharides? ),8a\ 9q\&IEv%7[%{%RhJ{fN~1@H)a=1Di)?V6br{CvdG8z:*zp0\Ac SbJ? PXRI^7wgGRf i=QJM)!WJAdsr9Q)@zv$Mp?#oa:FI$@Z -;t~$?zvCH1!WPqDw (jS{z=; zuQp>$C(7X>/tV s5hcD!8-)~uJB$0NmzIuBR3X"@I53]M}BF:HFuK$dEQesCaxw9h5$SLA[_m}4#S?0&g~g^>*'kHI$J(YUr03hv;y5tt &TRsB#C[E,mI=6XFv64on6NCD7og. @fl9w8 XgUT>{LBGD"?og oY]LCxn(n?(Kp{D\:bB#kV!45 d!n;$w d\|:f. If no amylase activity is present in the saliva, how many mg of starch will be detected in the first time point of the assay? 2017, 109, 962. This also means that the volumes could be easily adjusted in order to match the requirements of each particular enzyme analysis or study. Transfer 1500L of water and 1500L of universal buffer (control) or of amyloglucosidase solution (assay) into a 4mL amber glass vial. In: Dharumadurai, D. (eds) Methods in Actinobacteriology. In: Singh-Nee Nigam P., Pandey A., editors. 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 15 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. Xiao Z.Z., Storms R., Tsang A. Spectrochim. 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. The reagent has a dark orange colour and is stable for several days in room temperature. Forty-One Plant Extracts Screened for Dual Antidiabetic and Antioxidant Functions: Evaluating the Types of Correlation between -Amylase Inhibition and Free Radical Scavenging. 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. However, these methods share a common problem of being labor intensive, time consuming and require substantial amount of reagents and samples. 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. Before Describe what you expect to observe for the unheated saliva amylase assay. 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Maltose with glucose oxidase ( GOD ) and National funds ( FCT through grant no requirements... Available at https: //www.americanlaboratory.com/913-Technical-Articles/19109-Bioassays-2011/, accessed June 2022 salivary amylose enzyme assays were out.