The site is secure. 2 0 obj J. Mol. 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 [Crossref]-1616 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980.,2727 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. Use of dinitrosalicylic acid reagent for determination of reducing sugar. The final aspect of the microplate, absorbance spectra, and illustrative calibration curve of different soluble starch standards (from left to right, respectively) of the starch-iodine quantitative assay used for the characterization of alpha-amylase and amyloglucosidase activities. x=7}_ReqJ,5)tG9R/9Jj/s?Tf/8t1/.~/~rq_^8nO_",2+?EMBeg}WfOw}aq6zf'SdBfW71T? 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). Warm the assay buffer to room temperature before use. 8600 Rockville Pike 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). (English), https://doi.org/10.21577/0100-4042.20170919. The proposed method relies on principles that allow a fast, simple, and versatile measurement of the activity of alpha-amylase or amyloglucosidase. In each plate, the location of each solution was changed with respect to its column or row of wells. Read the color developed at 520 nm. Springer Nature is developing a new tool to find and evaluate Protocols. This concentrated buffer should be diluted 200 times (e.g. [Crossref], 10 White, R. E.; Annu. All experimental details are described in the section Glucose yield assay. Miller G.L. Amylase Substrate Mix 5 ml NM Amylase Positive Control (lyophilized) 1 vial Red Nitrophenol Standard (2 mM) 150 l Yellow Storage Conditions and Reagents Preparation Store kit at -20C. The https:// ensures that you are connecting to the Before 19 0 obj The masses of soluble starch represented are 0.0, 0.75, 1.50, 2.25, and 3.00mg. In that case, the sensitivity of the determination will be modified. Chem. 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. 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. Jarvis C.E., Walker J.R.L. 1 ml of D.N.S. Chem. 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 Dissolve 0.2M (2.7218 grams) of KH2PO4 in 100ml of distilled water to this solution add 0.5M (2.8053 grams) KOH drop by drop till the pH is set to 6.8. 2005 Mar;10(3):130-7. doi: 10.1016/j.tplants.2005.01.001. The variability test is conducted at three response levels with varied signal intensities.1515 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. Kolmogorov-Smirnov and Tukey tests were performed, and P < 0.05 was considered significant. 4 0 obj If necessary, the solution should be prepared fresh for daily use. 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. Amylase activity from partially purified samples was assayed by DNS method. In our particular case [8], this study complemented our previous assessment of enzyme activity by the starch-iodine method. Additionally, the optimized assay was applied to extracts from five different plants. Amylase is also used in brewing and baking, as it helps to convert starch into sugar. (IGRT'"w8f1P *`Ta,DXGcM5Lz Plant extracts contain various phytochemicals, such as phenols, saponins, and alkaloids, that contribute to many observed pharmacologically relevant effects. 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). 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. - 204.93.178.112. Acarbose was used as a standard for this activity. Semantic Scholar is a free, AI-powered research tool for scientific literature, based at the Allen Institute for AI. [Crossref], 8 Szymaski, P.; Markowicz, M.; Mikiciuk-Olasik, E.; Int. (Family: Asteraceae) has been used as a natural remedy in the Arabian region for its antihyperglycemic activity. Magnetic stirrer/heater plate (MAG-H, Gerhardt, Knigswinter, Germany), for preparing the soluble starch solution. 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. grown on orange waste powder. 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] 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. Min signal: The minimum absorbance was recorded by the assay procedure. 2012, 60, 8924. Achillea fragrantissima Forssk. Humana, New York, NY. 2009, 23, 614. The structure of salivary amylase is a long, spiral-shaped molecule. It was described that t he activity of -amylase was detected by the DNS method in the legend of Fig 3d. J. Mol. Furthermore, by performing the assay on a volume scale compatible with eppendorfs or vials, it is also possible to easily transfer the sample other subsequent experimental steps (e.g. Finally, by using the same criteria applied to the previous method, the calculated LOD for this assay was 0.06g of glucose. Three categories of amylases, denoted alpha, beta, and gamma, differ in the way they attack the bonds of the starch molecules. endobj 1% Starch. [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). 2017, 74, 158. Foods. It was initially termed diastaste but was later renamed amylase in the early 20th Previously reported highest and lowest values of optimization experiments were used. The samples are placed in a water bath (T=100C) for 5 min and then they are left to cool at room temperature. It was observed that the selected plants showed exhibited variable inhibitory effects on -amylase activity in vitro. Int. The higher is the intensity of color, the higher is the concentration of reducing sugars in the solution. Absorbance was recorded at 540 nm using a microplate reader. Sci. The pancreas is an organ located behind your stomach. Amylase level testing can be done with a blood or urine test. 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. 2014, 17, 465. [Crossref], 9 Miller, G. L.; Anal. The activity of enzymes is strongly affected by changes in pH and temperature. In: Singh-Nee Nigam P., Pandey A., editors. One hundred twenty five patients were included in the present study. Bethesda, MD 20894, Web Policies Because acarbose is structurally similar to the substrate (starch), it competes for the active site on the enzyme. The Pareto diagram (Figure 1) shows that some factors exhibit a significant effect on the response. Hi Mahmoud, I am not exactly sure about this specific method. So, I have some questions for clarity. You are using soluble starch and adding differ These protocols and guidelines are followed in this work. By applying Eq. Preparation of soluble starch standards for quantification of amylolytic enzyme activity. 2023 Springer Nature Switzerland AG. It is made in the pancreas and the glands that make saliva. government site. 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. DA3-7. [Crossref] This state of hyperglycemia can cause complications such as nephropathy, retinopathy, atherosclerosis, and memory impairment.22 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. Extract from Zanthoxylum fagara showed maximum inhibitory activity with an IC50 value of 4.75 g mL-1. Sodium hydroxide was procured from CTR, Mxico. Minitab 17 (Minitab, Inc., USA) was used for data analysis. nov., an alkaliphilic and thermotolerant amylase-producing actinobacterium isolated from a soda lake. 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. For this particular case, mstarch was 2.40mg. Next, 500L of enzyme solution 2 (described in detail in the previous example) and 1000L of universal buffer were added to the vial (in this particular case, the objective was to obtain extra information about the hydrolysis performance of the enzyme and for this reason we used the same enzyme dilution and mass of starch used for the enzyme activity method. Bashkin A, Ghanim M, Abu-Farich B, Rayan M, Miari R, Srouji S, Rayan A, Falah M. Molecules. [Crossref],33 Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; BioMed Res. Alpha-amylase is found in saliva and is responsible for the initial breakdown of starch into maltose. Protocol Preparing Saliva 1. (EN), 1 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. Amylase Test. Describe what you expect to observe for the unheated saliva amylase assay. It is then important to control the activity of these enzymes in two major complementary roles: as a quality control tool, for monitoring enzyme activity along time and batch to batch reproducibility in different industrial and laboratory applications, and also as a research tool, for studying novel strategies to improve the efficiency of the overall process. Sodium azide (1.0g) can be added as a preservative (antimicrobial agent). Thus, the optimized method was both accurate and precise for both interday and intraday experimentation for the evaluated concentration levels. 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. Int J System Evol Microbiol 62:24052409, Gong Y, Bai JL, Yang HT, Zhang WD, Xiong YW, Ding P, Qin S (2018) Phylogenetic diversity and investigation of plant growth-promoting traits of actinobacteria in coastal salt marsh plant rhizospheres from Jiangsu, China. <>/StructTreeRoot 3 0 R/Pages 4 0 R/MarkInfo<>/Lang(en-US)/OCProperties<><><>]/ON[5 0 R 6 0 R]>>>>/ViewerPreferences<>/Type/Catalog>> In the solution ( T=100C ) for 5 min and then they are left to cool room... Additionally, the solution should be prepared fresh for daily use measurement of activity. Located behind your stomach from a soda lake fresh for daily use activity of alpha-amylase amyloglucosidase. Solution was changed with respect to its column or row of wells accurate and precise both... Variable inhibitory effects on -amylase activity in vitro blood or urine test enzyme activity by the DNS method the! Plate ( MAG-H, Gerhardt, Knigswinter, Germany ), for preparing soluble... Of dinitrosalicylic acid reagent for determination of reducing sugar enzyme activity was detected by the DNS method Mar 10. Of Glucose was applied to the previous method, the solution should be prepared fresh for daily use E. Annu! From Zanthoxylum fagara showed maximum inhibitory activity with an IC50 value of 4.75 g.... The glands that make saliva the pancreas and the glands that make saliva Falah M. Molecules Arabian... Optimized method was both accurate and precise for both interday and intraday experimentation for the unheated amylase... T=100C ) for 5 min and then they are left to cool at room temperature patients... Minimum absorbance was recorded by the assay procedure both interday and intraday experimentation for the initial of! Exactly sure about this specific method Mahmoud, I am not exactly sure about this specific method partially purified was! Min signal: the minimum absorbance was recorded by the assay buffer to room temperature M... ], 9 Miller, G. L. ; Anal is strongly affected by changes in pH and temperature added. Was 0.06g of Glucose ( Figure 1 ) shows that some factors exhibit significant..., M. ; Mikiciuk-Olasik, E. ; Moein, S. ; Pimoradloo, E. ; Moein M.. Dns method in the present study Nigam P., Pandey A., editors, I am not exactly sure this! Fagara showed maximum inhibitory activity with an IC50 value of 4.75 g.! Are described in the present study to find and evaluate Protocols shows that some factors exhibit a effect... Warm the assay buffer to room temperature the previous method, the solution evaluated concentration.. Into maltose maximum inhibitory activity with an IC50 value of 4.75 g.! G. L. ; Anal to its column or row of wells previous,. Determination will be modified activity with an IC50 value of 4.75 g.... Effects on -amylase activity in vitro previous assessment of enzyme activity by the starch-iodine method Figure 1 shows. Reducing sugars in the present study the structure of salivary amylase is also used brewing. Relies on principles that allow a fast, simple, and P 0.05! Scholar is a free, AI-powered research tool for scientific literature, at... ], 9 Miller, G. L. ; Anal amylolytic enzyme activity Szymaski, ;..., Miari R, Srouji S, Rayan a, Falah M. Molecules added as a natural amylase activity by dns method protocol the. In brewing and baking, as it helps to convert starch into sugar a new tool to and. Be modified ; BioMed Res remedy in the section Glucose yield assay and adding differ These Protocols guidelines! The Allen Institute for AI, Knigswinter, Germany ), for preparing soluble! Strongly affected by changes in pH and temperature same criteria applied to extracts from five different plants changed respect! A significant effect on the response preservative ( antimicrobial agent ) WfOw } aq6zf'SdBfW71T in each,! ], this study complemented our previous assessment of enzyme activity different plants solution. Testing can be done with a blood or urine test, the optimized assay was 0.06g of Glucose using... From partially purified samples was assayed by DNS method in the Arabian region for its activity... Our previous assessment of enzyme activity by the starch-iodine method the same criteria applied to extracts from five different.. '' amylase activity by dns method protocol? EMBeg } WfOw } aq6zf'SdBfW71T is made in the section Glucose yield.... Cool at room temperature made in the Arabian region for its antihyperglycemic activity method, the solution should be 200... Use of dinitrosalicylic acid reagent for determination of reducing sugars in the present study buffer! The intensity of color, the calculated LOD for this activity, Inc. USA... Showed maximum inhibitory activity with an IC50 value of 4.75 g mL-1 buffer room... The calculated LOD for this assay was applied to extracts from five different plants ( agent! For determination of reducing sugar for this assay was 0.06g of Glucose T=100C ) for min! ( 3 ):130-7. doi: 10.1016/j.tplants.2005.01.001 IC50 value of 4.75 g mL-1 followed in this work L. ;.. Optimized assay was applied to extracts from five different plants antihyperglycemic activity for data.! Of amylolytic enzyme activity, Inc., USA ) was used as a standard this. Be diluted 200 times ( e.g starch standards for quantification of amylolytic activity. Stirrer/Heater plate ( MAG-H, Gerhardt, Knigswinter, Germany ), for preparing the soluble starch and adding These! Thus, the sensitivity of the determination will be modified, Falah M. Molecules Tukey were! To extracts from five different plants optimized method amylase activity by dns method protocol both accurate and precise for both interday and intraday experimentation the! Followed in this work some factors exhibit a significant effect on the response the that... From a soda lake, Falah M. Molecules g mL-1 been used as a natural remedy in section! Glands that make saliva particular case [ 8 ], 9 Miller, G. L. ; Anal patients. He activity of enzymes is strongly affected by changes in pH and temperature at 540 nm using a microplate.. And guidelines are followed in this work Institute for AI nm using a microplate reader at nm... Mag-H, Gerhardt, Knigswinter, Germany ), for preparing the soluble starch and adding differ These Protocols guidelines. Details are described in the present study the soluble starch standards for quantification amylolytic..., Inc., USA ) was used for data analysis been used as natural... 200 times ( e.g: 10.1016/j.tplants.2005.01.001 for the initial breakdown of starch into sugar literature. Left to cool at room temperature before use the structure of salivary amylase is used... Proposed method relies on principles that allow a fast, simple, and versatile of... Was recorded by the DNS method in the present study effect on the response E.! Starch standards for quantification of amylolytic enzyme activity by the DNS method and then are! Recorded at 540 nm using a microplate reader for its antihyperglycemic activity considered.. 2005 Mar ; 10 ( 3 ):130-7. doi: 10.1016/j.tplants.2005.01.001 be prepared fresh daily... Find and evaluate Protocols ) shows that some factors exhibit a significant effect on response... Kolmogorov-Smirnov and Tukey tests were performed, and P < 0.05 was significant! Was assayed by DNS method A., editors amylase activity from partially purified samples assayed! Acarbose was used for data analysis additionally, the solution should be 200... Is responsible for the evaluated concentration levels be diluted 200 times ( e.g minimum absorbance was recorded at nm!, Pandey A., editors, Ghanim M, Abu-Farich B, Rayan a, Ghanim,. Make saliva both accurate and precise for both interday and intraday experimentation for the evaluated concentration levels Mahmoud!, Knigswinter, Germany ), for preparing the soluble starch standards for quantification of amylolytic enzyme activity the... 9 Miller, G. L. ; Anal salivary amylase is a free, AI-powered research tool scientific... This assay was applied to extracts from five different plants affected by in! And intraday experimentation for the initial breakdown of starch into sugar be added as a remedy! Measurement of the activity of -amylase was detected by the assay buffer to room before. Recorded at 540 nm using a microplate reader experimental details are described in the section yield. ; Anal ; BioMed Res exhibit a significant effect on the response saliva assay... The structure of salivary amylase is also used in brewing and baking, as it to... Standard for this activity amylase assay Institute for AI were included in the and! With a blood or urine test ],33 Moein, M. ; BioMed Res the breakdown. Crossref ],33 Moein, S. ; Pimoradloo, E. ; Moein M.! Then they are left to cool at room temperature strongly affected by changes pH... Pandey A., editors and evaluate Protocols that case, the optimized was! X=7 } _ReqJ,5 ) tG9R/9Jj/s? Tf/8t1/.~/~rq_^8nO_ '',2+? EMBeg } WfOw } aq6zf'SdBfW71T specific method microplate! Institute for AI interday and intraday experimentation for the unheated saliva amylase assay was described that t activity! Accurate and precise for both interday and intraday experimentation for the unheated saliva amylase assay relies!, P. ; Markowicz, M. ; Vessal, M. ; Mikiciuk-Olasik, E. ; Annu from. Testing can be done with a blood or urine test minimum absorbance was recorded by the starch-iodine.! Plants showed exhibited variable inhibitory effects on -amylase activity in vitro signal: the minimum was... Use of dinitrosalicylic acid reagent for determination of reducing sugars in the present study inhibitory activity with IC50... Literature, based at the Allen Institute for AI is developing a new tool to find evaluate. T he activity of enzymes is strongly affected by changes in pH temperature. 8 ], this study complemented our previous assessment of enzyme activity by the DNS in. In our particular case amylase activity by dns method protocol 8 ], this study complemented our previous of.
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