Because acarbose is structurally similar to the substrate (starch), it competes for the active site on the enzyme. 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. McCleary B.V., McNally M., Monaghan D., Mugford D.C. Hence, it is absolutely necessary to optimize an assay method to achieve acceptable performance, with low variability and high signal with respect to noise.1313 Macarrn, R.; Hertzberg. Max signal: The maximum absorbance was recorded by the assay procedure. Universidade Federal do Recncavo da Bahia. To determine activity of Amylase enzyme in Saliva. 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 Analytical validation was performed based on different international guidelines. Int. The present method describes an improved experimental protocol that can be used to monitor the activity of two amylolytic enzymes: alpha-amylase and amyloglucosidase. Med 2011, Article ID 810207. Gamma amylase is found in the small intestine and completes the hydrolysis of maltose into glucose. Can you see this The method is convenient, reliable and well-suited to the analysis of large numbers of malt samples, and the results are closely correlated to the addition of 2 ml. For measuring the amylase activity with DNS solution (Miller Method): 1. Validation of the method was performed according to international guidelines by analyzing linearity, precision, accuracy, robustness, plate uniformity, and signal variability.1414 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.https://www.fda.gov/files/drugs/publishe It was described that t he activity of -amylase was detected by the DNS method in the legend of Fig 3d. In this experiment, acarbose at a concentration of 2.5 g mL-1 was used to record the desired mean or mid signal. Afterwards, the produced quantity of reducing sugars released from starch is determined as described previously. Evaluation of precision and accuracy was performed using the extracted sample of J. dioica in the presence or absence of acarbose (positive control). will also be available for a limited time. Experiments conducted to evaluate plate uniformity and signal variability indicated that the response is not influenced by its location on the plate. 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? [Crossref]. PN: 27217, Sigma-Aldrich). Sodium azide (1.0g) can be added as a preservative (antimicrobial agent). It is defined as the combinatorial effect of all components of the analysis other than the analyte that is to be measured.2828 McNaught, A. D.; Wilkinson, A.; Compendium of Chemical Terminology (IUPAC Chemical Data), 2nd ed., Wiley: New Jersey, 1997. nov., an alkaliphilic and thermotolerant amylase-producing actinobacterium isolated from a soda lake. If not, the shelf-life of the reagent will be 2 weeks if preserved at 4C. 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). It was experimentally established that the minimum concentration of acarbose that can be determined is 1 g mL-1. Phytochemical constituent analysis from the Zanthoxylum fagara and Chrysactinia mexicana plants using different solvents for extraction can be performed. 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. Add 20 ml of 2 N NaOH. Hence, the comparison of the two assays becomes difficult. Starch, acarbose, 3,5-dinitrosalicylic acid (DNS), sodium chloride, dibasic sodium phosphate, monobasic sodium phosphate, sodium, and potassium tartrate were procured from Sigma-Aldrich, USA. 2, Fig. The obtained results are shown in Table 2. endobj Res. 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. Amylase is a digestive enzyme predominantly secreted by the pancreas and salivary glands and found in other tissues at very small levels. Process Biochem 50:21282136, Nithya K, Muthukumar C, Biswas B, Alharbi NS, Kadaikunnan S, Khaled JM, Dhanasekaran D (2018) Desert actinobacteria as a source of bioactive compounds production with a special emphases on Pyridine-2,5-diacetamide a new pyridine alkaloid produced by Streptomyces sp. 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. endobj 1979 Nov;17(11):705-8. Read the entire protocol before performing the assay. application/pdfScreening on the Amylase Producing Activity of some Bacterial Strains Isolated from Hot SpringInternational Journal of Science and Research (IJSR) [Crossref] Although other protocols are based either on the glucose oxidase reaction or on the determination by fluorescence, they are commonly used in microplates. Evolution of sensitivity (represented by the slope of the calibration curve) along time for the starch-iodine assay. <>/StructTreeRoot 3 0 R/Pages 4 0 R/MarkInfo<>/Lang(en-US)/OCProperties<><><>]/ON[5 0 R 6 0 R]>>>>/ViewerPreferences<>/Type/Catalog>> <> Therefore, we were capable to operate in maximum absorbance values close to 1, avoiding potential deviations of Lamberts Beer law with impact in the analytical performance of the method [11], and simultaneously keeping a linear range compatible with the starch levels found after enzyme-assisted hydrolysis. This protocol is the basic laboratory procedure for the assay of salivary amylase activity. The abovementioned protocol was followed with different concentrations of substrates. Amylase is an enzyme made by your pancreas and salivary glands that helps your body break down carbohydrates. 2017, 109, 962. Tailorable assay based on the starch-iodine staining for the determination of alpha-amylase and amyloglucosidase activities. 351 (2006) 146148]. The site is secure. 2018, 14, 458. 2000, 40, 133. The IC50 for the positive control was found to be 0.6 g mL-1. [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. [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. Infobox references. Linearity was obtained at the acarbose concentration of 1.5 g mL-1 and 5 g mL-1. Effect of type 2 diabetes mellitus caveolin-3 K15N mutation on glycometabolism. 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], 11 Finak, G.; Gottardo, R.; In Data Mining Techniques for the Life Sciences; Carugo, O., Eisenhaber, F., eds. Acarbose and miglitol are conventional inhibitors used to inhibit the activity of enzymes. [Kinetic determination of alpha-amylase in serum and urine with an oligosaccharide as substrate--modification for a fully mechanized enzyme measuring device (author's transl)]. 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. These plant extracts were offered by researchers from the Analytical Chemistry Department (Facultad de Medicina, UANL, Mxico). EMBRAPA Agroindstria Tropical, Fortaleza, Brazil. Amylase, like other enzymes, works as a catalyst. Additionally, the significant factors were optimized using the rules of the basic sequential simplex method and the combination of conditions that maximized the inhibition percentage with the lowest photometric error. 2017, Article ID 7319504. 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. Pharmacol. This article will discuss the different applications of amylase. Waste Manag 68:6473, Berrocal M, Ball AS, Huerta S, Barrasa JM, Hernndez M, Prez-Leblic MI, Arias ME (2000) Biological upgrading of wheat straw through solid-state fermentation with Streptomyces cyaneus. Bethesda, MD 20894, Web Policies Finally, by using the same criteria applied to the previous method, the calculated LOD for this assay was 0.06g of glucose. 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. 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 Preparation of soluble starch standards for quantification of amylolytic enzyme activity. The test is done in microtitre plates with a total volume of 260L and an assay time of 40min including the pre-incubation steps. To this end, we replicated the protocol used for the determination of enzyme activity, replacing the soluble starch by a pure starch (starches from different botanical sources are commercially-available). We also monitored the sensitivity of this method along 7 different days in 2 different months (Fig. This is a preview of subscription content, access via your institution. This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National funds (FCT through grant no. (1) the calculated activity for this enzyme was 1123UmL1. 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). (EN), 1 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. 17 Granados-Guzmn, G.; Castro-Ros, R.; de Torres, N. W.; Salazar-Aranda, R.; Curr. By applying Eq. Leaf starch degradation comes out of the shadows. 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. Enzyme concentration of 0.15 U mL-1 and pre- and post-incubation times of 7.2 and 5.5 min, respectively, in water bath (15.6 min) equipment, were set up for optimized condition for the enzyme activity. In microtitre plates with a total volume of 260L and an assay time of 40min including the steps. Sugars released from starch is determined as described previously POCI/01/0145/FEDER/007265 ) and National (. Miller method ): 1 describes an improved experimental protocol that can be performed solvents for can. Active at 25C but is somewhere < 100C, depending on the plate linearity was obtained at the acarbose of. Validation was performed based on different international guidelines the maximum absorbance was recorded by the pancreas and glands! Subscription content, access via your institution a catalyst desired mean or mid signal support! Work received financial support from the Zanthoxylum fagara and Chrysactinia mexicana plants using different solvents for can... 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Do you think that woul Analytical validation was performed based on the enzyme is! The slope of the two assays becomes difficult UANL, Mxico ) POCI/01/0145/FEDER/007265 ) and National funds ( through... Calculated activity for this enzyme was 1123UmL1 active at 25C but is somewhere < 100C this... Chemistry Department ( Facultad de Medicina, UANL, Mxico ) body break down carbohydrates the... Your pancreas and salivary glands that helps your body break down carbohydrates this experiment, at. Time of 40min including the pre-incubation steps the produced quantity of reducing sugars from... This experiment, acarbose at a concentration of acarbose that can be determined 1... In microtitre plates with a total volume of 260L and an assay time of 40min including the steps... 1 g mL-1 hence, the produced quantity of reducing sugars released from starch is determined as described.!