The intra-day variation was below 1.6%. 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. The first approach relies on the measurement of reducing sugars, being the dinitrosalicylic acid (DNS) method [3] the classic approach. This also means that the volumes could be easily adjusted in order to match the requirements of each particular enzyme analysis or study. Tukeys test ( = 0.05) was used to evaluate the robustness of the individual methods. What are Polysaccharides? endstream Med 2011, Article ID 810207. An inhibition percentage near 50% was chosen as the best response, while the absorbance of the control at 0% inhibition (100% activity) resulted in the smallest photometric error. Microbiol Res 207:116133, Srinivas A, Rahul K, Sasikala C, Subhash Y, Ramaprasad EVV, Ramana CV (2012) Georgenia satyanarayanai sp. Hence, we kept the same volume ratio of the original method but we increased the assay to a total volume of 3.00mL according to our requirements, which were defined by the specifications of the US probe used to study the effect of US in the activity of amylolytic enzymes [7,8]. 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]. Rev. To each tube, 100 L of plant extracts with different concentrations (final concentration 6 g mL-1 to 200 g mL-1) and 100 L of -amylase (final concentration 0.15 U mL-1 in phosphate buffer) were added and incubated at 37 C for 7.2 min. When the enzymatic activity decreases, the formation of the product also decreases; therefore, the intensity of the color is reduced. Previously reported highest and lowest values of optimization experiments were used. 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. Pharmacol. Salivary amylase analysis is a simple procedure and the results obtained can be used as an additional criterion in the diagnostic process of diabetes. Beta amylase is located in the pancreas and produces maltose from the hydrolysis of the maltose molecule. Your email address will not be published. Therefore, our variables for Eq. The samples are placed in a water bath (T=100C) for 5 min and then they are left to cool at room temperature. sharing sensitive information, make sure youre on a federal Crops Prod. 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. However, because the incubation temperature is increased, more reactions between the enzyme and substrate occur. 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. Bethesda, MD 20894, Web Policies Toxicol. %PDF-1.5 J. Mol. [Crossref], 3 Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; BioMed Res. Would you like email updates of new search results? <>stream However, extracts from other plants had no effect on the enzyme activity, suggesting that plant extracts do not contain inhibitors of the enzyme -amylase. 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.). R. P.; Mol. The error percentage was calculated to be 5.46%, which indicates that the method is accurate. 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]. 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. 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. Chem. Biotechnol. Because acarbose is a reversible competitive inhibitor, an increase in substrate concentration results in the reversion of bound acarbose (Figure 2). 1% Starch. Enhanced consistence of reagent preparation. (Family: Asteraceae) has been used as a natural remedy in the Arabian region for its antihyperglycemic activity. {y)a?:(J F([[y#? 29 Zhang, J. H.; Chung, T. D.; Oldenburg, K. R.; J. Biomol. 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. 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 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. Amylase - blood. Int. Another potential application of the proposed method is the evaluation of the activity of amylolytic enzymes extracted from different natural matrices such as flours and microbial enzyme preparations. Briefly centrifuge vials before opening. This work is part of the Academic Masters Dissertation by student Milena Santos Aguiar linked to the Graduate Program of Microbiology and Biotechnology of Microorganisms (PPGBBM, UESC). 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. A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities. Three categories of amylases, denoted alpha, beta, and gamma, differ in the way they attack the bonds of the starch molecules. Transfer 50L of sample supernatant (or standard), and add 150L of GOPOD reagent to each well of the microplate. 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. Soluble starch standard solutions: prepare by stepwise dilution of the starch stock solution with water; prepare fresh daily. Some examples of amylolytic enzymes assessed by this method were: amyloglucosidase from Aspergillus niger (PN: A9913) (illustrative results with this enzyme will be shown in the next sections), and alpha-amylase from Bacillus licheniformis (PN: A3306, heat-stable), purchased from Sigma-Aldrich, amyloglucosidase Spirizyme Achieve and alpha-amylase from B. licheniformis Liquozyme SC DS (AA-2), supplied by Novozymes (Bagsvaerd, Denmark). [. The graph of inhibition percentage vs. inhibition concentration was plotted and checked for linearity (visually). In this experiment, acarbose at a concentration of 1.05 g mL-1 was used to record the desired maximum signal. phenol). The intra-day variation of sensitivity ranged between 2.0 and 6.3%, and the inter-day variation was 4.9% for the complete period. For the calculation of the glucose yield assay promoted by amyloglucosidase from Aspergillus niger (PN: A9913, Sigma Aldrich), 1500L of water were added to a vial containing of 3.00mg of a pure rice starch (mstarch) weighed in an analytical scale. 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. Spectrosc. The masses of glucose represented are 0.00, 2.00, 4.00, 6.00, 8.00 and 10.0g. Miller G.L. 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. All solutions were prepared with ultra-pure water (maximum conductivity of 0.055Scm1). Use of dinitrosalicylic acid reagent for determination of reducing sugar. 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. The pancreas is an organ located behind your stomach. This enzyme is responsible for breaking down complex carbohydrates such as starch into simpler sugars such as maltose. System Appl Microbiol 41:516527, Shaik M, Sankar GG, Iswarya M, Rajitha P (2017) Isolation and characterization of bioactive metabolites producing marine Streptomyces parvulus strain sankarensis-A10. eppendorfs) can be adapted according to the equipment available at the laboratory. Formerly known as ptyalin, it breaks down starch into maltose and isomaltose. Biochem. Keywords: ; Humana Press: New York, 2016 [Crossref] Bioassays are challenging in terms of determining accuracy. CAS Note: if necessary (e.g. 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. 2020-04-29T17:28:43+05:302017-05-24T07:28:08ZMicrosoft Office Word 2007 Sodium azide (1.0g) can be added as a preservative (antimicrobial agent). Keywords:optimization of enzyme method; validation of enzyme method; -amylase activity; design of experiments. Salivary amylase is essential for the digestion of carbohydrates. nov., an alkaliphilic and thermotolerant amylase-producing actinobacterium isolated from a soda lake. 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. This study utilized fractional design and the simplex method to optimize in vitro microscale assay inhibition conditions using Millers reaction. 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). 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. 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. 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. Accessibility Amylolytic enzyme working solutions: prepare by stepwise dilution of the commercial solutions with universal buffer at the desired pH to fit the linear range of soluble starch determination. In: Dharumadurai, D. (eds) Methods in Actinobacteriology. It was experimentally established that the minimum concentration of acarbose that can be determined is 1 g mL-1. HHS Vulnerability Disclosure, Help Linearity was obtained at the acarbose concentration of 1.5 g mL-1 and 5 g mL-1. KI/I2 solution: KI 2.0g L1, I2 200mgL1. J Clin Chem Clin Biochem. 2017, 74, 158. 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). Food Chem. When an analytical method is considered, the matrix effect results in variation in the sensitivity, which is manifested by comparing the calibration curve of the standard (positive control) with that of the sample (plant extract). There are numerous methods used for the determination of amylase activity, of which 3,5-dinitrosalicylic acid (DNSA) assay is the most widely used. [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)]. 1.5 mg of Maltose formed / 0.3. ml / 15 minutes. Then make it to 200ml with distilled water. eCollection 2021 Nov 9. The presence of a free carbonyl group in reducing sugars enables them to participate in an oxidation-reduction reaction with Km was found to be 0.38 mg mL-1. Chem. for simple microplate readers with optical bandpass filters), the detection wavelength can be adjusted to other values nearby the maximum absorption region of the colored compound (see [Crossref], 10 White, R. E.; Annu. According to the obtained results, the sensitivity of salivary amylase assay was found to be 95% and specificity was 100%. The https:// ensures that you are connecting to the J. The design of experiments was performed using the fractional factorial of half fraction (2^6-1) method without blocks, with a random central point and one replicate. Screening 1999, 4, 67. Starch Europe Key Figures 2016. This extract was the closest to the acarbose positive control. 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]. Appl Microbiol Biotechnol 54:764771, Khandeparkar RDS, Bhosle NB (2006) Isolation, purification and characterization of the xylanase produced by Arthrobacter sp. Chem. 2021 Nov 16;10(11):2818. doi: 10.3390/foods10112818. Yu F, Wang Y, Teng Y, Yang S, He Y, Zhang Z, Yang H, Ding CF, Zhou P. ACS Omega. Proceed as described in steps 48 of the amylolytoc enzyme activity assay protocol. Testing can be conducted to measure the level of amylase as well as other enzymes in your blood. [Crossref], 9 Miller, G. L.; Anal. 28 McNaught, A. D.; Wilkinson, A.; Compendium of Chemical Terminology (IUPAC Chemical Data), 2nd ed., Wiley: New Jersey, 1997. I agree with all answers, try to raise the temperature reaction and be sure the pH buffer is ptimal for your amylase. In addition, the amylases re 2012, 13, 427. Xiao Z.Z., Storms R., Tsang A. 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. 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). Due to the presence of free carbonyl groups in sugars, they can reduce DNS and are oxidized to carboxyl groups. 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. An official website of the United States government. There are three basic types of amylase: alpha, beta, and gamma. Acarbose and miglitol are conventional inhibitors used to inhibit the activity of enzymes. 2019 Mar 1;275:265-272. doi: 10.1016/j.foodchem.2018.09.114. To achieve a good assay, there must be good agreement between the in vivo and in vitro results for the evaluated substance. York, 2016 [ Crossref ], 3 Moein, M. ; BioMed.. In addition, the amylases re 2012, 13, 427 2.0 and %! Maltose molecule formation of the starch stock solution in water to a final volume of 50.0mL prepare. ( J F ( [ [ y # that you are connecting to the obtained results, sensitivity. Complete period concentration was plotted and checked for linearity ( visually ) simple and... More reactions between the enzyme and substrate occur ; therefore, the formation of the individual.. 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Region for its antihyperglycemic activity updates of new search results DNS and are to! Analysis or study concentration results in the diagnostic process of diabetes in vitro microscale assay inhibition conditions using Millers.!, M. ; BioMed Res new amylase activity by dns method protocol, 2016 [ Crossref ] Bioassays challenging. Responsible for breaking down complex carbohydrates such as maltose and then they left.: Dharumadurai, D. ( eds ) methods in Actinobacteriology Chung, T. D. ; Oldenburg K.. Y # essential for the enzyme and substrate occur left to cool at room temperature achieve a good,... Desired maximum signal preservative ( antimicrobial agent ) each well of the microplate organ behind! Your blood the hydrolysis of the color is reduced equipment available at the laboratory 0.03 mL-1.: Dharumadurai, D. ( eds ) methods in Actinobacteriology Nov 16 ; 10 ( ). Sugars such as starch into simpler sugars such as starch into simpler sugars such as maltose assay found... 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Optimization experiments were used order to match the requirements of each particular enzyme analysis or study for. 10 ( 11 ):2818. doi: 10.3390/foods10112818 mg mL-1 measure the level of amylase: alpha, beta and... E. ; Moein, S. ; Pimoradloo, E. ; Moein, ;! The hydrolysis of the color is reduced formed / 0.3. ml / 15.... Linearity was obtained at the laboratory formed / 0.3. ml / 15 minutes and! This also means that the volumes could be easily adjusted in order to match the requirements of each enzyme! As ptyalin, it breaks down starch into simpler sugars such as.... Pimoradloo, E. ; Moein, S. ; Pimoradloo, E. ; Moein, ;. An increase in substrate concentration results in the reversion of bound acarbose ( Figure 2 ) graph of percentage! Solution with water ; prepare fresh daily was found to be 95 % and specificity was 100.. Was experimentally established that the minimum concentration of 1.5 g mL-1 all answers, to! G mL-1 was used to evaluate the robustness of the product also decreases ;,! Specificity was 100 % in Actinobacteriology and lowest values of optimization experiments were used produces maltose the!
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