The robustness of the method was analyzed by estimating the response obtained by deliberately changing the conditions. [Crossref], 4 Rahimzadeh, M.; Jahanshahi, S.; Moein, S.; Moein, M. R.; Iran. centrifugation). Please enable it to take advantage of the complete set of features! Hence, the comparison of the two assays becomes difficult. In addition, the effect of substrate concentration on enzyme activity was analyzed. Chem. The final aspect of the microplate, absorbance spectra, and illustrative calibration curve of different glucose standards (from left to right, respectively) of the glucose oxidase/peroxidase assay used for the determination of the glucose yield after hydrolysis of pure starches catalyzed by amyloglucosidase. [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 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. Your email address will not be published. One negative control (absence of inhibitor) was set up to obtain 100% enzyme activity. 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. Amylase activity from partially purified samples was assayed by DNS method. 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. (function(d){var s=d.createElement("script");s.setAttribute("data-account","s8XDZXWqQY");s.setAttribute("src","https://cdn.userway.org/widget.js");(d.body||d.head).appendChild(s);})(document)Please ensure Javascript is enabled for purposes of website accessibility. Boric acid (PN: A79-212, 99.5%) and NaOH (PN: S/4920/60) were purchased from Thermo-Fisher Scientific (Waltham, MA, USA). Method. Acta Part A-Mol. endstream [1] Amylase was first described in the early 1800s and is considered one of the first enzymes in history to be scientifically investigated. Res. 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. It is an important enzyme and has wide range of functions like decomposing starch molecules in the human body. Amylase is a digestive enzyme predominantly secreted by the pancreas and salivary glands and found in other tissues at very small levels. The diagnosis of saliva amylase enzyme activity deficiency requires an oral test with a standard protocol. Universidade Federal do Recncavo da Bahia. eCollection 2021 Nov 9. Cultural and morphological characteristics of the selected bacterial were studied. KI/I2 solution: KI 2.0g L1, I2 200mgL1. Amylase is naturally produced in humans by the salivary glands and pancreas. Before 15 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. EMBRAPA Agroindstria Tropical, Fortaleza, Brazil. 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. [. You can decide for yourself whether you want to allow cookies or not. Chem. Biotechnol. In this experiment, acarbose at a concentration of 5 g mL-1 was used to record the desired minimum signal. 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. Mina Karimi-Avargani kindly mail me saidurga48@gmail.com. I need your help for calculation of data. In that case, the sensitivity of the determination will be modified. Linearity was determined using the graph of concentration and percent inhibition. Sci. Your email address will not be published. [Link], 5 Valentina, P.; Ilango, K.; Chander, S.; Murugesan, S.; Bioorg. A catalyst is a substance that hastens a chemical reaction but does not become part of the end product. Standard solutions of maltose (0-10 moles/l) are prepared in test tubes. official website and that any information you provide is encrypted 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. [Crossref]. Magnetic stirrer/heater plate (MAG-H, Gerhardt, Knigswinter, Germany), for preparing the soluble starch solution. J. Unable to load your collection due to an error, Unable to load your delegates due to an error. Amylase is the hydrolytic enzyme that breaks down many polysaccharides like Starch, Amylose, dextrins, and yields a disaccharide i.e., Maltose. There are three basic types of amylase: alpha, beta, and gamma. Influence of pH in the activity of an amyloglucosidase from Aspergillus niger (PN: A9913, Sigma Aldrich) measured by the described starch-iodine assay. (Family: Asteraceae) has been used as a natural remedy in the Arabian region for its antihyperglycemic activity. 13 Macarrn, R.; Hertzberg. Determination of kinetic constants was performed by determining the effect of substrate concentration on enzymatic activity. Additionally, the optimized assay was applied to extracts from five different plants. Sodium azide (1.0g) can be added as a preservative (antimicrobial agent). The test is done in microtitre plates with a total volume of 260L and an assay time of 40min including the pre-incubation steps. Effect of type 2 diabetes mellitus caveolin-3 K15N mutation on glycometabolism. The optimized and validated method was accurate, precise, and linear. Glucose oxidase / peroxidase (GOPOD) (K-GLUC) assay kit was purchased from Megazyme (Wicklow, Ireland). After 10min, the hydrolysis was stopped with the addition of 750L of 1molL1 HCl. Universidade Federal da Bahia. [Crossref]. and a-amylase activity. 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. 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. [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. 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. Briefly centrifuge vials before opening. 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. 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. It is made in the pancreas and the glands that make saliva. 96-well flat-bottom microplates (well volume 340L, Thermo Fisher Scientific). Accessibility Epub 2019 Nov 2. This section illustrates an example of the application of the method that includes the corresponding calculations for both methods herein described. Plate uniformity assessment is mandatory for all assays. Epub 2019 Aug 2. 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. [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). These findings demonstrated that our optimized and validated method is highly selective, enabling the identification of samples with or without potent activity. 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 work presents a protocol for the production of amylases by actinobacteria; amylases are important enzymes for different industrial sectors because they are able to perform in the hydrolysis of starch and/or starchy substrates. 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. [Crossref]. Place the vial in the block heater (or other alternative heating source) previously set at the target temperature of the study during the desired time of the assay (, Immediately stop the hydrolysis process by adding 750L of 1.0molL, Prepare the soluble starch standards as described in, Transfer 50L of the mixture (sample or standard), 150L of water, and 50L KI/I, Place the microplate in the plate reader, shake the plate (for 30s using the shaker function of the microplate reader) and read the absorbance at 580nm, at room temperature. Chem. Metabolic disorders, including hyperglycemia, characterize type-2 diabetes. Selected plant products could provide better alternatives due to their low cost and safer use. Experiments conducted to evaluate plate uniformity and signal variability indicated that the response is not influenced by its location on the plate. 5 ml of deionized water are added in each sample, followed by agitation. 29 Zhang, J. H.; Chung, T. D.; Oldenburg, K. R.; J. Biomol. Some of them are essential for the operation of the site, while others help us to improve this site and the user experience (tracking cookies). Synergy HT reader (Bio-Tek Instruments, Winooski, VT, USA) controlled by Gen 5 software (Bio-Tek Instruments), for measuring the absorbance of the wet-chemistry assays under microplate format. Med 2011, Article ID 810207. [Crossref],77 Yilmazer-Musa, M.; Griffith, A. M.; Michels, A. J.; Schneider, E.; Frei, B.; J. Agric. Amylase is an enzyme that helps digest carbohydrates. From these samples, salivary amylose enzyme assays were carried out. FOIA [5] for measuring the activity of alpha-amylase and amyloglucosidase using the starch-iodine assay principles. Methods in Actinobacteriology pp 495503Cite as, Part of the Springer Protocols Handbooks book series (SPH). 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. Epub 2018 Sep 19. Cool and dilute with 10ml of distilled water. 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. Amylase was first described in the early 1800s and is considered one of the first enzymes in history to be scientifically investigated. endobj 6, where we studied the influence of pH on the activity of amyloglucosidase from A. niger. For enzyme inhibition studies, the absorbance was recorded at a higher or maximum concentration of inhibitor (IC70). 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. ; Eli Lilly & Company and the National Center for Advancing Translational Sciences: Bethesda, 2004. phenol). Tailorable assay based on the starch-iodine staining for the determination of alpha-amylase and amyloglucosidase activities. 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. 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 Sci. This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. Salivary amylase is essential for the digestion of carbohydrates. It was observed that the selected plants showed exhibited variable inhibitory effects on -amylase activity in vitro. 4. The proposed method relies on principles that allow a fast, simple, and versatile measurement of the activity of alpha-amylase or amyloglucosidase. Keywords: endobj [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. By applying Eq. PN: 27217, Sigma-Aldrich). The Millers reactions were performed in 13 100 test tubes. The error percentage was calculated to be 5.46%, which indicates that the method is accurate. This experiment also counteracts problems associated with drift patterns, edge effects, and other systematic sources of variability. 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. Glucose yield (average values from two determinations) measured by the described glucose oxidase/peroxidase assay that resulted from the hydrolysis of four different pure starches mediated by an amyloglucosidase from Aspergillus niger (PN: A9913, Sigma Aldrich). Toxicol. 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. 5. The authors also acknowledge to Slvia Azevedo for the valuable technical assistance. (1) are: mcontrol=2.94mg, massay=0.133mg, time=10min, volume of enzyme used in the assay=0.500mL, dilution factor=2000. Med 2011, Article ID 810207. Mntele W., Deniz E. UVvis absorption spectroscopy: Lambert-Beer reloaded. The reaction was stopped by adding 200 L of DNS (final concentration 19.2 mmol L-1) and boiling in a water bath for 15.6 min. 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. 50mL of concentrated solution for a final volume of 1000mL) in order to be used in the extraction protocol. Factors significantly contributing to the response were selected by the Pareto graph. Transfer 50L of sample supernatant (or standard), and add 150L of GOPOD reagent to each well of the microplate. 8600 Rockville Pike Multi-Mechanistic In Vitro Evaluation of Antihyperglycemic, Antioxidant and Antiglycation Activities of Three Phenolic-Rich Indian Red Rice Genotypes and In Silico Evaluation of Their Phenolic Metabolites. Learn more 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. 2014, 17, 465. Note: if necessary (e.g. 2011, 44, 2076. Prepare enzyme extract filtered with syringe filter 3. Due to the presence of free carbonyl groups in sugars, they can reduce DNS and are oxidized to carboxyl groups. This extract was the closest to the acarbose positive control. The -amylase assay was performed using Millers method, i.e., the DNS method. 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]. Formerly known as ptyalin, it breaks down starch into maltose and isomaltose. Five plant extracts were used to determine -amylase inhibitory activity using the proposed method. Acarbose and miglitol are conventional inhibitors used to inhibit the activity of enzymes. Pol. These bonds can be easily broken by water, so saliva is necessary for the enzyme to work. R. P.; Mol. reagent is added in each tube and the mixture is agitated for a few seconds on vortex mixer. <>>> Yu F, Wang Y, Teng Y, Yang S, He Y, Zhang Z, Yang H, Ding CF, Zhou P. ACS Omega. Sodium hydroxide was procured from CTR, Mxico. 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. Absorbance was recorded at 540 nm using a microplate reader. [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. 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. To evaluate linearity, amylase inhibition studies were performed using different concentrations of the inhibitor acarbose (0.5 to 12 g mL-1). The monitoring of the activity of alpha-amylase and amyloglucosidase is an important tool for studying their role in the hydrolysis of starch. Required fields are marked *. D-Glucose (PN: 8337) and KI (PN: 105043) were purchased from Merck (Darmstadt, Germany). For the first time, a reliable, simple, rapid and high-throughput analytical method for the detection and quantification of -amylase inhibitory activity using the glucose assay kit was developed. Biochem. Save my name, email, and website in this browser for the next time I comment. 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). 8) [8]. The absorbance was measured using 96-well polystyrene plates with a flat bottom and a final volume of 200 L. A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities. Two plant extracts (Zanthoxylum fagara and Chrysactinia mexicana) inhibited -amylase activity. Screening 1999, 4, 67. 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). 2011, 76, H188. These plant extracts were offered by researchers from the Analytical Chemistry Department (Facultad de Medicina, UANL, Mxico). Based upon the plasma glucose value, all subjects were divided into three groups. It can also be naturally found in plants and animals. ASSESSMENT OF -AMYLASE INHIBITION ACTIVITY BY AN OPTIMIZED AND VALIDATED IN VITRO MICROSCALE METHOD. Med 2011, Article ID 810207. Most of the amylase in your body is made by your pancreas and salivary glands. 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. Microb Pathog 107:225233, Ottoni JR, e Silva TR, de Oliveira VM, Passarini MRZ (2020) Characterization of amylase produced by cold-adapted bacteria from Antarctic samples. Fig. The reduced 3-amino-5-nitrosalicylic acid is red-brown in color and can be quantified spectrophotometrically with an absorption maximum at 540 nm. [Crossref], 19 Satpathy, G.; Tyagi, Y. K.; Gupta, R. K.; Food Res. 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. endobj and transmitted securely. Extract from Zanthoxylum fagara showed maximum inhibitory activity with an IC50 value of 4.75 g mL-1. This article will discuss the various applications of amylase. It is an important enzyme and has wide range of functions like decomposing starch molecules in the human body. So the final concentration is 0.1M of 200ml Phosphate buffer. 26 Tundis, R.; Loizzo, M. R.; Statti, G. A.; Menichini, F.; Pharmazie 2007, 62, 473. Amylase is an enzyme responsible for breaking down carbohydrates, it is found in saliva and pancreatic juice, and it helps the body digest food. The multi-targets integrated fingerprinting for screening anti-diabetic compounds from a Chinese medicine Jinqi Jiangtang Tablet. The degree of inhibition was evaluated in terms of the concentration of the inhibitor that inhibited half of the enzyme activity (IC50). Basic Med. The graph of inhibition percentage vs. inhibition concentration was plotted and checked for linearity (visually). Most enzymes act specifically with only one reactant, called a substrate, to produce products. endobj Copyright 2016 Elsevier Ltd. All rights reserved. 2010, 75, H97. Amylase is an enzyme, or special protein, that helps you digest carbohydrates. government site. Amylasedigestsstarchbycatalyzinghydrolysis,whichissplittingbytheadditionofawatermolecule. The masses of glucose represented are 0.00, 2.00, 4.00, 6.00, 8.00 and 10.0g. Therefore, the glucose yield corresponded to the ratio between mglucose (483.4g) and the total mass of starch mstarch (2400g), which resulted in a value of 20.14%. Oliveira H.M., Pinheiro A.Q., Fonseca A.J.M., Cabrita A.R.J., Maia M.R.G. J Ferment Technol 54:267286, CAS Here we adopted the same detection wavelength of the original method (580nm) [5], which is associated with an amylopectin/amylose ratio of 80:20 [12]. Then make it to 200ml with distilled water. 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 is also crucial to accurately use well-defined conditions for laboratory transfer. 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 proposed method showed high precision in long-term use (RSD<6.3%). According to the obtained results, the sensitivity of salivary amylase assay was found to be 95% and specificity was 100%. Careers, REQUIMTE, LAQV, ICBAS, Instituto de Cincias Biomdicas de Abel Salazar, Universidade do Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal. The present method describes an improved experimental protocol that can be used to monitor the activity of two amylolytic enzymes: alpha-amylase and amyloglucosidase. This concentrated buffer should be diluted 200 times (e.g. (1) the calculated activity for this enzyme was 1123UmL1. results of inhibition of alpha amylase enzyme by the selected plant extracts have been represented in Figure 1. 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. Unfortunately, to date, many published studies cannot be reproduced; most of them are nonreplicable or simply erroneous. Importance of Assay of Salivary amylase enzyme, Estimation of Carbohydrates by the Anthrone Method, Assay of Acid Phosphatase enzyme activity from Potatoes, Assay of Urease Enzyme Activity (Enzymology Practical Protocol), Effect of Temperature on Amylase activity (Enzymology Protocol). By clicking accept or continuing to use the site, you agree to the terms outlined in our. Salivary amylase is produced by the salivary glands and released into the mouth when we eat. It has long been known that salivary gland dysfunction leads to glucose intolerance and ultimately to type 2 diabetes mellitus. Oliveira H.M., Correia V.S., Segundo M.A., Fonseca A.J.M., Cabrita A.R.J. 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). Describe what you expect to observe for the heated saliva amylase assay. The protocol applies to two new strains of actinobacteria, isolated in Northeastern Brazil, using residues from the processing of a local fruit of a palm tree (licuri) as a cultivation substrate for solid state fermentation (SSF). ),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. application/pdfScreening on the Amylase Producing Activity of some Bacterial Strains Isolated from Hot SpringInternational Journal of Science and Research (IJSR) In the case of amyloglucosidase, it is also possible to measure glucose yield using a protocol similar to the one applied to the enzyme activity. 2012, 37, 520. 2005 Mar;10(3):130-7. doi: 10.1016/j.tplants.2005.01.001. Max signal: The maximum absorbance was recorded by the assay procedure. 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.). 1959, 31, 426. This enzyme is responsible for breaking down complex carbohydrates such as starch into simpler sugars such as maltose.

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