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a sucrose standard curve was constructed by plotting absorbance at 540nm against the concentration (mg/mL). specifically the hydrolysis of sucrose into fructose and glucose. Enzymes are responsible for the catalysis of biomolecules. Concentrated HCl. The amount of absorbance at 540nm was measured using a spectrophotometer. Pipettes. 50°C. This is also effectively used in the handling of requirements for clinics in hospital laboratories. After observation and analysis. and the amount of acidhydrolyzed sucrose was calculated. invertase exhibits high activity over a broad temperature range of 50°C-70°C with optimum temperature at about 60°C. 0. the effects of temperature were carefully observed and analyzed to evaluate its effects on invertase activity.5dinitrosalicylic acid) is used as a reagent to determine sugar content especially glucose. Compounds tested (or Samples used) Baker’s yeast. A peak was observed on the graphs which indicated its optimum temperature. Optimum temperature is the temperature at which the enzyme is most active. taking into account that only a short period of time is required for the process to take place and the reagents are unreactive. Dinitrosalicylic colorimetric method is a test in which dinitrosalicylic acid (3. Celina Marie Wong Group 10 2F Medical Technology Biochemistry Laboratory ABSTRACT In this experiment. The DNS technique is employed in order to estimate sugar present in the samples. Sucrose standard solution (100mg/L).26) was isolated from Baker’s yeast which is classified as a hydrolase. John Henrick Uy. thus making it easier to reach product formation. Invertase was isolated from Baker’s yeast yielding an enzyme stock solution which was used for the procedures. A temperature/pH too high or low can lead to loss of enzyme function (denaturation). acidhydrolyzed sucrose curve. hydrolases for hydrolysis reactions. 70°C and 90°C). The complex process known as catalysis is the key which makes metabolism possible.  Through the proper execution of each method. . Ideally. Enzymes function as biological catalyst which increases the rate of chemical reaction without being altered in its chemical composition. EXPERIMENTAL A. Beakers. It performs its function by lowering the activation energy of a particular substrate. Spectrophotometry was also performed to get the absorbance at 540nm which was compared to the sucrose standard curve. invertases are enzymes that catalyze the hydrolysis of peptide bonds.5M KOH.1. isomerases for isomerization reactions and ligases (synthetases) for formation of bonds with ATP cleavage. Carl Jerome Umali. Invertase was then subjected to varying temperatures (20°C. Chrystel Marlowe Turla. Enzymes also act selectively by reacting only to one substrate. Test tubes. Dinitrosalicylic (DNS) colorimetric method was utilized to measure the activity of invertase. Dinitrosalicylic acid (DNS) reagent.DETERMINATION ON THE EFFECT OF TEMPERATURE ON INVERTASE ACTIVITY USING SPECTROPHOTOMETRY Kathleen Adryon Tan. Typically. Tertiary and quatenary structure are capable of forming pockets (active sites) which are responsible for holding specific substrates. Vienne Czarina Tongco. 60°C.  Invertase with a systematic name of βfructofuranosidase (EC 3.  Enzymes are classified according to their reaction mechanism namely: Oxidases (dehydrogenases) for reduction-oxidation reactions.  Enzyme activity is affected by two factors namely: pH and temperature which are related to the tertiary structure. Metabolism is defined as the physical and chemical processes that occur in a biological system that are necessary in the maintenance of the vital function required for the conservation of life. lysases for addition of double bonds or vice versa. Sucrose solution (10g/mL). 30°C.  INTRODUCTION All living things have the common characteristic of being able to undergo the process of metabolism.2. A separate graph was constructed by plotting temperature against amount of acidhydrolyzed sucrose (mg/ml). low cost and readily available. objectives which included extracting invertase from Baker’s yeast and determining the effects of changes in temperature on reaction rates of an enzyme-catalyzed reaction were precisely accomplished. An instrument known as a spectrophotometer was used to measure the absorbance of the invertase stock solution to be able to be plotted against the values of the amount of acidhydrolyzed sucrose in order to construct a sucrose standard curve which became a basis for the construction of the temperature vs. transferases for transfer of functional groups.
Solution was allowed to cool. but addition of a catalyst.50. components are separated from each other. a large amount of the sample was required. 6 test tubes were then prepared containing 1.00. 3mL of the diluted enzyme solution were added to all the tubes leaving each immersed in its respective water baths for another 5 minutes.5M KOH.1M buffer solution.019 4 0.50:0). Effect of Temperature on Invertase Activity Several water baths were laid out with each having varying temperatures (20°C. 1. A red-brown color indicated a positive result. 60°C. The solution was allowed to cool making frothing possible.15mL 0. Dinitrosalicylic acid was used to detect reducing sugars (free carbonyl group) and other reducing molecules to form 3-amino-5-nitrosalicylic acid under alkaline conditions which strongly absorbed light at 540nm. 30°C. Absorbance at 540nm were observed and recorded. A red-brown color characteristic was developed after 3mL of DNS reagent was added to the test tubes and submerged in 95°C water bath for 10 minutes. 70°C and 90°C).75.02 0. Sucrose Assay Using Dinitrosalicylic Colorimetric Method Series of test tubes were prepared with fixed ration of mL sucrose standard solution to mL distilled water (0:1.75:0. A hydrolyzedsucrose standard curve was constructed by plotting the absorbance at 540nm against concentration (mg/mL).001 2 0. 1.50.067 6 0.069 Table 1 Amount of hydrolyzed sucrose and Absrobance540nm 4. The reaction occurs at a slower rate. made the reaction occur at a rapid rate.00:0. All the test tubes were covered with marble/cotton during the water bath process to prevent the evaporation of solvent. pH 5 was added to have a diluted enzyme solution. 1.5mL sucrose solution and incubated separately for 5 minutes in each water bath.003 3 0. 0.0067 -0.50:1. Figure 1 Hydrolysis of sucrose Figure 1 shows that sucrose is formed by glucose and fructose connected by a glycosidic bond. Upon subjecting to hydrolysis.020 5 0. UV-Vis spectrophotometer.25. Test Tube Hydrolyzed sucrose (mg/mL) A540nm Blank 0 0.017 0. Procedure 1.0033 -0.Volumetric flasks.25:0.25:1. .01 0.20mL 0. 0. The resulting solution was immersed in 95°C water bath for 10 minutes to develop the red-brown color characteristic.000 1 0. 3 drops of concentrated HCl were added to all test tubes allowing it to be mixed and incubated at 90°C water bath for 5 minutes. it was subjected to the spectrophotometer for the determination of its absorbance at 540nm. Amount of acid-hydrolyzed sucrose was computed using sucrose standard curve constructed in the dinitrosalicylic colorimetric method. It was followed by the addition of 3mL DNS reagent. Hot plate. In a separate test tube. 2.013 0. After the solution has cooled. 50°C. Extraction of Invertase from Yeast Invertase was extracted by dissolving the 0. Only the supernatant was collected that served as the denatured enzyme stock solution. RESULTS AND DISCUSSION 3. Paraffin film.25g Baker’s yeast in distilled water to make a 250mL solution. The solution was allowed to stand for 20 minutes at room temperature. invertase. The solution was neutralized by adding 0. B. and then the supernatant was separated from the sediments that settled down that served as the enzyme stock solution. 0. Blank solutions were also prepared by the addition of denatured enzyme instead of enzyme stock solution. a second graph was constructed with a plot against temperature and acid-hydrolyzed sucrose (mg/mL). .80mL enzyme stock solution with 19. In order for a result to be detectable.25. After the determination of sucrose hydrolyzed. Preparation of Denatured Invertase Stock Solution 100mL of the enzyme stock solution was incubated in a boiling water bath for 10 minutes.
012 Amount of acid-hydrolyzed sucrose= slope x A540 Amount of hydrolyzed sucrose (mg/mL) of test tube 2: Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 1 (20°C): Amount of hydrolyzed sucrose (mg/mL) of test tube 3: Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 2 (30°C): Amount of hydrolyzed sucrose (mg/mL) of test tube 4: Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 3 (50°C): Amount of hydrolyzed sucrose (mg/mL) of test tube 5: Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 4 (60°C): .08 Absorbance.746 In figure 2.015 6 90 -0. the straight black line drawn was the best representation for the points plotted computed by getting linear regression of the graph using a scientific calculator.01 0.02 0.021 5 70 0.8986x R² = 0. 540 nm 0.06 0.0056 4 60 0. V1 being volume of the sucrose standard solution used varying for each test tube.019 0.746 2.052 0.0077 2 30 0. Computations: Amount of hydrolyzed sucrose (mg/mL) of blank test tube: y = 2.036 -0.0052 3 50 0.02 Test Tube Amount of hydrolyzed sucrose (mg/mL) of test tube 1: Acid-hydrolyzed sucrose(mg/mL) 1 20 0. Computations: Slope R2 y 0.403 0. but the graph didn’t give a linear-like appearance due to some errors committed by the students in performing spectrophotometry.005 0.013 0.1mg/mL.015 0.0048 Table 2 Amount of acid-hydrolyzed sucrose and Absorbance540nm based on temperature Computations: Temperature(°C) A540nm 0.04 0.025 Concentration mg/mL Figure 2 Amount of hydrolyzed sucrose and Absrobance 540nm curve Amount of hydrolyzed sucrose was computed using the formula C1V1=C2V2 with C1 being the concentration of the sucrose solution which was 0. C2 being the unknown and V2 being the total volume of the solution which was 7.5mL.02 0 0 0.8986x Amount of hydrolyzed sucrose (mg/mL) of test tube 6: -0.Hydrolyzed-Sucrose Standard Curve 0.014 0.
K.005 0 -0.. L. http://www. 8th ed.A. S. M.005 -0. to the equation derived (refer to table 2). Biochemistry.. 4 Theoretical effect of temperature on enzyme activity Referring to figure 3. Inc. From internet sources  http://www.L.(2012). Crouch. y. F.C.M. (2010). M. (2012). Liu. Quezon City: C & E Publishing.I..invertase. Singapore: Cengage Learning Asia Pte Ltd. the plot should have a bell-shaped graph showing increased enzyme activity with the increase of temperature up until 60°C wherein the enzyme activity started to decrease drastically and eventually became almost inactive which is a clear sign of denaturation (refer to figure 4). Chen. …Ysrael. R.html (accessed December 30).02 Concentration. REFERENCES From books  Campbell. West. a peak was observed at 60°C that indicated the optimum temperature for . de Guia. Daya..C. x. F. Laboratory manual in general biochemistry. 7th ed. D. Introduction to Analytical Chemistry. Philippines Cengage Learning Asia Pte Ltd. Farrow.Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 5 (70°C): Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 6 (90°C): The calculated linear regression equation above was used in order to come up with the value of acid-hydrolyzed sucrose.  Crisostomo.  Skoog. Ma.R.. and Farrell.015 0. A. S.O. Holler. jh Temperature. Effect of Temperature on Invertase Activity 0.htm (Accessed December 31).J. M.com/how_5221277_usedinitrosalicylic-acid. D. pg139-145. The curve constructed didn’t show the predicted graph based on theory because of some significant errors that included and not limited to: degrees of uncertainty caused by the students due to inexperience usage of the spectrophotometer.01 0 20 40 60 80 100 invertase.01 0. M.G. by substituting absorbance540nm to the variable.net/single.C. mg/mL 0.D. Gabona.....ehow. S. M. instrumental errors caused by the spectrophotometer due to the poor maintenance of the device and unmonitored temperature of the water bath during the experiment which led to higher/lower than what was specified on the instruction given having a big impact on the enzyme activity.. Theoretically. °C Figure 3 Temperature and amount of acid-hydrolyzed sucrose curve Fig.