This action might not be possible to undo. Are you sure you want to continue?
a sucrose standard curve was constructed by plotting absorbance at 540nm against the concentration (mg/mL). low cost and readily available. 70°C and 90°C). lysases for addition of double bonds or vice versa. Sucrose solution (10g/mL).26) was isolated from Baker’s yeast which is classified as a hydrolase. The complex process known as catalysis is the key which makes metabolism possible. Carl Jerome Umali. invertases are enzymes that catalyze the hydrolysis of peptide bonds.  Enzymes are classified according to their reaction mechanism namely: Oxidases (dehydrogenases) for reduction-oxidation reactions. Chrystel Marlowe Turla. A peak was observed on the graphs which indicated its optimum temperature. isomerases for isomerization reactions and ligases (synthetases) for formation of bonds with ATP cleavage. It performs its function by lowering the activation energy of a particular substrate. specifically the hydrolysis of sucrose into fructose and glucose.  INTRODUCTION All living things have the common characteristic of being able to undergo the process of metabolism. Invertase was isolated from Baker’s yeast yielding an enzyme stock solution which was used for the procedures. acidhydrolyzed sucrose curve. Tertiary and quatenary structure are capable of forming pockets (active sites) which are responsible for holding specific substrates.5M KOH. Enzymes are responsible for the catalysis of biomolecules. Beakers.5dinitrosalicylic acid) is used as a reagent to determine sugar content especially glucose. thus making it easier to reach product formation. the effects of temperature were carefully observed and analyzed to evaluate its effects on invertase activity.  Enzyme activity is affected by two factors namely: pH and temperature which are related to the tertiary structure.1. Vienne Czarina Tongco. Dinitrosalicylic colorimetric method is a test in which dinitrosalicylic acid (3. 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. Pipettes. Dinitrosalicylic acid (DNS) reagent. Dinitrosalicylic (DNS) colorimetric method was utilized to measure the activity of invertase. Typically. taking into account that only a short period of time is required for the process to take place and the reagents are unreactive.  Through the proper execution of each method. EXPERIMENTAL A. Concentrated HCl. Ideally. . Optimum temperature is the temperature at which the enzyme is most active. A temperature/pH too high or low can lead to loss of enzyme function (denaturation). Celina Marie Wong Group 10 2F Medical Technology Biochemistry Laboratory ABSTRACT In this experiment. Compounds tested (or Samples used) Baker’s yeast. 50°C. A separate graph was constructed by plotting temperature against amount of acidhydrolyzed sucrose (mg/ml). The DNS technique is employed in order to estimate sugar present in the samples. After observation and analysis. Spectrophotometry was also performed to get the absorbance at 540nm which was compared to the sucrose standard curve. John Henrick Uy. transferases for transfer of functional groups.DETERMINATION ON THE EFFECT OF TEMPERATURE ON INVERTASE ACTIVITY USING SPECTROPHOTOMETRY Kathleen Adryon Tan. Test tubes. 60°C. The amount of absorbance at 540nm was measured using a spectrophotometer. and the amount of acidhydrolyzed sucrose was calculated.2. 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. hydrolases for hydrolysis reactions. 30°C. Enzymes also act selectively by reacting only to one substrate. Sucrose standard solution (100mg/L). Enzymes function as biological catalyst which increases the rate of chemical reaction without being altered in its chemical composition. 0. Invertase was then subjected to varying temperatures (20°C.  Invertase with a systematic name of βfructofuranosidase (EC 3. invertase exhibits high activity over a broad temperature range of 50°C-70°C with optimum temperature at about 60°C. This is also effectively used in the handling of requirements for clinics in hospital laboratories.
0.50.00:0. Figure 1 Hydrolysis of sucrose Figure 1 shows that sucrose is formed by glucose and fructose connected by a glycosidic bond. The resulting solution was immersed in 95°C water bath for 10 minutes to develop the red-brown color characteristic. and then the supernatant was separated from the sediments that settled down that served as the enzyme stock solution.20mL 0. RESULTS AND DISCUSSION 3.50. pH 5 was added to have a diluted enzyme solution. a large amount of the sample was required. Blank solutions were also prepared by the addition of denatured enzyme instead of enzyme stock solution. 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. 1.01 0.1M buffer solution. B.5M KOH.80mL enzyme stock solution with 19. it was subjected to the spectrophotometer for the determination of its absorbance at 540nm. A red-brown color indicated a positive result. The solution was neutralized by adding 0. Paraffin film. UV-Vis spectrophotometer.017 0.25:1. In order for a result to be detectable.25g Baker’s yeast in distilled water to make a 250mL solution. Upon subjecting to hydrolysis. The solution was allowed to stand for 20 minutes at room temperature. invertase. 50°C. It was followed by the addition of 3mL DNS reagent. but addition of a catalyst. 0. Extraction of Invertase from Yeast Invertase was extracted by dissolving the 0. .001 2 0. After the determination of sucrose hydrolyzed. Effect of Temperature on Invertase Activity Several water baths were laid out with each having varying temperatures (20°C. All the test tubes were covered with marble/cotton during the water bath process to prevent the evaporation of solvent.25:0.75. 1. 3mL of the diluted enzyme solution were added to all the tubes leaving each immersed in its respective water baths for another 5 minutes.067 6 0.75:0. 1. The solution was allowed to cool making frothing possible.069 Table 1 Amount of hydrolyzed sucrose and Absrobance540nm 4. Procedure 1. After the solution has cooled. Absorbance at 540nm were observed and recorded.25. 30°C.Volumetric flasks.25.0067 -0.50:0).019 4 0.15mL 0.00. 60°C.003 3 0. 6 test tubes were then prepared containing 1.0033 -0. a second graph was constructed with a plot against temperature and acid-hydrolyzed sucrose (mg/mL). 2. In a separate test tube. The reaction occurs at a slower rate.013 0. Test Tube Hydrolyzed sucrose (mg/mL) A540nm Blank 0 0.020 5 0. components are separated from each other. 70°C and 90°C). Preparation of Denatured Invertase Stock Solution 100mL of the enzyme stock solution was incubated in a boiling water bath for 10 minutes. 0. Solution was allowed to cool. made the reaction occur at a rapid rate. Hot plate. Amount of acid-hydrolyzed sucrose was computed using sucrose standard curve constructed in the dinitrosalicylic colorimetric method.000 1 0. A hydrolyzedsucrose standard curve was constructed by plotting the absorbance at 540nm against concentration (mg/mL).5mL sucrose solution and incubated separately for 5 minutes in each water bath. Only the supernatant was collected that served as the denatured enzyme stock solution. 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.50:1. 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. 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. .02 0.
0077 2 30 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. V1 being volume of the sucrose standard solution used varying for each test tube.0056 4 60 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.1mg/mL. Computations: Slope R2 y 0.746 2.06 0. 540 nm 0.015 6 90 -0.036 -0.02 Test Tube Amount of hydrolyzed sucrose (mg/mL) of test tube 1: Acid-hydrolyzed sucrose(mg/mL) 1 20 0.8986x R² = 0.019 0.012 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): . but the graph didn’t give a linear-like appearance due to some errors committed by the students in performing spectrophotometry.5mL.04 0.02 0.403 0.0052 3 50 0.8986x Amount of hydrolyzed sucrose (mg/mL) of test tube 6: -0.08 Absorbance.0048 Table 2 Amount of acid-hydrolyzed sucrose and Absorbance540nm based on temperature Computations: Temperature(°C) A540nm 0.005 0.Hydrolyzed-Sucrose Standard Curve 0.021 5 70 0. C2 being the unknown and V2 being the total volume of the solution which was 7.015 0.013 0. Computations: Amount of hydrolyzed sucrose (mg/mL) of blank test tube: y = 2.052 0.746 In figure 2.014 0.01 0.02 0 0 0.
4 Theoretical effect of temperature on enzyme activity Referring to figure 3. to the equation derived (refer to table 2). a peak was observed at 60°C that indicated the optimum temperature for .. R.015 0. M.. M.html (accessed December 30). by substituting absorbance540nm to the variable.C. de Guia.D.K. L. 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. pg139-145.G. jh Temperature.. Philippines Cengage Learning Asia Pte Ltd. Gabona. M. Effect of Temperature on Invertase Activity 0. D.J.invertase. Quezon City: C & E Publishing. Introduction to Analytical Chemistry.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.02 Concentration. S. Ma.01 0.htm (Accessed December 31).. mg/mL 0. Theoretically. S. Inc.  Crisostomo. S.01 0 20 40 60 80 100 invertase...O. °C Figure 3 Temperature and amount of acid-hydrolyzed sucrose curve Fig. REFERENCES From books  Campbell. …Ysrael.C.005 -0. 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. and Farrell.com/how_5221277_usedinitrosalicylic-acid. 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). Liu.M.L. x. A.C. Singapore: Cengage Learning Asia Pte Ltd. 7th ed.. West. From internet sources  http://www.. Daya. M. y.net/single. F. Biochemistry...A. (2012). Farrow. M.(2012).005 0 -0. F. Chen. Crouch.  Skoog. (2010). Holler. http://www.I.R.ehow. Laboratory manual in general biochemistry. 8th ed. D.
This action might not be possible to undo. Are you sure you want to continue?
We've moved you to where you read on your other device.
Get the full title to continue listening from where you left off, or restart the preview.