Experiment on Factors Affecting Enzyme Amylase Activity - Lab Report Example

?Running head: Enzyme Amylase Activity Experiment on Factors Affecting Enzyme Amylase Activity In this experiment, the enzyme amylase was used. It was responsible for starch hydrolysis. A sample of starch was hydrolyzed in the presence of amylase, to shorter polysaccharides, glucose, maltose and dextrins. The extent of hydrolysis was based on the time of reaction. The product is glucose if starch was completely hydrolyzed. The absence or presence of starch in the solutions was tested using iodine. Iodine never reacts with glucose, instead, forms a blue to black complex with starch. Following addition of iodine to a solution of glucose, the only color observed was red or yellow (iodine color). Therefore, the faster the blue color is lost, the faster the enzyme amylase activity. The enzyme amylase can’t hydrolyze starch if it is inactivated, so the starch-iodine complex blue color will persist. The presence of glucose in the samples was tested using Benedict’s reagent. When blue solution of Benedict’s reagent is added to a glucose solution the color changes to green (at low concentrations of glucose) or reddish-orange (at higher concentrations of glucose). Starch can’t react with Benedict’s reagent, therefore the solution remains blue. The aim of the experiment was to look at how enzyme amylase activity is affected by pH, temperature and concentration of the enzyme. Introduction Enzymes are proteins in nature; they are catalysts for biological reactions. Like all catalysts, they speed up reactions by lowering the reaction’s activation energy without themselves being used up. Enzymes catalyze all biochemical reactions. They can be denatured in a variety of ways since they are proteins in nature, Therefore they work optimally under mild conditions. At body temperature and at a neutral pH, most of them have optimum activity. Enzymes also are known to be very specific; they act only on a specific substrate or one type of similar substrate molecules. This is because the enzyme active site is complementary to the polarity and shape of the substrate. Only one kind of substrate will “fit” into the active site. (Abu et al, 2005) Methods and Materials Preparation: Water baths of constant, low and high temperature were used in the experiment. A large water bath was set to 37°C.For low temperature bath, a 250-mL beaker, was half filled with tap water, and some ice was added to the water to attain between 0 and 5°C temperature. High temperature water bath was made by filling a 250-mL beaker to two-thirds full then heated to boil. The temperature of the bath was close to 100°C. 1% starch solution was used for every experiment part. Iodine reagent for each experiment was also used. Commercial amylase solution, clean droppers and a white spot plate were also needed in the experiment. For starch testing, few drops of starch were transferred to one well of the spot plate. One iodine reagent drop was added. Iodine and Starch was reacted to form a deep blue-black complex. For glucose testing, 1% glucose solution of 3 ml was added in a test tube. 2 ml of Benedict’s solution was added and heated in a boiling water bath for 3-4 minutes. The reaction formed a red-orange solid. Experiment 1: Effect of Enzyme Concentration Five test tubes were labeled as 1-5. 4 ml of 1 % starch was placed in every first four test tubes. 4 ml of amylase solution was placed in the fifth tube. All of the tubes were placed in water bath (37°C) for 5 minutes. 5 clean droppers were obtained then labeled from 1 to 5.Separate dropper were used for each mixture to avoid contamination. Tube 1 was the control and it never had any enzyme. The tubes were momentarily removed from the water bath and quickly 3 drops of the warmed amylase solution was added to tube 2, to tube 3, 6 drops of amylase was added and in tube 4, 10 drops of amylase was added. The tubes were mixed quickly by shaking gently then later put immediately back into 37°C water bath. The time at which the enzyme was added was recorded. Immediately, four drops of every reaction mixture (tubes 1-4) were transferred to separate wells on a spot plate with a separate clean dropper for each (The test tubes were kept in water bath). One drop of iodine reagent was added to each. Then the observations were recorded. Visual color reference table was used to assess the activity of the enzyme. After recording the observations, the spot plate was rinsed. Step 3 was repeated after 5 minutes of reaction time. Step 3 was repeated after ten minutes of reaction time. Glucose presence was confirmed with Benedict’s reagent in the test tubes that had hydrolysis. Benedict’s reagent was added to every test tube and then placed in boiling water bath for 4 minutes. Formation of a green to red or orange precipitate showed glucose presence. If the blue color of the solution persisted with no solid, then it indicated that glucose was absent, hence starch hydrolysis had not taken place. A graph of the enzyme activity vs. the quantity of amylase solution was plotted using the results at 10 minutes. Experiment 2: Effect of Temperature 4 ml of 1 % starch solution was placed in each of the 3 test tubes. 4 ml of amylase solution was placed in three separate test tubes (totaling to 6 test tubes: 3 had starch and 3 with the enzyme). Amylase and starch tubes were placed in the 37°C water bath. One of each tube was placed in boiling water bath and the other tubes, each in an ice-water bath. The tubes were kept in their baths for ten minutes to attain the water baths’ temperature. The temperature of the ice-water bath was recorded. The amylase solution was poured into starch solution, mixed, and then put back into the bath. The time was recorded. Step 10 was repeated for the 37°C bath. Step 10 for boiling water bath was also repeated. For every mixture, 4 drops of the mixture was transferred to a spot plate after 10 minutes of reaction. One drop of iodine reagent was added to each sample. The activity level and color of the enzyme was recorded in each case. The activity of the enzyme level vs. temperature graph was plotted. Experiment 3: Effect of pH Four test tubes were labeled as pH 2, 4, 7, and 10. 4 ml of the appropriate buffer was placed in each tube (with the corresponding pH). Amylase solution of 4 ml was added to each tube. 4 ml of 1% starch solution was added to each of the 4 additional test tubes. All the 8 of these tubes were placed in the 37°C water bath for 5 minutes to equilibrate the temperature. Four separate droppers were rinsed out. Each starch test tube contents were poured into a different test tube of amylase-buffer. The tubes were returned back to the 37°C water bath after mixing. 4 drops of each reaction mixture was transferred to a spot plate after 15 minutes had elapsed (using clean droppers). One drop of iodine reagent was added to each. The observations were recorded and the enzyme activity level was determined for each mixture. The spot plate was rinsed off with de-ionized water. Enzyme activity level vs. pH graph was plotted Part 4: Effect of Inhibitors 4 ml solution of amylase was placed in each of 3 test tubes. 10 drops of 1 % NaCl solution was added to the first tube. 10 drops of 95 % ethanol was added to the 2nd test tube. 10 drops of AgNO3 solution was added to the 3rd test tube. 4 ml of 1 % starch was added to each of the 3 separate test tubes. All the 6 test tubes were placed in the 37°C water bath for five minutes. After 5 minutes, a tube of starch solution was poured into each of the mixtures of enzyme-inhibitor. The tubes were mixed and returned to the water bath for fifteen minutes. 4 drops of each mixture was transferred to a spot plate after 15 minutes, (with a clean dropper). 1 drop of iodine reagent was added to every sample. The observations of enzyme activity level were recorded. Results The enzyme-starch mixture was collected at different times. A drop of iodine reagent was added to each sample. The formed color indicated roughly how much starch was hydrolyzed. High enzyme activity was indicated by very short time of starch hydrolysis. Low or inactive enzyme activity was indicated by blue-black color which was seen after a long time. The relative enzyme activity was assessed as follows: Experiment 1: Effect of Enzyme Concentration Figure 1: shows color observed on separate wells on a spot plate with different concentrations of the enzyme. Test Tube Iodine test for starch Amount of starch remaining Enzyme activity level 1(control) Dark blue-black all None (0) 2(3 drops) Blue Most Low (1) 3(6 drops) Light brown some Moderate (2) 4 (10 drops) Gold None High(3) Table 1: Shows the effect of enzyme concentration on enzyme activity. Figure 2: Show a graph of the enzyme activity vs. the amylase solution amount Experiment 2: Effect of temperature Key: B-Boiling water, W-warm, C-cold water Figure 3: shows color observed on separate wells on a spot plate with different temperatures of the enzyme. Test Tube Iodine test for starch Amount of starch remaining Enzyme activity level 1(Boiling water) Dark blue-black all None (0) 2(warm water) Gold None High(3) 3(cold water) Light brown some Moderate (2) Table 2: Shows the effect of temperature on enzyme activity. Figure 4: Show a graph of enzyme activity level vs. temperature Experiment 3: Effect of pH Figure 3: shows color observed on separate wells on a spot plate with different pH of the enzyme. Test Tube Iodine test for starch Amount of starch remaining Enzyme activity level 1(pH 10) Dark blue-black all None (0) 2(pH 7) Gold None High(3) 3(pH 4) Light brown some Moderate (2) Table 2: Shows the effect of pH on enzyme activity. Figure 4: Show a graph of enzyme activity level vs. pH Discussion Visual tests were used to evaluate the enzyme activity in every part of the experiment. The enzyme-starch mixture was collected at different times a drop of iodine reagent was added to each sample. The formed color indicated roughly how much starch was hydrolyzed. High enzyme activity was indicated by very short time of starch hydrolysis. Low or inactive enzyme activity was indicated by blue-black color which was seen for a longer time. The presence of glucose in the samples was tested using Benedict’s reagent. When Benedict’s reagent blue solution was added to a glucose solution, the color changed to green (at low concentrations of glucose) or reddish-orange (at higher concentrations of glucose). Starch can’t react with Benedict’s reagent, therefore the solution remained blue. Work Cited Abu E. A., Ados A., James D. B. Raw starch degrading amylase productionby mixed culture of Aspergillus riger and Saccharomyces cerevisae grown pomase. Afr. J. Biotechnol. 4 (8): 785-790. 2005. Print. Read More