THE SECRETS BEANO REVEALS ABOUT ENYMATIC ACTIVITY

Alaina Weinheimer February 20th, 2013 CHEM 113B, Section 104 Partner: Rachel Thomas TA: Kate ORourke

Introduction: In the human body, thousands of chemical reactions occur every single moment. According to the Collision Theory, which is based on the Kinetic Molecular Theory, reactions occur when two molecules of reactant make contact. In order of these reactant molecules to collide, each must have the minimum kinetic energy, or activation energy, and the proper orientation when they make contact1. Some reactions in the human body can take an extremely long time in order for the reactant molecules to overcome the activation energy and make proper contact. To increase the rate of reactions, body cells contain enzymes that catalyze, or promote, these reactions. If enzymes did not exist, bodily reactions would occur at such a low rate that life would not be able to sustain2. Enzymes are proteins that contain an active site for a specific substrate, or reactants, of the reaction. The enzyme lowers the activation energy and the reaction occurs. The product leaves the enzyme and the enzyme finds new substrate molecules. With the reactants together in close proximity at the proper orientation in the enzyme, decreasing the activation energy, enzymes increase the rate of reaction3. Many factors contribute to the rate of enzymatic activity, which effects the rate of reaction. Some of these factors include temperature, pH, substrate concentration, and water activity3. The effect of increasing temperature on the rate of reaction is based on the Kinetic Molecular Theory. The Kinetic Molecular Theory states that the kinetic energy of a molecule is directly related to temperature. If temperature increases, the speed of the molecule increases. When the molecules of reactant have a higher kinetic energy, moving at a faster speed, contact with an enzyme is more frequent causing the reaction to occur faster. Conversely, if the temperature is decreased, the reactants move slower, contact is less frequent with the enzyme,

causing the reaction to occur much more slowly4. In short, enzymes are more active at higher temperatures and less active at lower temperatures. Varying substrate concentration relates to enzymatic activity. If there is more substrate, or reactant, more reactant is present to bind to the enzyme and react. Thus, enzyme activity increases when substrate concentration increases, which consequently increases the rate of reaction. Varying enzyme concentration also affects the rate of reaction. With a lower enzyme concentration, only so much substrate can bind to the fewer enzymes, causing the reaction rate to decrease. With a higher enzyme concentration, more substrate can bind to the abundant enzymes, causing the reaction rate to increase5. In summary, varying substrate and enzyme concentration impacts enzyme activity, which directly impacts the rate of reaction. In this experiment the enzymatic activity of the enzyme in Beano was analyzed. Beano contains the enzyme alpha-galactosidase, which speeds up the breakdown of sugars called oligosaccharides, which are present in foods such as peas, onions, and legumes. The enzyme, alpha-galactosidase breaks down these oligosaccharides into galactose and sucrose. The sucrose is then broken down by sucrase into alpha-glucose and fructose6. Many people lack the enzyme alpha-galctosidase in their small intestine. This enzyme is located largely in the large intestine, where bacteria, that have alpha-galactosidase, ferment the oligosaccharides and release gas as a waste product. This gas can create a pressure on the lower stomach that may be uncomfortable to some. To decrease the pressure by decreasing the amount of gas, people consume Beano, which contains the alpha-galactosidase. By consuming Beano, the enzyme enters the small intestine and can break down the oligosaccharides before they reach the large intestine7. In this experiment, the enzymatic activity of Beano was measured based on the varying factors. The activity was measured by a glucometer, which can only detect beta-glucose. Beta-

glucose has a different structure than alpha-glucose, which is the end product of the reaction the enzyme induces. The location of the alpha and beta hydroxyl groups is in different places. Luckily, in solution, alpha-glucose readily converts to beta-glucose, allowing the enzymatic activity of Beano to be measured by the glucometer6. The glucometer used in this experiment, the ReliOn Ultima (Wal-Mart Stores, Inc.), is designed to measure the levels of glucose in blood. Blood contains many complex molecules. The glucometer is designed to factor in those other molecules in its reading. In this experiment, the substrate of the enzymes in Beano was a pea extract. Because this experiment involved a water-based solution of pea extract and Beano, not blood, the glucometer had to be calibrated to the water so that there was a better indication of how much glucose is in the solution. Beanos enzyme activity was measured for varying substrate concentration and varying temperature in this experiment. To test for the effect of varying substrate concentration on enzyme activity, three different solutions of pea extract were tested for glucose levels over intervals of two minutes: 25%, 50%, and 100% pea extract. It is expected that the 100% pea extract will lead to higher enzymatic activity, and faster rate of reaction than the lower concentrations of pea extract, because in the 100% pea extract there are more substrate molecules to interact with the enzymes. To test the effect of temperature on enzymatic activity, the glucose concentration of solutions containing 50% pea extract were measured at 10C, 25C, and 40C over two minute intervals. It is expected that the reaction carried out at 40C will yield the fastest rate of reaction than the other two temperatures because at high temperatures, molecules move faster. Other factors that werent tested for, but were noted was varying enzyme concentration and varying pH. It is expected that with increasing enzyme concentration, enzyme activity increases, which increases the rate of reaction. Considering that the enzyme alpha-

galactosidase is located in the human instestine, which is an acidic environment, it is expected that there is more enzyme activity at low pH than at high pH6. In short, this experiment measured the enzyme activity of Beano with many factors.

Procedure: This experiment was carried out using the steps in the PSU Chemtrek6. To start, the glucometer was calibrated to aqueous solutions containing varying concentrations of glucose. Next, the test for the effect of varying substrate concentration on the enzyme activity began. A solution was prepared by adding 1.000mL of 100% pea extract and a stir rod to a 20mL vial that was placed on a stir plate to mix the solution. 1.000mL of Beano solution was added to the vial. After two minutes of mixing, the solution in the vial was measured for glucose concentration by the placing a little sample of solution on the glucometer. After another two minutes passed, the glucose concentration was measured again. The glucose concentration was measured in two minute intervals until the glucometer had five numeric measurements, which happened after 10 minutes passed in this case. This process was repeated for a solution of 50% pea extract with Beano and 25% pea extract and Beano. Once all of the measurements were recorded, they were graphed and a line of best fit was found for each concentration of pea extract over time, which reflected the rate of reaction, the enzyme activity. To test the effect of temperature on enzyme activity, special environments had to be prepared for the vials. To achieve 40C, a Styrofoam cup was cut to fit the vial. The vial with 1.000mL of 50% pea extract and a stir rod was put into the Styrofoam cup. A separate cup was filled with hot tap water and adjusted to be 40C. That water was then carefully poured into the Styrofoam cup that contained the vial, without any water entering the vial. 1.000mL of Beano

was added to the vial. After two minutes intervals of mixing, the glucose concentrations of the vial solution were measured until five numeric measurements were read. To achieve a 10C environment for the solution of Beano and pea extract, the water bath in the Styrofoam cup of 40C was replaced by tap water cooled by ice to be 10C. A new solution of 50% pea extract and a stir rod were added to a clean 20mL vial. 1.000mL of Beano was added to the vial and the glucose concentrations were measured again in increments of 2 minutes until the glucometer read five numeric measurements. The glucose concentrations at the different temperatures of 40C , 25C and 10C were plotted against time. The glucose concentrations measured at 25C were used from the readings obtained during the testing of varying substrate concentration.

Results: Glucometer Calibration Data Table 1. Calibration of Glucose Readings in the ReliOn Ultima Glucometer Caption: This table shows the glucometer readings of increasing concentrations of glucose in milligrams per deciliter. These values were then used to create a calibration curve, Graph 1. Glucose Concentration (mg/dL) 50 100 150 200 250 Reading (mg/dL) 62 223 281 323 407

Graph 1. Glucometer Readings vs. Glucose Concentrations Caption: The glucose concentrations read by the glucometer were plotted against the known concentrations of glucose to find a line of best fit. The line of best fit shows the relationship between the known glucose concentrations and the reading of the glucometer. Glucometer Readings vs. Known Glucose Concentration
140 Glucometer Reading (mg/dL) 120 100 80 60 40 20 0 0 2 4 6 8 10 12 Known Glucose Concentration (mg/dL) y = 13.5x - 25

Table 2. Glucose Concentration in Pea Extract and Beano Solutions Over Time Caption: This table shows the concentrations of glucose over two minute intervals in vials containing solutions of Beano with three different concentrations of pea extract: 100%, 50%, and 25%. Time (Minutes) 2 4 6 8 10 12 14 Glucose in 100% Pea Extract (mg/dL) low 32 56 74 116 137 Glucose in 50% Pea Extract (mg/dL) low 23 58 77 99 134 Glucose in 25% Pea Extract (mg/dL) Low Low 23 24 51 59 83

Graph 2. Glucose Concentration vs. Time Caption: This graph shows the amount of glucose concentration in each of the pea extract solutions over time. The line of best fit represents the initial rate of reaction in each solution.

Glucose Concentration vs. Time

180 160 140 120 100 80 60 40 20 0 -20 0 -40 Glucose Concentration (mg/dL) y = 13.5x - 25 y = 13.15x - 27 100% Pea Extract 50% Pea Extract 25% Pea Extract

y = 7.75x - 29.5 5 10 15

Time (minutes)

Table 3. Rate of Reaction in 25%, 50%, and 100% Pea Extract Solutions Caption: This table shows the rates of reaction in vary pea extract solutions. Pea Extract Concentration 25% 50% 100% Rate of Reaction 7.75 13.15 13.5

Table 4. Glucose Concentration in 50% Pea Extract Solution at 10C, 25C, and 40C Over Time Caption: This table shows the concentration of glucose over time when the reaction between Beano and pea extract is carried out in three different temperatures: 10C, 25C, and 40C.

Time (minutes) 1 4 6 8 10 12 14 16 18

10C Low Low Low Low 21 32 42 51 67

25C Low 23 58 77 99 134

40C Low 31 91 149 22 250

Graph 3. Glucose Concentration vs. Time Caption: This graph shows the glucose concentrations of vials in three different temperatures, 10C, 25C, and 40C over time. The line of best fit represents the initial rate of reaction between the Beano and the 50% pea extract solution.

Glucose Concentration (mg/dL)

Gluose Concentration vs. Time

500 400 300 200 100 0 0 -100 5 10 Time (minutes) y = 5.55x - 35.1 15 20 y = 13.15x - 27 y = 28.45x - 79 10C 25C 40C

Table 5. Temperature and Reaction Rate Caption: This table shows the varying temperatures the 50% Beano and pea extract solution were placed in and the rate of the reaction. Temperature (C) 10C 25C 40C Rate of Reaction 5.55 13.15 28.45

Discussion: Overall this lab supported the hypotheses on the factors on the rate of reaction and enzyme activity. Regarding the calibration of the glucometer to the pea-extract and Beano solution, the glucometer read measurements much higher than the known concentrations. For instance, the known concentration of glucose was 200 mg/dL, but the glucometer read 323 mg/dL. The correlation between the glucometer reading the known concentrations of glucose was positive and linear. This shows that the glucometer overshot the actual amount of glucose in the water-based media because it is designed to facto in the complex molecules in blood media into its glucose reading. The hypotheses regarding enzyme activity were affirmed. The hypothesis that increasing the substrate concentration would increase the enzyme activity, increasing the rate of reaction was averred. At low levels of substrate concentration, 25% pea extract, the initial rate of reaction was about twice as slow as the initial rate of reaction when there was a substrate concentration of 50% pea extract. The initial rates of reaction were not exactly proportionally faster or slower based on concentration. 100% pea extracts reaction rate, 13.5, was not double

that of 50% pea extracts reaction rate, 13.15. The time it takes for the reaction to go to completion may not be proportional between the solutions. After completion, however, after all of the substrate is reacted with the enzyme, there would be proportional amount of glucose in each pea extract solution. This is because there are only proportional amounts of substrate in solution. For instance, there is less substrate in the 25% pea extract solution than in the 100% pea extract solution. Thus, after completion, there would be less glucose in the 25% pea extract vial than in the 100% pea extract vial. Varying the temperature by 15C played a major role on the initial rates of reaction between the enzymes in Beano and the pea extract. The results confirmed the hypothesis that at higher temperatures, enzyme activity increases, which increases the rate of reaction. When the reaction was carried out in 10C conditions, the initial rate of reaction was roughly 2.4 times slower than when the reaction was carried out at 25C. When the reaction was carried out at 40C, the reaction occurred at a rate 2.2 times faster than at 25C. This shows enzymatic activity is directly related to temperature. With increasing temperature, the Beano enzymes interacted with the pea extract substrate to produce glucose faster. Raising the temperature 15C had approximately the same quantitative effect on the rate of reaction as lowering the temperature 15C. This further confirms that enzyme activity and reaction rates are very closely and directly related to temperature. Unfortunately, throughout this lab there was some room for error. There may have been some inherent instrumental error in the glucometer that could have produced inaccurate readings beyond the inaccuracy of media discrepancy. Also, the timing of glucose measurements could have led to some error in results. The time that the glucose was measured was not always two minutes on the dot. Sometimes, a few extra seconds passed and those seconds could have led to

different results. Another major room for error was in the water bath solutions. Maintaining an environment of 40C is not easy. The heat could have escaped enough to lower the bathwater enough to impact the rate of reaction. Conversely, the 10C bathwater was not exactly 10C the whole time. The warmth of the room increased the bathwater temperature which would have caused the reaction to occur faster than at exactly 10C. In short, there was a noticeable amount of room for error in this experiment. However, the results still confirmed the hypotheses. Conclusion: Overall, this experiment fulfilled the predictions about factors affecting enzymatic activity through the reaction of Beano and pea extract in glucose appearance. Beano is a common medicine taken by people who experience uncomfortable pressure in their lower stomach from bacteria breaking down the oligosaccharides in their large intestine because their small intestine lacks the enzyme alpha-galactosidase that breaks down the oligosaccharides. Beano contains the enzyme alpha-galactosidase. When Beano is consumed, the alphagalactosidase in Beano are released into the small intestine and the oligosaccharides can be broken down in the small intestine. In this experiment, factors on the enzyme alphagalactosidase were measured by mixing Beano with pea-extract. It was found that increasing the substrate concentration, the pea extract, increased the initial rate of reaction because there was more substrate for the enzyme to interact with. It was also found that increasing temperature of the Beano and pea-extract solution increased enzymatic activity because the substrate molecules and enzyme molecules moved around faster, increasing their rate of collision, thus increasing the rate of reaction. In summary, this experiment showed that the enzyme activity of alphagalactosidase in Beano follows that Collision Theory and the Kinetic Molecular Theory in that

enzyme activity increased with increasing temperature and that enzyme activity increased with increasing concentration. Citation: 1. "Collision Theory." Salve Regina University Department of Chemistry. Salve Regina University. Web. 28 Feb 2013. <http://www.salve.edu/academics/departments/chm/faculty.asp&xgt;. 2. Cooper, Geoffery. The Cell: A Molecular Approach. 2nd. Sunderland: Sinauer Associates, 2000. Web. 3. Enzymes." The Royal Society of Chemists. The Royal Society of Chemists. Web. 20 Feb 2013. <http://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm>. 4. . "Factors Effecting Enzyme Activity." Ohio State University Department of Biochemistry. Ohio State University. Web. 28 Feb 2013. <http://class.fst.ohiostate.edu/fst605/605p/EnzymeFactors.pdf>. 5. Bender, David. "The Effect of Substrate Concentration on Enzyme Activity." University College London Dept of Chemistry. University College London. Web. 28 Feb 2013. <http://www.ucl.ac.uk/~ucbcdab/enzass/substrate.htm>. 6. Keiser, Joseph. PSU ChemTREK. Hayden McNeil. (Spring 2013): 6-1 6-15 7. Ganiats, T.G., Norcross, W.A., Halverson, A.L., Burford, P.A., Palinkas, L.A. "Does Beano prevent gas? A double-blind crossover study of oral alpha-galactosidase to treat dietary oligosaccharide intolerance." J-Fam-Pract. 1994 Nov; 39(5): 4415 8. Weinheimer, Alaina. Chem 113B Notebook. 9. Thomas, Rachel. Chem 113B Notebook.

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