Aim and Prediction: The aim of this experiment is to find out the optimum pH of amylase. This will be done by putting potato cubes in solutions of different acidity/alkalinity which will represent different environments where the amylase works.
The potato cubes contain starch, thus it will prove the presence of reaction between amylase and starch if the potato cubes’ final mass decreased from the initial mass. The mass of each group of potato cubes will be measured and compared after the same duration of time. Our prediction for this experiment is that the amylase will catalyze starch in potato better in acidic solutions. This prediction is due to prior knowledge that amylase is present in the acidic organs, such as stomach. Background Information: Enzymes are globular proteins which function as biological catalysts. They speed up biochemical reactions, also known as metabolism, within the living organisms.
The substance in which an enzyme helps to catalyze is called the substrate. An enzyme is able to catalyze only one chemical reaction due to enzyme-substrate specificity. The substrate in this experiment is starch, broken down by the enzyme, amylase. As enzymes are biological catalysts, there are several factors which influences the activity of enzymes.
One of these factors is the pH, which determines the acidity or alkalinity of a solution. A solution with lower pH (from pH 1 to pH 6) means that it has a higher concentration of hydrogen ions. A reduction of one unit in the pH scale makes a solution ten times more acidic, thus the difference of acidity or alkalinity is significant between pH units. Enzymes have optimum pHs at which their rate of collision with the substrate is the highest. The difference of the pH from an enzyme’s optimum controls whether or not the activity of the enzyme increase or decrease.
The structure of the enzyme may be changed if the pH of its environment goes beyond a certain level. Due to this, the enzyme may be denatured, which means that the structure of its active site change and it is not able to bind with its substrate anymore. Every enzyme has different pH optimum and there apparently is a wide range, due to the difference of pH in different environments where the enzymes work in. This experiment will determine the optimum pH of amylase. Amylase is an enzyme which breaks starch down into sugars. It is found in the saliva in the mouth where the process of digestion begins and it is produced in the salivary gland and also in the pancreas.
Research Question: What is the effect of pH on amylase’ rate of reaction for breaking starch down in a potato cube thus changing its mass? Materials and Apparatus: ? Amylase powder ? 50ml of pre-prepared hydrochloric acid solutions of the following concentrations: – 0.2 – 0.6 – 1.0 ? 50ml of pre-prepared sodium hydroxide solutions of the following concentrations: – 0.2 – 0.6 – 1.0 ? Distilled water ? Potatoes ? Spatula ? Three 25ml measuring cylinders ? Seven 150ml beakers ? Forceps ? Stopwatch ? Digital mass balance ? Ruler (All uncertainties for the apparatuses used are listed on page 4 of this document.) Risk Assessment: All solutions of HCl and NaOH should be carefully handled. Scalpels must also be carefully used. Lab coats, safety goggles and gloves are necessary for this experiment. Procedures: 1. A potato was cut into 21 small cubes of 1x1x1. 2. The potato cubes were divided into 7 groups of 3.
3. The masses of each group of potato cubes were recorded using a digital mass balance. 4. 3 HCl solutions of different acid concentrations (0.2 0.6 1.0 ) were prepared, the total volume of each one being 50ml. 5. 3 NaOH solutions of different base concentrations (0.2 0.6 1.0 ) were prepared, the total volume of each on being 50ml. 6. 50ml of distilled water was prepared as a control.
7. The seven substances were transferred individually into 150ml beakers. 8. The beakers were labelled with markers to avoid confusion. 9. Half spatula of amylase powder was added into each beaker. 10. A group of potato cubes was put into each beaker and the stopwatch began. 11. After 10 minutes, the potato cubes were taken out of each solution using forceps.
12. The mass of each group of potato cubes were recorded again using the digital mass balance. 13. The procedure above was repeated three times for an average data to be obtained. Control / Variables: Independent Variable pH (acidity/alkalinity) of environment of enzyme (determined by HCl/NaOH solution concentration in mol/dm^3) 1. 0.00 (distilled water) 2. 0.20 3. 0.60 4.
1.00 Dependent Variable The change in mass of the potato cubes Controlled Variables Reason for control Method of control 1 Total volume of 50cm^3 To make sure that pH is the only variable changing. Usage of measuring cylinder 2 Shape of potato blocks To ensure that every potato blocks have the same surface area to volume rati. A ruler was used to cut out the pieces of potatoes 3 Temperature of experiment To make sure that pH is the only variable changing. The experiment was held in the laboratory where temperature was kept constant by air conditioning 4 Enzyme concentration To make sure that pH is the only variable changing. Half spatula of amylase powder was added into each beaker 5 Time taken To make sure that enzymes in the solutions had equal time to catalyze their substrates. The experiment began at the same time and the potato blocks were left in each solution for 10 minutes Table 1.1 and 1.2: Showing the independent variable, dependent variable and controlled variables.
Uncertainties: Apparatus Uncertainty for each Measuring cylinder ±1ml Balance ±0.1g Ruler ±0.05mm Table 2: Listing all uncertainties of the apparatuses used. Results: Concentration of HCl solution (mol/dm^3) Trial Initial mass of potato cubes (g) (±0.1g) Final mass of potato cubes (g) (±0.1g) 0.00 (control) 1 2.5 2.3 2 2.5 2.3 3 2.5 2.4 0.20 4 2.5 2.2 5 2.5 2.1 6 2.5 2.2 0.60 7 2.5 2.5 8 2.5 2.5 9 2.5 2.5 1.00 10 2.5 2.5 11 2.5 2.5 12 2.5 2.5 Table 3: Showing the raw data collected of the masses of potato cubes before and after becoming catalyzed by amylase in different concentrations of HCl solution. Concentration of NaOH solution (mol/dm^3) Trial Initial mass of potato cubes (g) (±0.1g) Final mass of potato cubes (g) (±0.1g) 0.20 1 2.5 2.5 2 2.5 2.5 3 2.5 2.5 0.60 4 2.5 2.5 5 2.5 2.5 6 2.5 2.5 1.00 7 2.5 2.5 8 2.5 2.5 9 2.5 2.5 Table 4: Showing the raw data collected of the masses of potato cubes before and after becoming catalyzed by amylase in different concentrations of NaOH solution. Graphing raw data: Graph 1: Mass of potato cubes before and after becoming catalyzed by amylase in different concentrations of HCl solution.
Graph 2: Mass of potato cubes before and after becoming catalyzed by amylase in different concentrations of NaOH solution. Data Processing: Sample calculations 1. Calculating the mass change: e.g. 2. Finding the percentage mass change: e.g. 3. Finding the mean percentage change: e.g.
4. Finding the standard deviation: The standard deviation for the percentage change in masses was obtained by inputting the results in the STDEV function in Microsoft Excel. Concentration of HCl solution (mol/dm^3) Trial Change in mass (g) (±0.2g) % Change in mass Mean % Change Standard deviation 0.0 1 -0.2 -8 -6.7 2.3 2 -0.2 -8 3 -0.1 -4 0.2 4 -0.3 -12 -13.3 2.3 5 -0.4 -16 6 -0.3 -12 0.6 7 0 0 0 0 8 0 0 9 0 0 1.0 10 0 0 0 0 11 0 0 12 0 0 Table 5: Displaying the processed data which are the change in mass, the mass change in percentage, the mean percentage mass change and the standard deviation of the results. Graph 3: Effect of HCl concentration on potato cube mass. The error bars show the standard deviation of each change in mass. Conclusion: The results of experiment display the pH of the environment in which amylase works best in. The change of mass was greatest at the solution of HCl, which had the concentration of 0.2 mol/dm^3; from this we can predict that the optimum pH of the amylase used is around pH 6.
The results also show that there is no change to the potato cubes’ mass at the HCl solution concentration above 0.2 mol/dm^3 and at all NaOH solutions. The explanation to this is that amylase denatures at pH way below or above its optimum. Evaluation (Limitations): 1. The concentration of acidic/alkaline solution may not necessarily follow the pH predicted in the conclusion. Thus it would be better to use a universal indicator to predetermine the pH of solutions before the actual experimentation or to prepare solutions with accurate and known pH. 2. The unit to measure the amount of amylase powder (half spatula) might have caused a human error. Using a digital mass balance to measure the mass of each amount may be a better idea to ensure that all solutions are provided with the exact same amount of amylase powder.