ENZYME LAB PRESENTATION By May, Nam T., Por, Parn, Mook, Mix (10-9)

Objective To study how pH, temperature, ionic conditions, substrate concentration affects enzyme activity

Something you should know before : -The enzyme that is used in this experiment is ‘ urease ’, which contains in the soya bean. -Soya suspension, in this experiment, is used as an enzyme for urea. -The substrate is the urea. -Enzyme urease breaks down urea to ammonia and carbon dioxide. CO(NH2)2 + H2O  2 NH3 + CO2 Urea + water  ammonia + carbon dioxide

Something you should know before (cont.) : -Ammonia that is produced has a high pH (basic). -We can detect ammonia by using the red cabbage indicator and also by smelling.

Hypothesis -Drop citric acid  change to red -Drop sodium bicarbonate  change to blue -Drop distilled water  no change Procedure 1: Investigation of urease enzyme and Indicator

Result Test tube 1- Citric Acid Test tube 2- Sodium Carbonate Test tube 3- Distilled Water Color (initial)Purple Color (final)RedDark greenish blue Purple

Answer questions 1. Why is it important to have three reference tubes? Each reference tube contains acid, base and distilled water, which is neutral, respectively. They are a control for reaction between red cabbage indicator and acid, red cabbage indicator and base, and the same indicator and water. They show the different results after the indicator reacts with acid and base.

2. Can you predict what color of the solution will be if the urease enzyme converts urea to ammonia? I can predict that the color of the solution will be blue or green, as the result, when red cabbage indicator reacted with sodium carbonate, which is basic, it became blue or green.

Procedure 2: Investigating the effect of urease from soya beans on urea. Hypothesis -The test tube which contains both urease and urea will be the only one test tube that changes the color.

Result Test tube 1- Urea Test tube 2- Urea + Soya Test tube 3- Distilled water + soya Color changePurpleGreenLight purple Odor(smell)No smellLight smellStrong smell

Answer the questions 1. What are the purposes of procedure 2? (Think about the total contents in each tube, and what can the results from each tube tell us.) The purpose of procedure 2 is to test the enzymatic working, which shows that if there is only the enzyme (only urease) or only the substrate (only urea), the enzymatic reaction cannot occur. The reaction will occur when there are both the enzyme and the substrate fitting each other such that the test tube 2 represents.

2. Why do we need to add an enzyme into a test tube without urea even though we can predict that no enzyme activity can occur? Because we want to make sure that we can use this indicator to test the pH changing. If we add the enzyme into the test tube without urea (test tube 3) but the color changes then we cannot use this indicator to test in this experiment. Although we can predict that no enzyme activity can occur but we have to double-check the result.

Hypothesis -The color of the solution of both test tube with 5% NaCl and with 10% NaCl will change to green. Procedure 5: Effect of the concentration on urease

Result Test tube 1- 5% NaCl Test tube 2- 10% NaCl Test tube 3- Saturated NaCl Color changeClear greenClear greenish blue Opaque blue Odor(smell)Light pungent A bit strong pungent

Answer the questions 1.How does ionic condition affect the urease activity? How do you know? The ionic condition affects the rate of the urease activity. The rate of the activity depends on the ionic concentration; if the concentration of the ionic substances increases, the rate will increase. However, too much concentration can stop the reaction as well that means the enzyme will not cause the reaction anymore. We can know that from the color of the solutions from the results. When the activity occured, the color of the solution turned green. If the solution is blue, the activity did not occur.

2. From your results, what ionic condition would you use and avoid if you have to conduct an experiment using urease enzyme and why? We would use the 5% NaCl ionic condition and avoid the saturated NaCl ionic condition, because, according to the experiment, the 5% NaCl ionic condition could use the most efficiently, it could cause the faster activity; whereas the more than 5% NaCl can lower the rate of the activity. Moreover, the saturated can stop the activity because of the too much concentration of the ionic substances, NaCl.

3. Your special task is to try and deactivate the enzyme with table salt. We can deactivate the enzyme by adding a lot of salt. Much concentration of the salt can deactivate it.

What were you able to learn from this experiment? -Temperature, pH, ionic condition, substrate concentration are all the factors that affect enzymatic reaction. -Enzyme can work in only its properly condition. -Two ways to detect the reaction; indicator and smell -Red cabbage indicator can be used to detect ammonia.

What were you able to learn from this experiment? (cont.) -Urease is an enzyme of urea that can be found in soya beans, as in the experiment, the enzyme activity causes when we added soya. -When urease breakdowns urea, ammonia and carbon dioxide are the products. -How to work in group

Did your results support the prediction made before conducting the experiment. If it did not, then explain why. => All the results have supported our prediction or hypotheses.

Any experimental errors? How can you avoid these errors in the future? The contaminated urea  Do not suck the chemical directly from the bottle  Do not pour the rest chemical back into the bottle  Keep the chemical correctly, close the lid tightly  Check the chemical before starting the experiment

Thank you for your participation