Introduction: (Initial Observation)

Enzymes play many important roles in our our body and have many industrial applications as well. One role of enzymes in our body is digesting different foods. For example Lactase is the enzyme in the small intestine that digests lactose (the naturally occurring sugar in milk), and Amylase is an enzyme that digest starch.
Because of importance of enzymes activity in different chemical and biochemical reactions, it is important to know the factors that affect enzymes reaction rate. In this way we will be able to optimize and control the enzymes activity for its medical and industrial applications.

Reaction rate is the speed at which the reaction proceeds toward equilibrium. For an enzyme catalyzed reaction, the rate is usually expressed in the amount of product produced per minute.

In this project we will study the effect of temperature, pH and enzyme concentration on the rate of enzymes activity.

If certain range of pH can reduce the rate of enzyme activity, it simply means that we may have digestion problem by eating foods in that specific range of pH. Just knowing this is a valuable outcome of this project.

Information Gathering:

Study about the enzymes in animal and plant cells. Find out about the specific catalytic action of each enzyme that you study. Read books, magazines or ask professionals who might know in order to learn about the mechanism of enzymes activity as a catalyst. Keep track of where you got your information from.

Enzymes are proteins composed of long chains of amino acids held together by peptide bonds. They are the catalysts, which make biochemical reactions possible. Without them, life would not exist. Enzymes increase the rate of a reaction, but are not themselves consumed or produced by the reaction. These catalysts are very specific and will not catalyze any reactions but those suited specifically to the enzyme. Catalase is an enzyme, found in almost all cells, which detoxifies hydrogen peroxide, a poisonous byproduct of cellular metabolism. Without catalase, a cell would die because hydrogen peroxide would oxidize the other enzymes. Catalase is therefore vital to living organisms. The substrate, hydrogen peroxide, is reduced by catalase to water and oxygen by the following reaction:

2H2O2 + catalase ——–» 2H2O + O2 + heat energy +catalase (enzyme)

Although hydrogen peroxide would be spontaneously reduced, this would occur at such a slow rate that cells would die before the breakdown took place. This spontaneous breakdown is why hydrogen peroxide is commercially sold in dark brown bottles with an expiration date.

COMMON MISCONCEPTIONS:

Enzymes are depleted in the course of a chemical reaction.

Enzymes are altered in the course of a chemical reaction.

Enzymes cause a reaction to occur rather than simply increase the reaction rate.

Additional enzyme or substrate will always increase the rate of a reaction instead of reaching a point and remaining constant.

Enzymes are not affected by temperature or pH changes.

Reaction rate is independent of temperature.

There are 5 important factors that can have profound influence on the reaction rate of enzymes.

• enzyme concentration
• substrate concentration
• pH
• temperature
• inhibitors and activators

The enzyme, which functions as a natural catalyst for a biochemical reaction, is not changed or used up during a reaction. For example, during fermentation, enzymes manufactured by yeast cells convert molecules of sugar into molecules of ethanol, but the yeast enzymes are not diminished in the process. This is why small amounts of commercial enzyme products yield such large results, thus being more economical to use compared to other processing methods.

Enzymes [for the most part] are derived from fungal and bacterial organisms, with some products coming from plant sources, e.g., papaya, pineapple, etc., or from animal tissues, e.g. pancreatic lipase.

Enzymes are very specific in the jobs that they perform. For instance, amylase enzymes only work on starch; protease enzymes only work on protein; and so on, thus allowing enzymes to contain features that will provide great benefits in industrial processing. In other cases several enzymes are used together to achieve the desired end result.

Take a look at Enzyme Nomenclature to see how thousands of different enzymes perform thousands of different tasks.

REAL WORLD APPLICATIONS:

There are now detergents containing lipases and proteases for such compounds as chlorophyll and blood.

There are laboratories hard at work genetically engineering bacteria that will produce enzymes to digest crude oil for use in ocean oil spills.

The dairy industry has developed products that contain the enzyme lactase for people who suffer from lactose intolerance.

Hydrogen peroxide is commonly used as a disinfectant. On keratinized epithelial tissue, there is no reaction, but when hydrogen peroxide comes into contact with the catalase of the living cell, the reaction proceeds.

Question/ Purpose:

What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.

The purpose of this project is to determine how enzyme concentration, temperature or pH affect the rate of a reaction involving a specific enzyme as a catalyst.

This is a 3 in one project. You may choose to study only one of the above factors.

Identify Variables:

When you think you know what variables may be involved, think about ways to change one at a time. If you change more than one at a time, you will not know what variable is causing your observation. Sometimes variables are linked and work together to cause something. At first, try to choose variables that you think act independently of each other.

Independent variables are Enzyme concentration, pH and temperature.

Dependent variable is the rate of a chemical reaction involving a specific enzyme

Controlled variables are the type of enzyme, type and concentration of substrate, light and experiment procedures. When you are experimenting or studying one of the above variables (Enzyme concentration, pH and temperature), two other variables are also among controlled variables.

Hypothesis:

Based on your gathered information, make an educated guess about what types of things affect the system you are working with. Identifying variables is necessary before you can make a hypothesis. Following are sample hypothesis:

Higher enzyme concentration results higher rate of reaction.

Low temperatures slow down the rate of chemical reactions and the rate of enzymes activity. Enzymes activity will increase by increase in temperature.

The rate of Enzymes activity is at highest when the pH is 7 or neutral. Higher or lower pH may cause unexpected chemical reactions with enzymes and denature and destroy enzyme.

Experiment Design:

Design an experiment to test each hypothesis. Make a step-by-step list of what you will do to answer each question. This list is called an experimental procedure. For an experiment to give answers you can trust, it must have a “control.” A control is an additional experimental trial or run. It is a separate experiment, done exactly like the others. The only difference is that no experimental variables are changed. A control is a neutral “reference point” for comparison that allows you to see what changing a variable does by comparing it to not changing anything. Dependable controls are sometimes very hard to develop. They can be the hardest part of a project. Without a control you cannot be sure that changing the variable causes your observations. A series of experiments that includes a control is called a “controlled experiment.”

Experiment 1:

The Effect of Enzyme concentration on the activity of enzyme

Introduction:

Catalase is an enzyme found in all living cells. It makes Hydrogen Peroxide decompose into water and oxygen.

You can represent this in the equation: 2H2O2 =2H2O + O2

Enzymes: Enzymes are able to increase the rate of reaction without actually being consumed in the process. In all, enzymes are very efficient. Small quantities at low temperatures are able to produce results, which would require high temperatures and a violent reaction from any normal chemical means. Although increases in temperature may speed up the reaction, enzymes are unstable when heated.

There are three important definitions that are used when talking about enzymes:

• The substance that enzymes act on is the substrate.
• The substance formed by the reaction is the product
• The site on which the enzyme takes place is called the active site.

Enzyme function can be explained by the Lock and Key Hypothesis: the active site of an enzyme (the lock) has a specific shape in which only the precise amount of substrate (the key) will fit – forming an enzyme-substrate complex. Therefore producing a product.

Note: Enzymes are also used in fermentation where yeast is used to produce alcohol.

Aim of the Experiment

To see how different concentrations of yeast affects how much oxygen is given off in 1 minute, when 10cm3 of yeast (which contains catalyse) is mixed with 10cm3 of hydrogen peroxide

List of Material

• 90cm3 Yeast (Check the concentration. You may need to change your procedure based on your yeast concentration. The sample that I used was a 1% yeast)
• 90cm3 Hydrogen Peroxide
• 1 Stopwatch
• 1 Burette or graduated cylinder
• 1 Water Bath
• 1 Boiling tube
• 1 Delivery tube.

Plan

For my experiment I shall be using the concentration of yeast as a variable. I will use the enzyme concentrations of 1%, 0.5% and 0.1% so that there is a range of high to low results. I will repeat each test 3 times to make sure the results are reliable.

Safety: Risk Assessment

Risk	Preventative Actions
Hydrogen Peroxide irritant	Wear goggles, Care when dispensing, Mop up spillages, wash of spillages quickly.
Tube breakages	Wear goggles, clear up immediately after breakage occurs.

Predictions (More descriptive Hypothesis)

I predict that as I increase the concentration of yeast the amount of oxygen produced should increase proportionally:

This is because I know from my background knowledge that this shall happen because as the enzyme concentration rises, the number of active sites that are available to interact with the substrate also rises, this raises the rate of product formation.

Bibliography

The Living World by Michael Roberts – Publisher: Nelson

Encarta Encyclopaedia ’96 – Publisher: Microsoft

Procedure:

Start by making different concentrations of yeast. You may choose (1%, 0.5% & 0.1%). Do this by mixing the yeast with an amount of water. If you want to make 10ml of the 0.5% concentration of yeast using your 1% sample, then you would need 5ml of water and 5ml of yeast (As the yeast you are using comes at a 1% concentration). To make the 0.1% concentration you would need 1ml of yeast and 9ml of water. You would not need to add water for the 1% concentration as it already starts at this strength, therefore use 10ml of yeast.

After making these concentrations and putting them each in their separate boiling tubes* I would then set up my apparatus as shown in the diagram below.

An upside down measuring cylinder or burette filled with water is used to collect and measure the produced oxygen.

I would then put 10ml of hydrogen peroxide into one of the boiling tubes, quickly put a cork with a delivery tube on the top of the boiling tube, place the u-bend of the tube into the upside down measuring cylinder and time three minutes with a stop-watch.

 

After three minutes measure how much oxygen has been produced by the reaction and record it in your results table. Repeat this experiment with each mixture three times (nine results in total) to insure that there are no inconsistent results.

Need a control?

An identical test tube containing hydrogen peroxide with not enzyme may be used as control. Control is your way of showing that the release of oxygen was not caused by an unknown environmental condition and only enzyme has caused the release of oxygen.

Sample Results

1^st Set Of Results

Concentration/%	Oxygen Produced/cm3
1	46.0
0.5	33.5
0.1	4.2

2^nd Set Of Results

Concentration/%	Oxygen Produced/cm3
1	42.3
0.5	34.2
0.1	5.1

3^rd Set Of Results

Concentration/%	Oxygen Produced/cm3
1	44.7
0.5	33.8
0.1	5.4

Table of Average Results

Concentration/%	Oxygen Produced/cm3
1	44.3
0.5	33.8
0.1	4.9

Analysis

I found out that the concentration of the yeast did affect the amount of substrate that was produced; this can be shown by these graphs:

 

 

 

In my experiment the rate of reaction was fastest at the 1% concentration and became slower the lower the concentration was. This is because as the concentration of yeast is increased, there are more active sites that are available to interact with the substrate, which increases the rate of product formation, as there are more active sites to produce the product. There were no differences from my prediction and my actual results, this was because I used the right amount of each substance and my experiment went well enough to produce the results I expected.

How do I test other factors?

You need to design a modified version of this experiment in order to test the effect of pH or temperature on enzyme activity.

Effect of pH on the rate of Enzyme activity:

Modify this experiment and use the same concentration of enzyme in all your three boiling tubes. Reduce the pH of one tube by adding a few drops of acetic acid. Increase the pH of another tube by adding a few drops of ammonia solution. Do not add anything to the third tube. Label the tubes with “Low pH, High pH and Normal”. The rest of experiment and recording data is the same.

Effect of temperature on the rate of Enzyme activity:

Modify this experiment and use the same concentration of enzyme in all your three boiling tubes. Place one tube in warm water, another tube in cold water and the third tube in normal room temperature. Label the tubes with “Warm, Cold and Normal”. The rest of experiment and recording data is the same.

Experiment 2:

Introduction: There are many different methods of measuring the rate of enzyme activity. In this experiment you measure the time the amylase enzyme takes to break down starch. You use iodine as an indicator to show the presence of starch. This general experiment can be performed at different temperatures, different pH or at different enzyme concentrations in order to determine the effects of those factors.

Apparatus

• 20ml Solution of Starch (1%)
• Dropping pipette
• Dimple tile or any similar white surface
  (You can use white plastic spoons as dimples)
• Stopwatch
• Iodine solution
• 2ml Solution of Amylase (1%)

Method

• Put one drop of iodine into each well on a spotting tile.
• Into dimple number 11 put one drop of starch. (This should be the color of the iodine at the start of your experiment.)
• Into dimple 12 put one drop of water. (This shows the color your experiment should go when there is no starch left.)
• Put 5ml of starch into a test tube.
• Put 1ml of amylase enzyme solution into another test tube.
• Start the stopwatch at the same time as you add the amylase to the starch.

 

 

• At the times given in the results table take out ONE drop of the mixture and put it into the dimple tile.
• Mix your enzyme-starch mixture and repeat step 7

 

 

 

Results

Materials and Equipment:

List of material can be extracted from the experiment section.

Results of Experiment (Observation):

Experiments are often done in series. A series of experiments can be done by changing one variable a different amount each time. A series of experiments is made up of separate experimental “runs.” During each run you make a measurement of how much the variable affected the system under study. For each run, a different amount of change in the variable is used. This produces a different amount of response in the system. You measure this response, or record data, in a table for this purpose. This is considered “raw data” since it has not been processed or interpreted yet. When raw data gets processed mathematically, for example, it becomes results.
Place your results table and your observations here.

Calculations:

No calculation is required.

Summary of Results:

Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.

It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.

Place your results tables here.

Conclusion:

Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.

Related Questions & Answers:

What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.

Possible Errors:

If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.

If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.

References:

List of References

http://courses.washington.edu/conj/bess/enzyme/enzyme.html

http://structbio.vanderbilt.edu/chazin/classnotes/biochem.5012/Biochem-5012.5A.ppt