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Catalysis Using Enzymes in Pineapple.

Catalysis Using Enzymes in Pineapple.

Introduction: (Initial Observation)

For many years I was wondering why a goat can eat and digest paper and wood (Cellulose), while sheep and human can not. Later I saw a meat tenderizer in a supermarket and I was wondering what is in a meat tenderizer and how does it work. Finally when I first heard about DNA sequencing, I was wondering how can scientists cut a DNA molecule from a certain spot.

Surprisingly, later I learned that the answer to all my questions are Enzymes.

Goat digestive system produce enzymes that can digest cellulose and break it to sugar, while human lacks such enzyme. Also that is the enzyme inside a meat tenderizer that breaks meat (protein molecules) and softens the meat. Enzymes are also used to cut DNA molecules.

Because of the importance of enzymes, we need to know about the factors that may modify the effects of enzymes.

For example we need to know if heat, light or acids may alter the enzyme activity and stop its effects. In this project you will study the effect of heat on a specific enzyme. Similar procedures can be used to test the effect of UV light, Enzyme concentration and pH.

Dear

This project guide contains information that you need in order to start your project. If you have any questions or need more support about this project, click on the “Ask Question” button on the top of this page to send me a message.

If you are new in doing science project, click on “How to Start” in the main page. There you will find helpful links that describe different types of science projects, scientific method, variables, hypothesis, graph, abstract and all other general basics that you need to know.

Project advisor

Information Gathering:

Find out about enzymes and their function. Read books, magazines or ask professionals who might know in order to learn about the sources of enzymes and their structure. Keep track of where you got your information from.

Following are samples of information that you may find:

All living cells produce enzymes which catalyze metabolic reactions. An enzyme is an organic catalyst that alters the rate of a specific chemical reaction but which remains unchanged at the conclusion of the reaction. Enzymes are classified according to the substrate on which they function or the reaction they catalyze.

The enzyme that is investigated in this experiment is one that is produced in pineapple and hydrolyzes certain kinds of proteins called gelatins. The gelatin used in this experiment is derived from skin, bones, and/or connective tissue of animals. These proteins, when dissolved in hot water and allowed to cool, form a semi-solid or gel state; hence the name gelatin. Hydrolyze, here, refers to breaking up the protein polymer in such a way as to prevent its forming this gel state. The manufacturers of gelatin containing desserts customarily warn the consumer against adding fresh or frozen pineapple or kiwifruit to the dessert to prevent this reaction.

The hydrolyzing enzyme from pineapple is denatured by heat. Denature, here, means to alter in such a manner that the enzyme is no longer able to catalyze the reaction. Enzymes can also be denatured by changes in pH, subjection to detergents or radiation, etc.

  1. Gelatin is made from a protein called collagen which comes from the joints of animals. Gelatin may be dissolved in hot water. As the dissolved gelatin mixture cools, the collagen forms into a matrix that traps the water; as a result, the mixture turns into the jiggling semi-solid mass that is so recognizable as Jell-O™.
  2. Pineapple belongs to a group of plants called Bromeliads. Kiwi, papaya, and figs are other types of Bromeliads. The enzyme in pineapple juice that is responsible for the breakdown of collagen is bromelin. The process of canning pineapple denatures the bromelin, rendering it incapable of catalyzing the break down of gelatin.

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 study the effect of heat on enzymes. We suspect that heat may denature the enzyme and alter it in such a manner that it will no longer be able to catalyze the reaction.

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.

The independent variable for our experiment is heating time. The dependent variable is the catalysis action of enzyme. We control and maintain constant all other variables that may affect the activity of the enzyme. Constants include the temperature, light, pH and experiment procedures.

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 some sample hypothesis:

  • I think heat has no affect on the enzyme in a pine apple or other fruits. My hypothesis is based on the fact that water contents in fruits and vegetables does not allow the heat to exceed certain level. Also many enzymes in our warm body, process and digest our food. So some excess heat should not be able to effectively alter enzymes.
  • Heat can denature and modify the enzyme in a way that it will no longer be a catalyst. My hypothesis is based on my observation that cooked and canned pine apple can be added to jell-o with no harmful affect.

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.”

Introduction: Pineapple contains an enzyme that hydrolyzes certain proteins called gelatins. In this experiment, I add pineapple, which has been subjected to various treatments (Heat treatment), to gelatin to determine if the treatment has altered the enzyme’s activity. If pineapple enzyme breaks gelatin molecules, our gelatin will not gel.

Procedure:

  1. Cut each slice of drained canned pineapple into four approximately equal pieces. Peel and cut the fresh pineapple into slices and pieces which are about the same size as the canned pineapple pieces. Cut the dried pineapple into pieces the same size as the others. (So now you have identical pieces of fresh, canned and dried pineapple for your experiment.)
  2. In a 2-L beaker, heat approximately 500 mL of water to boiling for each set of 10 samples.
  3. Weigh out ten 17-g samples of gelatin and place one sample in each of ten 150-mL or 250-mL beakers.
  4. Carefully add 40 mL of boiling water to each sample. Stir until dissolved. With stirring, add 40 mL of cold water to each sample.
  5. Begin to heat another 500 mL of water in the 2-L beaker on the hot plate.
  6. Label ten beakers as follows:
    2 – canned pineapple
    2 – sun-dried pineapple
    2 – fresh pineapple
    1 – fresh pineapple heated 0.5 min.
    1 – fresh pineapple heated 1.0 min.
    1 – fresh pineapple heated 1.5 min.
    1 – fresh pineapple heated 2.0 min.
  7. Place appropriate pieces of pineapple into each of the first six beakers of gelatin. At the same time, put a 1-L beaker containing the remainder of the fresh pineapple in the beaker of boiling water on the hot plate and start to time immediately. At the appropriate times, remove a piece of the pineapple and put it in the corresponding beaker. (So you are heating up samples of fresh pineapple for 0.5 up to 2 minutes)
  8. Place all the samples in a cool place. A refrigerator will hasten the jelling process and make it easier for you to distinguish between samples that have jelled and those that have not. Observe the samples the next day.

Discard the remains of the pineapple and the gelatin in the locally approved manner for food wastes.

Caution should be used in preparing the pineapple pieces and in heating and transferring both the water and the pineapple. Students should not eat the samples unless equipment used only for food preparation has been used throughout the experiment and the samples have been chilled in a refrigerator used solely for food.


A Different Experiment: If you need numeric results for your experiment, you can modify the above procedure as follows. In this new procedure you first make the gels and let them harden in a cold place; then you place different pieces of different pineapple on the gel and see what happens. In this way you can separate the liquefied portion of the gel and measure the percentage of the gel that liquified.

Procedure:

  1. Cut each slice of drained canned pineapple into four approximately equal pieces. Peel and cut the fresh pineapple into slices and pieces which are about the same size as the canned pineapple pieces. Cut the dried pineapple into pieces the same size as the others. (So now you have identical pieces of fresh, canned and dried pineapple for your experiment. You also need samples that are heated for certain amount of time)
  2. Prepare samples of pineapple that are heated 30 seconds, 60 seconds, 90 seconds and 120 seconds.
  3. Make 10 identical beakers (or cups) of gel. Let them solidify in a refrigerator. Label the ten beakers as follows:
    2 – canned pineapple
    2 – sun-dried pineapple
    2 – fresh pineapple
    1 – fresh pineapple heated 0.5 min.
    1 – fresh pineapple heated 1.0 min.
    1 – fresh pineapple heated 1.5 min.
    1 – fresh pineapple heated 2.0 min.
  4. Place appropriate pieces of pineapple into each of the first six beakers of gelatin.
  5. Place all the samples in a cool place. Make hourly observations until some of the gels liquefy.
  6. Separate and weight the liquefied portion.

Record your results in a table like this:

 

Type of Pineapple Liquefied portion
Canned pineapple 1
Canned pineapple 2
Sun-dried pineapple 1
Sun-dried pineapple 2
Fresh pineapple 1
Fresh pineapple 2
fresh pineapple heated 0.5 min.
fresh pineapple heated 1.0 min.
fresh pineapple heated 1.5 min.
fresh pineapple heated 2.0 min.

You can later use your results table to draw a bar chart. Each bar represents one type of pine apple. The height of the bar represents the activity of enzymes.

Need a control?

You must make sure that gels are liquefied by enzymes, not by an unknown or outside environmental factor. For this reason you may keep another cup of gel and put no pine apple on it. That will be called your control experiment. If gel in the control cup melts, then a warm room or any other factor such as bacteria on the air may have caused it. In this case you will need to repeat your experiments.

Note 2: The activity of enzymes can also be tested by hydrogen peroxide.

 

Materials and Equipment:

Chemicals

gelatin – one 6 oz box of Jell-O Brand gelatin dessert will be enough for 10 samples
canned pineapple slices
fresh pineapple*
sun-dried pineapple rings*

Equipment

150-mL or 250-mL beakers*
stirring rods*
hot plates
slotted spoon or tongs
2-L beaker*
1-L beaker*
100-mL graduated cylinder
balance

*Modifications/Substitutions:

  1. Frozen pineapple may be substituted for the fresh pineapple if fresh is not available. Freezing does not denature the enzyme.
  2. Sun-dried pineapple is available at some grocery stores and at health food stores.
  3. If samples are to be eaten following the experiment, equipment that has never been used for chemical experimentation should be used. The following equipment could be substituted for normal labware: clear plastic cups, Popsicle sticks, 2-qt saucepan, 1-qt jar, and measuring cup.

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.

Calculations:

No calculations is required for this project.

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.

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.

You could also study the effects of pH, detergents, and microwave radiation as a means of denaturing the enzymes. Perhaps fresh pineapple could be allowed to ferment to see if the fermentation process altered the enzymes’ function.

Possible Errors:

If for any reason the results of your experiment was totally unexpected, send a message to your project advisor to discuss possible errors.

References:

Hechtlinger, A., Biochemistry Units for the High School Biology Teacher, Parker Publishing Co., Inc., West Nyack, NY, 1973, p. 93.
This reference mentions, very briefly, the possibilities of using pineapple to study enzymes.

Lehninger, A.L., Principles of Biochemistry, Worth Publishers, Inc., New York, 1982, p. 158.
This reference describes, in some detail, the chemical make-up of enzymes and their functions. Any good, college level biochemistry text would be an applicable substitute.