Expt 057 -- Enzyme Activity

Extracts of fresh fruits contain enzymes. Some contain enzymes that prevent the gelling of gelatin. A study of extracts on the formation of a gel from unflavored gelatin is undertaken.

Background

• Gelatin is a protein obtained from the partial hydrolysis of collagen -- a structural protein found in tough, fibrous tissues such as animal hooves. As solutions of gelatin cool, the protein chains become entangled, trap and adsorb water, and form a gel.
• Proteases are enzymes that catalyze the reaction of water with the peptide bonds that hold amino acids together in protein polymers. Proteins are required for the digestion of proteins.
• Some plants, especially in their fruits, have high concentrations of protease enzymes. A protease catalyzes the hydrolysis of the gelatin protein, degrades the long chains which entangle to form a gel, and thereby prevents gelling.

Safety

Wear goggles and apron. Burns are possible. Handle the gelatin solution with caution.

Procedure

1. Use calibrated plastic delivery pipettes to measure volumes of solutions. Fill four wells in a 24-well plate with the following:
   • 0.5 mL buffer + 0.5 mL apple extract
   • 0.5 mL buffer + 0.5 mL kiwi extract
   • 0.5 mL buffer + 0.5 mL pineapple extract
   • 1.0 mL buffer
2. Cool these mixtures on ice water or in a refrigerator.

   !!!Click here to See Picture.

3. While cooling, add 1.0 mL of gelatin solution to each of the four wells. Try to add them as quickly as possible.

   !!!Click here to See Movie.

4. Stir all wells as quickly as possible, so they will all begin to gel at the same time.

   !!!Click here to See Movie.

5. Stir the mixtures periodically with half a toothpick. See if the toothpick can stand up in the well.

   !!!Click here to See Movie.

6. Gelling should begin in 10-15 min if the plate is kept in ice water.

   !!!Click here to See Movie. Apple(left) and Kiwi juice are shown in this movie.

   !!!Click here to See Movie. Pineapple juice(left) and buffer are shown in this movie.

7. Note and record which solutions gel first, and which do not gel at all.

   !!!Click here to See Picture.

Questions

1. Define the terms catalyst and enzyme.
2. Describe the role played by the enzyme in gelling.
3. Predict what will happen if the fruit extract is boiled before it is added to the well.

Handout

Name ___________________________ Class ________

Teacher__________________________

SmallScale 057 Enzyme Activity

Makeup students should watch the movies and record observations for the experiments.

Juice Observations

Curriculum-

This topic fits when catalysts, enzymes, or biochemistry are discussed. The experiment works well for applied chemistry classes. A variety of extensions are possible -- controlling temperature, pH, etc.

Safety-

Wear goggles and apron. Burns are possible. Handle the gelatin solution with caution.

Time-

Teacher Preparation: 20 minutes

Class Time: 40 minutes

Materials-

• 4 mL of 0.1 M phosphate buffer pH 7 (Mix 100 mL of 0.2 M Na₂HPO₄( 5.361 g of Na₂HPO₄•7H₂O in 100 mL) with 100 mL of 0.2 M NaH₂PO₄(1.379 g of NaH₂PO₄•H₂O in 100 mL).)
• 5 mL of Gelatin -- Add 2 g of gelatin to 35 mL of nearly boiling water using the markings in the beaker. Keep gelatin solution in hot water bath so that it does not set before it is added to the fruit juices. (This gelatin mixture is more concentrated than a typical recipe mixture.)
• 1 mL each juice extracts: grind approximately 10 g of fresh fruit with a mortar and pestle; add about 10 mL of buffer solution.
• Place a cotton plug in a funnel;filter the mixture through the cotton plug (or cheese cloth).
• Fresh pineapple is an excellent fruit to test. Fresh pineapple is never used in gelatin deserts, only canned pineapple.
• toothpicks
• 24-well plate
• cotton plug

Disposal-

The gels may be discarded with ordinary solid trash. Liquids used in this experiment may be discarded safely at the sink.

Answers-

Q1. Define the terms catalyst and enzyme.

A1. A catalyst speeds the rate of a chemical reaction.

Q2. Describe the role played by the enzyme in gelling.

A2. The enzyme plays no role in gelling. Instead, it degrades the long protein polymer molecules that are required for gelling to occur.

Q3. Predict what will happen if the fruit extract is boiled before it is added to the well.

A3. Proteins are used inactivated (denature) by boiling. If the fruit extract is boiled, it is likely that the proteases will be destroyed, and that gelling can occur. The proteases are more sensitive to heat than gelatin.

CoopLearn-

Students can be asked to bring fresh fruits from home. Different fruits can be compared.

Key Words 1-

enzyme, catalyst, protease, protein, gel, hydrolysis, buffer, pH

Elements-

C