Abstract

Objective

The goal of this project is to monitor enzyme activity by measuring the amount of apple juice released by pectinase.

Introduction

Enzymes are the workhorses of biochemistry. Enzymes are proteins that catalyze (speed up) specific chemical reactions—increasing reaction rates by factors of at least a million.

Pectinase is an enzyme that catalyzes the breakdown of pectin, a component of the cell wall in fruits such as apples and oranges. Pectinase is used commercially to aid in extracting juice from fruit. By enzymatically breaking down the cell wall, pectinase releases the juice from within the cells. Pectinase is also used for clarifying the extracted juice.

In this project, you can apply pectinase to fruit under controlled experimental conditions in order to investigate the enzyme activity of pectinase. You can monitor the enzyme activity by measuring how much juice is produced under the various experimental conditions. Some possible conditions to investigate are duration of enzyme treatment, enzyme concentration, and temperature.

Terms, Concepts, and Questions to Start Background Research

To do this project, you should do research that enables you to understand the following terms and concepts:

• pectin,
• plant cell wall,
• enzymes,
• substrate,
• pectinase,
• cellulase,
• amylase.

Questions

• What is the source of pectinase enzyme?
• How does pectinase promote the release of juice from apples?
• What are the substrates for the enzymes cellulase and amylase? Do you think that these enzymes would increase juice production?

Bibliography

• NCBE, 2006. "In a Jam and Out of Juice," National Centre for Biotechnology Education, University of Reading, U.K. [accessed July 7, 2006] http://www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/INAJAM/PDF/JAM03.pdf.
• Wikipedia contributors, 2006a. "Cell Wall," Wikipedia, The Free Encyclopedia. [accessed July 7, 2006] http://en.wikipedia.org/w/index.php?title=Cell_wall&oldid=61797174.
• Wikipedia contributors, 2006b. "Enzyme," Wikipedia, The Free Encyclopedia. [accessed July 7, 2006] http://en.wikipedia.org/w/index.php?title=Enzyme&oldid=62644117.
• Wikipedia contributors, 2006c. "Pectin," Wikipedia, The Free Encyclopedia. [accessed July 7, 2006] http://en.wikipedia.org/w/index.php?title=Pectin&oldid=61390030.

Materials and Equipment

To do this experiment you will need the following materials and equipment:

• apples,
• sharp knife for cutting apples,
• balance for weighing out apple pieces, such as the Fast Weigh MS-500-BLK Digital Pocket Scale, 500 by 0.1 G available at Amazon.com
• pectinase (available online from biological supply companies such as Carolina Biological and Fisher Scientific; you may also be able to find it locally at a store with home winemaking supplies),
• paper coffee filters,
• plastic wrap,
• disposable plastic spoons for stirring,
• two 1 mL syringes or pipets,
• two small funnels,
• two 100 mL graduated cylinders,
• two 100 mL beaker (or clean glass jars),
• water bath (can be as simple as a styrofoam box or insulated chest, big enough to hold the beakers),
• distilled water,
• timer or clock.

Experimental Procedure

Safety Note. Do not drink or cook with the juice produced in this experiment. The concentration of pectinase used will be much higher than is used in commercial juice production, and the fruit and enzyme have not been handled aseptically.

1. Chop the apples into cubes that are roughly 5 mm on a side. (Use care with the knife!) It is important to chop the apple into very small pieces—added surface area helps the enzyme break down the pectin in the plant cell walls, releasing more juice.
2. Use the balance to weigh equal amounts of chopped apple (about 50 g) into each beaker.
3. Prepare the pectinase enzyme according to the manufacturer's instructions.
4. Add 4 mL of diluted enzyme to one beaker, and add 4 mL of distilled water to the other beaker. Label the beakers ("pectinase" and "water").
5. Stir the chopped apple pieces in each beaker with a separate plastic spoon. Be sure to wet all of the pieces. Cover the beakers with plastic wrap.
6. Put both beakers into a 40°C water bath for 25–30 minutes. Your water bath can be as simple as a styrofoam box or insulated cooler, with water at 40°C. The water should come up to the level of the chopped apples, but you don't want so much water that the beakers float and tip over. After removing jars from water bath, you may want to use a wooden spoon to gently stir/squeeze the apple pieces in each.
7. Use the paper coffee filters in funnels to filter the juice from the apple preparations into 100 mL graduated cylinders (see Figure 1). Again, label the cylinders so you can keep track of the contents. Record the amount of juice in each cylinder at 5 min intervals.

   Figure 1. Use paper coffee filters in funnels to filter the juice from the apple pieces. Collect the juice in graduated cylinders, recording the amount collected at 5 min intervals (NCBE, 2006).

8. Make a graph showing the total volume of juice (mL) produced by each treatment vs. the time (hours).

Variations

The basic procedure above can be adapted for many different investigations. Here are a few possibilities, you can probably think of others yourself:

• What is the effect of varying the enzyme concentration? Use 2- or 3-fold serial dilutions of a concentrated enzyme solution to find out.
• You could also vary the duration of the enzyme incubation in the water bath, or the temperature of the water bath.
• Try the experiment with different types of apples. Do some apples produce more juice than others? Do background research to find out why. Is it the age of the apples, how they are stored prior to juice extraction, or some other factor?
• What happens if you add another enzyme, like cellulase or amylase (either alone or in combination with pectinase)? Are the results the same for all varieties of apples?
• Apples aren't the only fruit that has high levels of pectin! Try this experiment on some other pectin-rich fruits and vegetables: apricots, guavas, plums, citrus fruits (particularly the peels), and carrots.
• For a more advanced Science Buddies project on enzymes, see: Enzyme-Catalyzed Reactions -- What Affects Their Rates?.

• For more science project ideas in this area of science, see Biotechnology Techniques Project Ideas.

Credits

Andrew Olson, Ph.D., Science Buddies

Last edit date: 2011-11-15 09:00:00