The Digestion of Pectin by the Enzyme Pectinase

Introduction

After cellulose, the polysaccharide found in plant cell walls, pectin is one of the most abundant carbohydrates on Earth. Pectin is a naturally occurring polysaccharide molecule found in all fruits. Pectin is found between the cellular "bricks" in the fruit and acts as inter-cellular "mortar" or "cement" holding the bricks tightly together in a gel-like suspension. Complete removal of this binding layer causes the fruit tissue to fall apart. This is what has happened to fruits or vegetables which have gone soft (or gotten rotten). During ripening, the structure of pectin is altered by naturally-occurring enzymes, such as pectinases, in the fruit. Pectin becomes more soluble and its grip on the surrounding cell walls is loosened so that the plant tissue softens.

Pectinases were some of the first enzymes to be used at home and they were utilized in the commercial preparation of wines and some fruit juices during the 1930s. The enzyme pectinase attacks the bonds of the pectin molecules causing them to lose their "glue-like" properties. This allows the fruit particles to settle out and release the juice. The resulting product is clear juice with few particles, which allows rapid separation of juice and pulp.

Enzymatic juice extraction from apples was introduced 30 years ago and today some 5 million tons of apples are processed into juice annually throughout the world. Scientists have perfected this process by determining the amount of enzyme, the treatment time and temperature required for optimal production of juice. In this experiment, you will be testing the effects of temperature on pectinase activity. You will be using pectinase to make apple juice from applesauce.

Method

1. The applesauce has been placed at three different temperatures: 4°C (refrigerator), 25°C (room temperature), and 37°C (incubator). Your instructor will assign one temperature to your group. You will perform the following procedures in duplicate for your temperature (i.e., you will be doing 2 trials). Because of the time required for the incubations, it will be best to set up your 2 trials simultaneously.

2. Set up a filtering apparatus by placing a funnel into a 100 mL graduated cylinder. Place filter paper into the funnel and wet the filter with tap water.
3. Measure 100 ml applesauce into a 250 mL beaker. For the 4°C applesauce, keep the beaker in an ice bath during the experiment. For the 37°C applesauce, keep the beaker in the incubator or in a 37°C water bath. The 25°C applesauce can be kept on the lab bench.
4. Add 1.0 mL pectinase to your applesauce and stir with a glass rod. Allow the pectinase and applesauce to incubate together for exactly 20 minutes, stirring once after 10 minutes of incubation.
5. After 20 minutes, filter the applesauce into the graduated cylinder through the pre-wetted filter paper/funnel set-up.

6. Allow the apple juice to drain into the graduated cylinder for exactly 10 minutes. (Stir the applesauce in the funnel CAREFULLY after 5 minutes.) Record the volume of apple juice collected after 10 minutes in the spaces below.

7. Before disposing of your apple juice, bring your graduated cylinders to the front of the room to compare the clarity of your juice with the clarity of apple juice produced at other temperatures. Record your observations.

Temperature: ________        Volume of juice produced - Trial 1:___________        Trial 2:                      

Table 1. Volume of Juice Produced by Pectinase on Applesauce at Different Temperatures.

Writing Assignment

Write a lab report including a title, abstract, results, discussion and conclusion.

The results should consist of  Table 1 and a graph which shows the average volume of apple juice produced as a function of the temperature.

For your discussion, you should consider the following questions.

1. What was the effect of the temperature on the activity of the pectinase enzyme? What volume of apple juice would you expect if the applesauce-pectinase mixture were at 100°C? 0°C?
2. What was the effect of temperature on the clarity of the apple juice produced? Make an inference about how temperature and enzyme activity might have an effect on the clarity of the juice.
3. How do the results compare between your group and the overall class average for your temperature? Explain any discrepancies.
4. Do the results of this exercise agree with your understanding of how enzymes function?

Your conclusion should be a simple statement about the effect of temperature on pectinase enzyme activity.

Lab Introduction and Method modified from "In a Jam and Out of Juice" (December 2000) www.ncbe.reading.ac.uk