The use of pectinase in fruit juice production
Introduction
Pectinase is an enzyme which catalyses the breakdown of pectin in the cellulose cell wall in the plant cells. Pectin is a sticky polysaccharide which is embedded in the cell wall of the plant cells to hold cellulose together in place. It is the structural carbohydrate which gives the plant cell mechanical support and prevents the cell from bursting when water enters into the cell by osmosis. It is also insoluble due its structure and arrangement of its monomers which are simple sugars. The presence of pectin in the cellulose cell wall prevents the cell contents from leaving the cell. This minimises the yield of the fruit juice when the manufactures try to extract juice from fruit.
Pectinase, like many enzymes, is a globular protein and its structure is maintained by hydrogen bonds, disulphide bridges and the ionic bond. Enzyme has a specific site called the active-site where substrate molecules bind to form enzyme-substrate complex. The shape of the active-site is so specific that only substrate which has a complementary shape can fit in. This is called lock and key mechanism and suggests that one enzyme can only catalyse one reaction. However, enzyme slightly changes its shape when substrate binds to its active- site. This process is known as an induced fit. Enzyme speeds up the rate of reaction by lowering the activation energy. So more enzyme-substrate complexes are formed per unit time and thus the rate of reaction increased.
In this experiment, we are looking at the effect of pectinase in apple juice production.
Hypothesis
The apple sauce mixed with pectinase will yield greater volume of apple juice than the one which is not mixed with pectinase.
Reason
Pectinase hydrolyses the pectin into soluble sugar and glutaronic acid. During the reaction, pectin binds to the active-site of pectinase and forms enzyme-substrate complex. This formation leads to the break down of cellulose cell wall and therefore the cell contents are released out of the cells. So with the presence of pectinase, the yield of the fruit juice is increased. This also increases the sweetness of the juice because soluble sugars are produced at the end of the reaction.
Method
- The experiment was carried out according to the procedure.
- Two test tubes of the same mass of apple sauce were prepared. One test tube contained 1 cm3 of pectinase and 1cm3 of buffer solution of pH 5.5. The other test tube did not contain pectinase but 1 cm3 of the same buffer solution. The contents of this test tube acted as a control because it indicated that buffer solution has no effect on the apple sauce on its own which in turn showed that pectinase is the one which is responsible for the greater volume of apple juice produced. The test tube which did not contain the pectinase would produce very little or no apple juice.
- Enzymes are sensitive to the change in temperature. At very low temperature, the kinetic energy of molecules will be so small that the rate of reaction will be very slow. At very high temperature, the heat energy supplied will starts breaking the bonds holding the tertiary structure of the enzyme. Finally, the enzymes will loose their specificity and become denatured and can no longer catalyse the reaction. So it is important to keep the temperature as constant as possible at the optimum temperature. It is the temperature at which enzymes work most efficiently that is the rate of reaction is the fastest. In this experiment, the temperature was kept at 20°C (room temperature) which is assumed to be the optimum temperature for pectinase.
- The pH of the solution is also need to be constant at the optimum pH for pectinase. Change in pH alters the ionisation of R-groups in amino acid in enzymes. At extreme pH, the bonds holding the tertiary structure will starts to break and so will the shape of the active-site. So the enzyme is said to be denatured because it can no longer bind with substrate molecules to form enzyme-substrate complex. In this experiment, pH was kept constant by using buffer solution which is pH 5.5. This is assumed to be the optimum pH for pectinase, pH at which pectinase works best and the rate of reaction is the fastest.
- The concentrations of enzyme and substrate also affect the rate of enzyme controlled reaction. These also need to be kept under controlled. Increase in concentration of enzyme or substrate will increase the rate of the reaction and vice versa. By using the same mass for substrate (apple sauce) throughout the experiment, the variation was controlled.
- The mixtures in the both test tubes were allowed to stand for 5 minutes before pouring the contents in each tube into separate measuring cylinders. This was done to allow the pectinase to breakdown pectin in cellulose cell wall and so the juice is releasesd. This was also one of the variables which were kept under controlled to give a fair experiment.
- The volume of apple juice was measured every minute from both mixtures after being transferred to filter funnels in two graduated measuring cylinders. The time interval was the same for both mixtures so that the volume of apple juice produced from two mixtures can be compared.
- Different pipettes were used for pectinase and buffer solution to avoid the contamination for both test tubes.
