Enzymes are proteins, which act as the acceleration devices of biochemistry. This means that they are used to catalyze (speed up) reactions. Enzymes are specifically designed for a target substrate, according to their size, shape and chemical charges that precisely bond to its substrate molecule. The section of the enzyme that binds to the substrate is called the active site and an advantage of enzymes being so specific is that they will not interact with or disturb other components present during the reaction.
The diagram shows the reaction between pectinase and pectin and also how they are specific to each other. The enzyme that I will be using during my investigation is pectinase, which is the specific enzyme used in the breakdown of pectin, a polysaccharide substrate found in the cell walls of many plants. In plants the pectin naturally functions as a glue to hold the cell wall together. Pectins are large polysaccharide molecules, similar to starch except that the repeating unit (residue) of pectin is galacturonic acid instead of the glucose in starch.
Moreover, pectins are made up mostly of chains of several hundred galacturonic acid residues. This causes fruit cell walls to have a complex structure of interwoven polymers, which no single enzyme can fully break down. The complete breakdown of a cell wall would most likely need a complex cocktail of carbohydrase enzymes. Commercial pectinase is usually a mixture of enzymes such as polygalacturonases and pectin lyases. Furthermore, pectins are known to be very good at binding water, so, for example in jam or jelly, pectin is what makes it set.
In the structure of the cell, the middle lamella, the part between neighbouring cells is rich in pectin, this means that pectin also helps to glue cells together. Pectinases are obtained commercially from fungi, particularly Aspergillus. Pectinase is in a particular class of enzymes and is known as a hydrolase, these are enzymes that are present when catalyzing reactions between water and a substrate. During a process like this water is bonded to certain molecules and molecules are also broken up into smaller units (as shown in diagram).
Simply, pectin, along with cellulose and proteins, is found in the cell walls of such fruits as apples and oranges and the enzyme pectinase breaks down pectin. So it makes sense to say that pectinase breaks down plant cell walls, which, evidently produces more fruit juice, this way, they are widely used in the fruit juice industry, where they are commonly used to help extract, clarify and modify fruit juices. The commercial uses of pectinases include the clarification of wines/juices and aiding the disintegration of fruit pulps.
This is done by reducing the large pectin molecules into smaller units and the eventually into galacturonic acid, here, a compound has been made soluble in water. Factors affecting the enzyme pectinase Changes in temperature can affect pectinase; this is because all molecules are in motion, except when at absolute zero. For example, as the temperature increases, the kinetic energy of the molecules has been increased. So, in the case of enzyme catalyzed reactions, the speed of both the enzyme and substrate molecules have been increased.
As this happens, the chances of collisions forming enzyme-substrate complexes also increases, this an increased rate of reaction, which would result in more pectin being catalysed in a given time, therefore leading to an increased rate of juice production. However, the temperature can only increase up to a certain point called the optimum temperature. For most enzymes, this is generally below 50i?? C and around 35i?? C, as the graph shows. Beyond this point, the rate of reaction begins to decrease.
This is because an increasing temperature causes more and more hydrogen bonds to break, which changes the shape of enzyme molecules. Moreover, after an enzyme has passed its optimum temperature, it has been denatured and can never work as if new again as fewer enzyme molecules can fit with the substrate molecules at their active site. The reaction continues to decrease until the entire enzyme is denatured and the reactions come to an end. Variations in pH will also affect the activity of pectinase. Just like enzymes have an optimum temperature, they also have an optimum pH.
However, this value is different for each enzyme. For example, gastric acid in the stomach, where the conditions are clearly acidic, and the optimum pH level will be low, at pH2. In the case of pectinase, which I will be using during my experiment, the optimal activity of this enzyme is at pH5. 5 (as shown in the graph). At low pH levels pectinase becomes inactive but this is a not a permanent problem such as high temperature, since the enzyme can be restored back to its optimum pH where normal activity will resume.
However, an extreme pH may cause an irreversible alteration to the enzyme. This may be a change in its protein structure, causing binding problems between substrate and enzyme. This graph shows that as the enzyme concentration increases, the rate of reaction also increases, so long as there is an excess of substrate, at constant concentration, available in order for the enzyme to continue working. This is a linear or directly proportional relationship. They both increase because of the greater presence of active sites that can catalyze substrates, thus causing an increase in activity.
Increasing the substrate concentration will increase the rate of reaction to a certain extent, as long as the concentration of the enzyme remains constant. This is because the more pectin molecules there are, the more enzyme-substrate complexes formed, which therefore, leads to more juice being produced. It is to a certain extent because at some point, increasing the substrate concentration will not affect the rate of reaction because the substrate molecules will have occupied all active sites within the enzyme; this point is labelled as Vmax on the graph. Collision Theory
Whenever I have mentioned that an increase in kinetic energy leads to an increase in the rate of reaction, the collision theory has been the basis of my idea. The collision theory states that chemical reactions are the result of reactants colliding with each other, with enough energy to form a chemical bond. When these bonds are formed, they are known as successful collisions. The energy in question is caused by different factors, such as, temperature or concentration. This energy causes molecules to move faster than usual (increased kinetic energy) and also brings a higher chance of collision.
Activation Energy Activation energy is defined as the energy that must be overcome or the minimum energy needed in order for a chemical reaction to occur. As I have previously explained, energy is needed for collisions to happen. Without a catalyst, there is only a small portion of particles with sufficient energy to react. Adding an enzyme does not actually lower the activation energy but, instead would provide an alternative route for the reaction to occur. This alternative route has a lower activation energy. So, many more particles can then react by taking this route.
Therefore, in this experiment, pectinase would decrease the time taken to break down pectin in the cell walls of the apple pulp. Aim This practical offers a number of possibilities for individual investigations, for example, I can study the effects of the enzyme’s concentration, the type of apple to be used or even the type of fruit altogether. I have chosen to use apple as my fruit and also to focus on the temperature of the reaction. So, the aim of my investigation is: To investigate the effects of the enzyme pectinase, more specifically the volume of juice produced at different temperatures, when acting on apple pulp.