Experiment to Investigate the effect of the size of Applie Pieces used on the Yield of Juice Produced by Apples Using the Enzyme Pectinase

 From my preliminary experiment I have therefore learnt that the enzyme pectinase has quite a significant effect on the yield of apple juice produced from the apple pieces. From doing my preliminary experiment, I have also acknowledged the fact that the measuring cylinders provided to do my experiment only have scales starting at 10ml3 and it was quite hard to work out exactly how much apple juice was being produced. In effect of realising this, I will add 10ml3 of water to each measuring cylinder before filtering the apple juice and this will make it much easier to work out exactly how much apple juice is being produced.

HYPOTHESIS

Pectins are a group of polysaccharides found in the matrix of plant cell walls. Pectin is linked to the cellulose in the plant cell wall to form protopectin (which has the ability to absorb large amounts of water by binding water by hydration). The main function of pectins is therefore to help keep the cell walls of plants intact by absorbing water and in effect the cell sap is retained. Due to this function and the chemical nature of pectins, they therefore limit the extraction of juice from the fruit pulp. However, when the pectins are degraded, their water binding capacity and their contribution to cell wall integrity are both eliminated, therefore more juice can be extracted from the fruit pulp.

Pectinase is an enzyme widely used in the fruit juice industry to help extract, clarify and modify fruit juices. The enzyme acts on the pectins and degrades them to shorter molecules of galacturonic acid, by breaking the link between the pectin and cellulose. By degrading the pectins, the plant cell wall is therefore softened which in effect helps increase the production of fruit juice.

Enzymes are catalysts, which are substances that speed up chemical reactions that would normally happen very slowly. Enzyme molecules have a complicated three-dimensional shape due to the particular way the amino acid chain that makes up the protein is folded. This  gives the enzyme its catalytic ability. A few of the amino acids on the surface of the molecule fold inwards to make a specific indentation, called the active site, into which a particular substrate can fit. Once the enzyme and the substrate are joined they form an enzyme-substrate complex. The formation of an enzyme-substrate complex makes it possible for substrate molecules to be brought together to form a product. The product is released and the enzyme is free again to take part in another reaction. Generally, there is only one active site on each enzyme molecule and only one type of substrate molecule will fit into it. This idea is known as the lock and key theory and is illustrated in the diagram below:

It has, however, been discovered that competitors for an active site (similar in shape to the substrate) could fit even though they are larger than the substrate. This means that the substrate and active site are a little flexible. It is believed that when the enzyme and substrate form a complex, structural changes occur so that the active site moulds around the substrate (the substrate induces the active site to change shape). The reaction will take place and the product, being a different shape to the substrate, moves away from the active site. The active site then returns to its original shape. This theory is known as the induced fit theory and is illustrated in the diagram below:

Reactions take place because the products have less energy than the substrates. However, most substrates require an input of energy to get the reaction going and this is called the activation energy. Enzymes work by lowering the activation energy required to proceed a reaction and therefore help increase the rate of reaction. There are, however, many factors which effect the efficiency of enzymes and therefore the rate of reaction.  The key factors are temperature, pH and concentration.

Temperature has an immense affect of the efficiency of enzymes. Enzymes have a unique optimum temperature (around 40oC) which they work best in. Below the optimum temperature, the molecules involved in the reaction will be provided with  less kinetic energy meaning that the numbers of collisions between enzyme and substrate will decrease, as the speed of molecular movement is decreased. Any temperatures above the optimum will cause the enzymes to be denatured - bonds holding the structure together will be broken and the active site loses its shape and will no longer work. Therefore during reactions involving enzymes, the temperature has to be greatly considered.

The pH condition in which an enzyme is made to work also has a great effect on the efficiency of the enzyme. As with temperature, enzymes have an optimum pH (usually a small range). If the pH changes much from the optimum range, the chemical nature of the amino acids can change. This may result in a change in the bonds and so the tertiary structure may break down, causing the active site to be disrupted and the enzyme to be denatured.

Concentration is another important factor effecting the efficiency of enzymes. Firstly the enzyme concentration has to be considered. At low enzyme concentration there is great competition for the active sites and therefore the rate of reaction is low. As the enzyme concentration increases, there are more active sites and the rate of reaction will increase. However, increasing the enzyme concentration beyond a certain point has no effect because the substrate concentration becomes the limiting factor. The substrate concentration has also got to be considered. At a low substrate concentration there are many active sites that are not occupied, therefore the rate of reaction is low. When more substrate molecules are added, more enzyme-substrate complexes can be formed and the rate of reaction increases. Eventually, there will be a point when increasing the substrate concentration further will have no effect as the active sites will be saturated so no more enzyme-substrate complexes can be formed.

As proven in my preliminary experiment, the enzyme pectinase significantly increases the amount of fruit juice produced from apple pieces. This, as explained above, is due to the fact that pectinase degrades the pectin, making the cell wall of the plant softer and therefore allowing the fruit juice to flow more easily. If I did an experiment investigating the effect of pectinase on different sized apple pieces (of the same shape), I predict that the smaller the apple pieces, the greater amount of apple juice produced. This is because the smaller the apple piece, the larger the surface area which would mean more binding sites of the substrate exposed to the enzymes.  If more binding site are exposed to the enzymes, there is more chance of enzyme-substrate complexes being formed (i.e. between the pectinase and pectin) and therefore the rate of reaction will increase which can be displayed by the fact that more fruit juice will be produced. My predictions are displayed on the graph below:

APPARATUS LIST

• Whole apples (of the same make)
• Cork barer
• Knife
• Ruler
• Pectinase enzyme (Novo Pectinex, 1cm3)
• Distilled water
• Glass stirring rods (x2)
• Coffee filter papers (x20)
• 1cm3 syringes (x2)
• Filter funnels
• 100cm3 measuring cylinders
• 100cm3 beakers
• Water bath set at 40oC
• Stop clock

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DIAGRAM

METHOD

• Using a cork barer, cut out as many cylindrical pieces from a medium-sized apple as possible.
• Cut each cylindrical apple length into 3mm long pieces, using a ruler to measure.
• Place half the apple pieces into a beaker (use a weighing scale to check that exactly half is used).
• Add 2cm3 of diluted pectinase enzyme to the beaker using a syringe
• Stir the contents of the beaker using a glass rod.
• The beaker should then be incubated in a water bath set at 40oCfor 20 minutes.
• Filter the juice from the apple pieces into a measuring cylinder originally with 10cm3 of distilled water in it (to make easier to take readings – explained in preliminary work) using a coffee filter paper in funnels.  
• Record the volume of juice obtained from the apple pieces at 5-minute intervals for 40 minutes (use a stop clock to measure the time).
• The experiment was repeated 2 more times using apple pieces of 3mm in length and then 3 experiments were carried out for the following apple sizes: mush (mashed up using a potato masher),  6mm, 9mm and 12mm. Also a control was carried out for each apple length using distilled water instead of the pectinase (separate glass rods and syringes were used to prevent contamination).

VARIABLES

There a 3 key variable to this experiment which are explained in detail in the hypothesis –temperature, pH and concentration. To make sure that the experiment is fair and reliable, these key variables must be controlled throughout the experiment. Apart from the three key variables above, there are also a few more things which could have been changed in the experiment that would have effected my results:

• The amount of  pectinase enzyme used. More enzyme would mean more active sites for the substrate to form enzyme-substrate complexes in and therefore a greater rate of reaction.
• The time the beakers containing the apple pieces and pectinase were left in the water bath. This would have effected the experiment because a longer time in the water bath would mean more kinetic energy provided to the molecules involved in the reaction and therefore the number of collisions between enzyme and substrate will increase.
• The apparatus
• The plan

RISK ASSESSMENT

        In this investigation, there are many areas where care must be taken. You have to be extremely careful when cutting the apple pieces into precise lengths with the knife as it is very sharp and could cause a serious cut. Do not wave the knife around because it could cut someone else or it could poke someone in the eye. Take care when handling the glass equipment (i.e. the beakers, measuring cylinders, funnels and glass rods) because if dropped, they will smash and sharp glass can also be very harmful. Also be careful when using the water bath as there are precautions to be taken with all electrical appliances. Care must also be taken when handling the pectinase. If in contact with the skin, wash immediately to prevent it from possibly entering the mouth. Finally, try not to spill any solution on the ground because someone could slip on it but if some liquid is spilt, wipe it up straight away.

FAIR TEST

• The three key variables (described above) must be controlled throughout the experiment because as explained in the hypothesis, they have a great effect on the efficiency of enzymes and if slightly altered, could therefore effect the pectinase. The temperature was controlled by putting each beaker in a water bath set at 40oC for 20 minutes before actually filtering the apple juice. The pH and concentration of everything were kept constant throughout the experiment.

• The same amount of apple pieces (i.e. same amount of apple juice) was put in each beaker and this was controlled by weighing exact amounts of apple pieces.
• The size of the apple pieces were measured accurately using a ruler to ensure that e.g. all apple pieces in the 3mm test were actually 3mm in length.

• Distilled water was used instead of tap water to ensure that no unwanted substances would interfere with the  control experiment i.e. distilled water is purer.

• Each apple length was repeated three times and an average was taken. If the three sets of results were marginally different, I would know that something had gone wrong in the experiment. Also, repeating each concentration gives me a wider set of results which would give me a much more accurate figure when averaged.
• The volume of the diluted pectinase enzyme/distilled water used was the same for each experiment.
• The diluted pectinase enzyme used was of the same concentration for all experiments.
• The stop clock was started as soon as all the apple pieces were in the filter funnels; therefore each experiment got the same time to produce the apple juice– 40 minutes.
• The readings on the measuring cylinder were noted down very carefully because it is quite easy to make a mistake when reading off such small scales. Also, the measuring cylinders originally contained 10cm3 of acid to make reading off the scale much more easy.
• A measuring cylinder with a large scale was used to measure out the amount of apple juice produced because it much more accurate.