The use of pectinase in fruit juice production

I predict that there will be no juice produced to the apple sauce that had no enzyme and juice will produce to the apple sauce that had pectinase. A control is carried out in order to compare the rate of reaction between with pectinase and without pectin’s.

Pectin → sugars + galataronic acid

Pectin is a substance which helps to hold pant cell walls together. As a fruit ripens the plant produces proteolytic enzymes, which convert the insoluble protpectin of the unripe fruit into more soluble forms, causing the fruit to soften. When fruits are mashed and pressed to form juices these more soluble forms of pectin enter the juice, making it cloudy and causing the colour and flavour to deteriorate.

Enzymes are specific in the reactions they catalyse, much more so than inorganic Catalysts. Normally, a given enzyme will Catalyse only one reaction, or type of reaction. The enzyme has an active site that helps it to recognise its substrate in a very specific way. Just like a key only fits into a specific lock, each enzyme has its own specific lock; each enzyme has its own specific substrate. This is called the lock and key theory. The enzymes never actually get consumed in the process; they just increase the rate of reactions.

When enzymes denature the heat starts to destroy their shape and structure. The shape of the enzyme is so important to its working that any change in the shape of the molecules will make them less effective or stop them working completely.

Variables

In order to create a fair test, I considered the variables that affect the activity of the enzyme, pectinase. Variables that were likely to disrupt the results were controlled, and the variables being investigated were varied accurately so that their effect could be measured precisely.

i) Temperature - As temperature increases, molecules move faster (kinetic theory). In an enzyme catalysed reaction, such as the digestion of pectinase into sugars and galataronic acid, this increases the rate at which the enzyme and substrate molecules meet and therefore the rate at which the products are formed. As the temperature continues to rise, however, the hydrogen and ionic bonds, which hold the enzyme molecules in shape, are broken. If the molecular structure is disrupted, the enzyme ceases to function as the active site no longer accommodates the substrate. The enzyme is denatured.

This experiment will be carrying out at room temperature, however to control this variable and vary it accurately, water baths were used to maintain the temperature of the enzyme at set intervals to obtain an accurate range of temperatures. This ensured that the temperatures of the enzyme and substrate molecules were kept constant so that results were more accurate.

pH - Any change in pH affects the ionic and hydrogen bonding in an enzyme and so alters it shape. Each enzyme has an optimum pH at which its active site best fits the substrate. Variation either side ...

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pH - Any change in pH affects the ionic and hydrogen bonding in an enzyme and so alters it shape. Each enzyme has an optimum pH at which its active site best fits the substrate. Variation either side of pH results in denaturation of the enzyme and a slower rate of reaction.
In this experiment, the pH should be kept constant using a pH 5.5 buffer, selected to maintain a pH level suited to the enzyme by being equal to the natural environment of the enzyme pectinase.
Substrate Concentration – mass of the apple sauce. When there is an excess of enzyme molecules, an increase in the substrate concentration, produces a corresponding increase in the rate of reaction. If there are sufficient substrate molecules to occupy all of the enzymes´ active sites, the rate of reaction is unaffected by further increases in substrate concentration as the enzymes are unable to break down the greater quantity of substrate, as the amount of enzyme is the limiting factor. At this point the graph tails off.
To control the substrate concentration, identical quantities of the substrate were used for each reading. Each measurement of substrate was taken from the same 2 decimals place balance and the apple sauce was weighed to exactly 20g. To ensure that this was measured precisely, a 4decimals palce balance should be use to increase the accuracy.

Method

Put 20g of apple sauce into each of two beakers.
To one add 1cm3 of buffer, pH5.5, and to the other add 1cm3 of the enzyme pectinase in buffer, pH5.5, although the volume of two solution would be different, however this is done to keep mass constant.
Mix carefully and leave to stand for 5 minutes.
Transfer the contents of the beakers to separate filter funnels and collect the juice produced in a graduated measuring cylinder.
Take reading of the juice collected.

Results

Table to shows apple juice in cm3 collected over time in minutes

Table to shows initial rate of the enzyme pectinase when breaking down pectin into sugars and galataronic acid

Analysis

The hypothesis is proved that the break down of pectin with pectinase was successful and there was no reaction with the apple sauce that exclusive of pectinase. The pectinase causes disulphide bonds to break. These bonds help to maintain the shape of the enzyme molecules. Once these bonds are broken, pectin turns into two simpler molecules – sugars and galataronic acid.

From the Table to shows apple juice in cm3 collected over time in minutes, a trend was observed that there was a rapid increase of volume at the first two minutes of the experiment, this suggests that the enzyme reacted straight away once it was added into the apple sauce. This is because there is plenty of substrate available. However the increase of volume has slowed down when the experiment was carried to the 6th minute. From an increase of 0.75 cm3 at the first two minutes. The volume has only increased 0.25 cm3 starting from the 6th minute. This indicates that there is limited substrate hence it takes longer for the enzyme to look for a ‘free’ substrate.

The properties of enzymes can explained in relation to the lock and key mechanism of enzyme action. The region that contact with the substrate is where the reaction is taken place and it is called the active site. The active site of an enzyme can be used again and again; therefore enzymes work efficiently at very low concentrations.

Provided that other conditions such as temperature and pH are suitable for the reaction, and provided there are excess substrate molecules, then the rate of reaction is directly dependent on the concentration of enzyme molecules. Similarly, if the amount of substrate is limited, then this will limit the rate of reaction. Thus the substrate concentration can also affect the rate of reaction. However, if the substrate is in excess, then a point will be reached in the reaction where all the active sites are being used and increasing the substrate concentration will have no effect on the rate of reaction.

If the solution is made more concentrated, it means there are ore active sites between the substrates which increases the rate of reaction.

There is no reaction with the solution that does not contain any pectinase, this is because the pectin will only break down when a related enzyme is added into the solution in order to break the bonds. Since there was no enzyme pectinase present, hence the volume in cm3 of apple juice produced was zero all the way through.

A line of best fit was drawn onto the graph. Overall, the graph formed a slight curve. As the gradient of the curve get steeper at the beginning of the experiment, this suggests that the rate of reaction was increasing rapidly. The rate of reaction at each minute was calculated and all the results are recorded in the results section of the write up. From the calculations, an overall trend was observed that the rate of reaction had a rapid increase then a sharp decrease following by gradually decreasing. There was a rapid increase from 0 to 0.75 cm3min-1 between 0 minute to the first minute intervals. However the rate of reaction starts to decrease from the second minute. There was a sharp decrease of 0.125 cm3 min-1 between minute 1 to minute 2 intervals. After that, there was a gradually decrease of roughly 0.05 cm3. A graph showing the rate of reaction of the enzyme pectinase when breaking down pectin into sugars and galataronic acid is also drawn onto the back of the graph which shows the volume of apple juice in cm3 over time in minutes.

Error bars provide a simple and clear way to represent the uncertainty of each point on a graph. Error bars are drawn onto the graph, which shows the volume of apple juice in cm3 over time in minutes. There were big error bars from minute 1 to minute 4 intervals ; they all have a range of 0.5cm3 within the average. Then the error bar was minimised to within a narrower range of 0.25cm3 at 4th and 8th minute intervals. There was no error bar at 5th, 6th and 7th minute intervals, which indicates that the experiment results were agreed by both of the groups. The bigger the error bars, indicates the more inaccurate of the results that were collated hence the results were unreliable and which would effect our prediction of the outcome of the experiment.

Evaluation

There were insufficient sets of results in order to make this experiment reliable since there were only two sets of results. To improve the accuracy of this experiment, repeats should be done.

The apple sauce that was prepared had a big impact on the experiment.