Apple Pectinase free essay sample

The aim of this experiment is to see the effect of different Pectinase concentrations have on the production on apple juice. Pectinase is an enzyme, which breaks down pectin, a polysaccharide found in plant cell walls. This enzyme is mainly commercially used to speed up the process of fruit juice production as the cell walls of plants are broken down more quickly. Therefore by changing the Pectinase concentrations, the results may show the effects it may have on how much apple juice will be produced. Hypothesis The apple pulp mixed with any of the pectinase concentrations will yield greater volume of apple juice than the one which is not mixed with pectinase. Also as the concentration of the Pectinase increases in concentration, there will be more apple juice produced. However, after a certain Pectinase concentration, the volume of apple juice produced would not be proportional to the increase in enzyme concentration. This is because there is an excess of active sites in the Pectinase for the pectin in the cell walls of the apple to react with and therefore the apple juice produced will not increase further. The enzymes have specific shapes so that they can catalyse reactions. Every enzyme has an active site, which is the part where it joins to its substrate to catalyse the reaction. If the substrate doesn’t match its active site the reaction wont catalyse. This is called the lock and key mechanism. If, however, there are more active sites than substrate then no matter how high the concentration is it wont affect the production of apple juice. Since I am only varying the concentration of the enzyme (pectinase) I will have to make sure that all other variables are maintained (e. . Temperature, PH and the amount of apple pulp). This is because enzymes are affected by temperature, pH and concentration. Equipment| Why I am using this equipment| Apple pulp| I am using apple pulp as an example of a fruit to determine the maximum amount of juice that can be extracted using different concentrations of the enzyme pectinase. I will be using 20g of apple pulp for each percentage concentration of pectinase solution| Pectinase solution| I am using the enzyme pectinase to break down the pectin in the cell wall so that the fruit juice can be extracted. I will be using 5 different percentage concentrations of pectinase solution (0%, 25%, 50%, 75%, 100%) to see which concentration extracts the maximum amount of apple juice. | Filter paper| I am using filter paper in the experiment to filter the apple juice into the measuring cylinder, separating it from the apple pulp. | Glass rod| I am using a glass rod in order to stir the apple pulp and pectinase solution together in a beaker so that the pectinase is evenly mixed throughout the apple pulp. Beakers| I am using beakers into which I will put the apple pulp and pectinase solution so that effective stirring can take place. | Measuring cylinder| I am using measuring cylinders so that the apple juice extracted using the pectinase can be measured accurately volumetrically. Therefore, I can then measure how many milliliters of apple juice have been produced from the different percentage concentrations of pectinase. | Filter funnel| I am using a filter funnel so that I can hold the filter p aper containing the mixture of apple pulp and pectinase solution. The filter funnel also fits well in the measuring cylinder. Syringe | I am using a syringe to measure accurately the correct volume of pectinase solution (5cm3) to be added to the apple pulp at different concentration of pectinase enzyme. The concentrations are: 0%, 25%, 50%, 75% and 100%. | Thermometer| I am using a thermometer to monitor the temperature of the apple pulp and pectinase mixture and noting down any changes in temperature. | Stopwatch| I am using a stopwatch to ensure that when the pectinase is added to the apple pulp it is only incubated for precisely 10 minutes for each of the pectinase concentrations. After 10 minutes the mixture is poured into the filter paper and the stopwatch is restarted and stopped at 5 minutes to see what volume of apple juice is has been delivered in 5 minutes into the measuring cylinder for each of the pectinase concentrations used. | Controls Control| How it will be controlled and why? | Temperature| Temperature increases enzyme activity by increasing the  kinetic energy and reduces the activation energy for enzyme catalysis. At extreme temperatures (40oC and above) the enzyme activity decreases and stops due to enzyme  denaturation. This results in the irreversible change in shape of the enzyme active site and the substrate can no longer fit into the active site of the enzyme But, generally higher temperature tends to increase enzyme activity. Therefore, I have to  ensure that the temperature remains constant for each experiment.. To ensure this is  in system for each experiment, a room temperature will be dictated for each experiment. Then, with the use of the  thermometer, the solutions, before, during and after experimentation, will have their  temperature measured to ensure a fair experiment. | Volume of pectinase| In the experiment I will be keeping the volume of pectinase solution the same. I will do this by using a syringe, which will accurately measure out 5cm3 for each of the different percentage concentrations of pectinase solutions. If different volumes of pectinase solutions are used then the overall concentration of pectinase in the apple pulp will change and will affect the results. Therefore it is important to have the same volume of pectinase for the different concentrations of pectinase used| PH| The pH is an important factor to take into account. This is because all  enzymes have optimum pH for maximum enzyme catalysis. Above and below the optimum pH enzyme catalysis decreases . Therefore, it is important to have the same pH for all the experiments. I will check the pH of all the pectinase enzyme concentrations before each experiment commences so a fair experiment is conducted. | Concentration of pectinase| The concentration of pectinase is the dependent variable in this experiment. I will use the different percentages of pectinase solutions so that I can see from my results which percentage produces the most amount of apple juice. I will be using 5 different percentages of pectinase, which are 0%, 25%, 50%, 75% and 100%. | Apple age| It is important that the apple pulp used comes from the same stock. This is because if for some experiments the apple pulp was from young unripe apples (little juice synthesized by plant) then little juice may be extracted even after adding pectinase than from ripe apple where more juice as been synthesised by the plant. Therefore, apples which are the same age and from the same stock should be used in all experiments. | Type of apple| The type of apple must be the same because some apples may be naturally juicer than other apples. Therefore it may produce more apple juice than another apple affecting the results of the experiment. We are trying to see how the concentration of pectinase affects the amount of apple juice produced and not the type of apple. I will make sure the apple pulp has come from the same type so that the experiment is controlled. Time| The time must be accurately controlled because the longer the pectinase is in contact with the apple pulp more juice will be released until eventually no more juice can be liberated as all the pectin has been digested by the pectinase. Since we are investigating the rate at which the different enzyme concentrations affect juice extraction, the time of incubation of pectinase with the apple pulp must be constant for each of the experiments conducted. I am therefore using a stopwatch to ensure that when the pectinase is added to the apple pulp it is only incubated for precisely 10 minutes for each of the pectinase concentrations. After 10 minutes the mixture is poured into the filter paper and the stopwatch is restarted and stopped at 5 minutes to measure the volume of apple juice in the measuring cylinder If the timings are different for each of the repeats then this may affect the amount of apple juice liberated from the pulp and the volume collected in the measuring cylinder. | Risk| How it will be managed| Spillages| Any spillages should be notified to the teacher. Then it should be cleaned up so that there is no risk of someone slipping and causing an injury. Breakages| Any breakages should again be notified to the teacher so that the breakage can be cleared up and the any injuries are prevented. | Eating/drinking| In the lab no eating or drinking should take place because the food/water could get contaminated and affect the health of the person eating or drinking. Also the apple juice produced should not be drunk as it may still contain pectinase, which could also affect the health of the person drinking the juice. | Pectinase | The pectinase solution can be an irritant so if it has touched the skin or eyes make sure to wash thoroughly with water. Wear goggles to prevent splashes to the eyes occurring. | Overall Plan 1) Pour each of the 20g of apple pulp into 5 empty and clean beakers. 2) Use syringes to measure out 5cm3 of each of the different percentage concentrations of pectinase solutions into the 5 beakers containing apple pulp. 3) As soon as the pectinase solutions have been syringed into each of the beakers start the stopwatch and time for 10 minutes 4) As soon as the stopwatch has started use the stirring rod to mix the apple pulp and the pectinase solution together for 10 minutes. Stirring should be continuous to allow good mixing to occur. 5) Read the temperature of the mixture using thermometer and record it by placing the thermometer in the mixture for 30 seconds. 6) After the 10 minutes of vigorous stirring, stop the stopwatch and pour the mixture into the filter paper contained in the filter funnel which is resting on the measuring cylinder. Make sure that all of the mixture has been poured into the funnel. 7) As soon as the mixture has been poured onto the filter paper start the stopwatch and time for 5 minutes to allow the filtrate (apple juice) drip into the measuring cylinder. The results from both the primary and secondary are graphically represented in Figure 1 and shows the average volume of apple juice produced (cm3) in 10 minutes versus percentage pectinase concentration. From Figure 1 both the primary and secondary data clearly show that upon adding 25% pectinase solution to the apple pulp there is a large increase in the volume of apple juice produced. For the primary data volume increase is 6. 4cm3 (7. 7 – 1. 3) and for the secondary data 8cm3 (9. 5 – 1. 5). For the primary data subsequent increases in pectinase concentration do not yield anymore apple juice and show a slight decrease in the volume of apple juice production when compared to 25% concentration The results for 50%, 75% and 100% concentration of pectinase are anomalous as I would have expected some increase or an equal volume of apple juice produced when compared to 25% concentration pectinase, the graph should have plateaued out. However, the decrease in volume for 50%, 75% and 100% relative to 25% are very small and can be accounted for by experimental error as these experiments are conducted by 5 different groups in the class. The secondary data from Figure1 shows an initial similar trend to the primary data in that a large volume of apple juice produced when 25% concentration of pectinase solution was added relative to no pectinase (0%). The trend shows a gradual increase in apple juice volume as the concentration of pectinase increases if the anomalous result for 75% is ignored. This anomalous result at 75% could be accounted for by experimental error as the overall trend indicates increase in volume with increasing concentration. The increase in volume of apple juice by increasing the concentration from 5% to 100% is only 0. 8cm3. Therefore, increasing pectinase concentration from 25% to 100% only gives a very small volume increase in apple juice. Both the primary and secondary evidence show that the volume of apple juice produced increases greatly when 25% concentration pectinase is added to apple pulp. Accounting for experimental error the primary data shows no more volume of apple juice produced when increasing pectinase concentration. The secondary data shows a similar trend except that there is a very slight increase (0. cm3) in apple juice volume when pectinase concentration is increased fourfold from 25% to 100%. The difference in the two primary and secondary data is probably due to a combination of experimental error and experimental conditions employed during the experiment. Conclusion based on evidence My hypothesis for the above experiment was: a)The apple pulp mixed with any of the pectinase concentrations will yield greater volume of apple juice than the one which is not mixed with pectinase and b) as the concentration of the Pectinase increases in concentration, there will be more apple juice produced. However, after a certain Pectinase concentration, the volume of apple juice produced would not be proportional to the increase in enzyme concentration. The primary and secondary evidence both support part a) of my hypothesis. There is a large increase in volume of apple juice when pectinase is added to apple pulp. Pectinase aids in the extraction of juice from apples. It does this by breaking down and digesting pectin, which is found in the cellulose fibers of apples. Pectin is also found within the layers of the apple’s cell wall. The pectinase enzyme breaks down the pectin, causing each of the apple’s cells to release greater amounts of juice than just squeezing pulp. Part b) of my hypothesis is partially supported by the primary data collected in the classroom and wholly supported by the secondary data which was generated by the technician. The data can be explained by enzyme kinetics and substrate concentration. Pectinase is an enzyme which specifically breaks down pectin and therefore has specific shape that only fits pectin and the site where the reaction occurs on the pectinase is called the active site. Pectin specifically fits into the active site and is broken down. As with all enzymes the activity of an enzyme is dependent on factors such as pH, temperature and enzyme concentration and substrate concentration. Temperature was monitored during the reaction and was 20oC for all the experiments. The pH was not measured and will be discussed later. The concentration of the pectinase was the independent variable and the apple pulp concentration remained constant at 20g for all the pectinase concentrations. When an enzyme (pectinase) concentration is low then reaction rate will be low with the substrate molecules competing for the active site. As the concentration of the enzyme increases then more active sites are available for the substrate and the reaction rate will be high. If the enzyme concentration is increased even further then there would be more active sites than substrate then, the rate of reaction would not increase but plateau out and the limiting factor would be the substrate. In our experiment the graphs show that there was an initial high enzyme activity (large volume of juice produced) and then the activity plateaued out at higher pectinase concentrations for the primary results (taking experimental error in account). For the secondary data results were similar except that increasing enzyme concentration only increased enzyme activity slightly as seen by the small increase in volume in juice production. The results show that the substrate was the limiting factor as increasing the pectinase concentration meant there was not enough substrate and the rate could not be increased and this suggests that virtually all the pectin that could have been digested by pectinase to release the juice was achieved with the 25% concentration of pectinase. Evaluation of conclusion My conclusion is based on the primary and secondary data generated during this experiment and is supported by mechanisms of enzyme activity and factors affecting enzyme activity. The addition of 25% concentration pectinase to apple pulp increases apple juice production by a factor of approximately six when compared to no pectinase added for both primary and secondary data. Therefore, this large increase is due to the pectinase which is known to digest pectin in cell walls. As a result of pectin digestion in cell walls the cell wall integrity diminishes resulting in release of more juice. As the pectinase concentration increases increase in juice production relative to 25% is not significant and is supported by the secondary data. Primary data shows a small decrease in juice production beyond 25% concentration but this may be due to experimental error. The conclusion explains this by stating that the substrate (apple pulp) is the limiting factor and there are more enzyme active sites than substrate. If there was a further large increase in juice production at 50%, 75% and 100% then the conclusion could not explain the results in terms of substrate limitation and excess enzyme active sites. The collected evidence can be improved by conducting the experiment with more pectinase percentage concentrations between 0% and 25%. For example 5%, 10% 15% and 20% and also the time of experiment could be for different time intervals, for example 20 minutes and 30 minutes. The lower concentrations would show that juice production and hence enzyme activity is linear and proportional to concentration of enzyme and that the substrate is not the limiting factor. Enzyme catalysis follows zero order kinetics when substrate, pH and temperature are not limiting. Increasing the time would show that no more juice is produced when the enzyme is incubated for longer and that would support the conclusion that the substrate is exhausted and no more pectin can be digested by the enzyme. Evaluation of method The method is a very simple method and can produce results which can be interpreted. The strengths of this experiment relies on the availability of reagents for example apple pulp and enzyme and general laboratory equipment. The experiment can be easily performed and repeated. Also there are few variables which can skew results and therefore difficult to draw conclusions. The hypothesis can be supported by the results as only the enzyme concentration is changed and everything else is controlled. However, the weakness can influence the results for example temperature and pH must be maintained for all experiments as these can have a large impact on juice production and hence enzyme activity. Ineffective stirring can also affect the results as the pectinase may not have been mixed in properly and therefore unable to digest pectin effectively and this can lead to lower volume of juice being produced. The anomalies in the data points can be attributed to the above factors and this has reflected in the hypothesis where I expected to see a slight increase in juice production at the higher concentrations of pectinase and not a decrease in juice production relative to the 25% concentration. The secondary data supports my hypothesis but this experiment was conducted by the technician and not by the class. The methodology can be improved by firstly incubating the pectinase and apple pulp in a water bath a constant temperature for all experiments. The pH of the solution should be checked using a pH meter and pH adjusted using appropriate buffers. The stirring of the mixture should not be manual but mechanical using a stirrer therefore eliminating human error. This may have caused the reduction in juice production at 50%, 75% and 100% in the primary data and hence the data did not quite support the hypothesis. Each concentration data point should be repeated by the same individual as this would eliminate further errors. By controlling these variables more reliable results would be obtained with very few anomalies. Variation in pH and temperature can dramatically affect the results and can impact the hypothesis. For example, lower temperature would reduce enzyme activity and this would result in lower juice production at all concentrations of pectinase. This would impact the hypothesis in that I would not be able to conclude that the substrate was the limiting factor and vacant enzyme active sites. This would also be true for changes in pH.