HOW IS PECTINASE MADE INDUSTRIALLY?
This is done using Biotechnology; the micro organism is first grown in bulk in a fermenter, which is a large vessel in which the micro organisms are grown. Pectinase is made using batch fermentation and is secreted from the cells as an extra cellular enzyme, which can be purified more easily. The advantage of Pectinase compared with some other enzymes is the fact that it can be used after purification with no need to be immobilised.
PICTURE OF A FERMENTER (Picture taken from Reference 7 )
Microbes are preferred to plants and animals as sources of enzymes because:
- They are generally cheaper to produce.
- They have simple nutritional requirements.
- Their enzyme contents are more predictable and controllable.
- Reliable supplies of raw material of constant composition are more easily arranged.
- Plant and animal tissues contain more potentially harmful materials than microbes, including phenolic compounds (from plants), endogenous enzyme inhibitors and proteases.
- They have high growth rates.
- They can be grown economically in bulk in fermenters under controlled conditions.
- They produce a lot of extra cellular enzyme; because these leave the cell, they are easier to recover and purify.
- Production can be altered to suit demand.
- They can be genetically engineered relatively easily, and mutant varieties can be easily produced, to improve performance.
I am going to investigate the question: How does a Temperature change affect Pectinase?
Like most other enzymes, Pectinase has an optimum temperature of 35 degrees, at which it works best. Temperatures below and above this optimum temperature have considerable effects on the way enzymes work. At low temperatures, near of below freezing point, enzymes work at a very slow rate but as the temperature is increased to the optimum, the rate at which the enzyme works on pectin increases. This can be explained using kinetic theory.
Kinetic theory is the idea that when a substance is heated, its molecules are supplied with more kinetic energy and therefore move around faster. In this experiment, as the temperature increases, Pectinase works on breaking down pectin at a much faster rate.
At high temperatures, although the chance of pectin breakdown is increased, the chances of breaking down the three-dimensional structure of Pectinase are also increased. At temperatures just above the optimum, the enzyme temporarily loses some of its structure and then regains it. At high temperatures, the weak interactions that hold enzymes together, are shaken apart and results in the denaturation of the enzyme, at which the structure is lost and the enzyme becomes totally ineffective.
PREDICTION/ HYPOTHESIS
Using all the information I have gathered on Pectinase and based on my own knowledge of enzymes, I predict that the most amount of fruit juice is going to be produced at a temperature of between 30-40 degrees. I chose this temperature because this is the optimum temperature for the enzyme Pectinase, at which it works most effectively and it therefore would be able to breakdown pectin more effectively at this temperature.
I predict that as the temperature increases, the amount of juice produced will increase at a proportional rate until between 40-50 degrees where the amount of juice will start to decrease. I think that at a higher temperature, which I predict will be 70 degrees, the pectinase will have denatured to an extent that no fruit juice will be produced.
I predict my graph of results to look quite similar to the one shown below:
(GRAPH FROM REFERENCE 7 )
THE EXPERIMENT
The independent variable in this experiment will be temperature. This will be what I controlled during the experiment. The variable dependant on this will be the amount of fruit juice produced. All the other variables had to be kept constant to give a fair test. These controlled variables were concentration of pectinase, amount of Pectinase, pH and amount of fruit used. The same type of apparatus also has to be used throughout or this could arise in inaccurate results.
APPARATUS
A pilot study was carried out beforehand to determine what amount and concentration of Pectinase would work effectively on the fruit (Plum). Following this study, I decided to use 2 Cm3 of 2% Pectinase solution for the main experiment. The main experiment will be conducted at a temperature range of 0-80 degrees at 10 degrees intervals to obtain a sufficient number of dissimilar results.
PECTINASE- 60ml- I was given the chance to use ready-made Pectinase solution or to make my own Pectinase using Pectolase. I decided to make my own Pectinase solution because in my opinion, this would be fresher.
WATER BATHS- To control the temperature. I chose this because it works using a thermostat system so the desired temperature should be maintained and this is more accurate than a water bath above a Bunsen burner.
DIGITAL BALANCE- More specifically a 3-decimal balance to improve accuracy when measuring the fruit pieces.
FRUIT-PLUMS- I chose this fruit because of its high pectin and juice content.
Measuring cylinders, 50 cm3
1cm3 syringes (for measuring out the enzyme)
Filter funnels and filter paper
100cm3 beakers
Cling film
Thermometer- for assurance that the water bath is at the required temperature
Stock clock (digital)
Knife
Glass rods
METHOD
- The plums were cut into small pieces, roughly at the same size.
- 100g of the fruit was weighed and placed in the beaker.
- 2cm3 of the 2% Pectinase solution was added to the beaker containing the fruits, using a syringe.
- The contents of the beaker were stirred with a glass rod.
- The beakers were covered with cling film (to prevent evaporation of juice) and placed in the water bath (starting temperature 10 degrees) to incubate, for 20 minutes (using timer).
- The juice was filtered from the fruit pieces using filter papers in filter funnels placed in measuring cylinders.
- After 10 minutes the volume of juice obtained was recorded.
This process was repeated for each of the temperatures- 20,30,40,50,60,70,80 degrees.
For the 0 degrees temperature, ice water was used instead using the same procedure.
DIAGRAM OF APPARATUS
SAFETY/RISK ASSESSMENT
I did not carry out a risk assessment as I am not using any chemicals or hazardous substances. My chosen apparatus is safe so there are no major concerns. Nevertheless I am still going work as safe as I can.
- Safety goggles must be worn throughout.
- A lab coat must be worn throughout the experiment.
- Hands must be dried when operating the water baths.
- The juice produced must not be tasted as this might not be suitable for consumption. It’s better to be safe than sorry.
RELIABILITY
Reliability is the amount of confidence that can be placed in the set of observations .To ensure that the results are reliable; some factors must be kept constant throughout the experiment. These factors are
Amount and Concentration of Pectinase- kept constant for all experiment and has to be measured accurately for a fair test.
Amount of fruit- This also needs to be kept constant throughout.
The same type of apparatus needs to be used throughout.
The experiment was carried out twice to enable an average to be taken.
I used a range of nine temperatures for better comparison of results. These were at 10 degree intervals to include extreme and intermediate temperatures.
RESULTS
The following results were obtained.
Table showing amount of juice produced at different temperatures.
Looking at the table I can see the trend that I had already predicted. Initially the amount of juice produced increases as the temperature increases which mean the efficiency of Pectinase increases with these temperatures. This can be seen more clearly on the graph.
The peak shown on this graph is at 40 degrees then there is a sharp fall in the amount of juice produced between the 40-50 degrees temperature range. The amount of juice produced then continues to decrease to the temperature of 80 degrees.
This graph supports my prediction that the optimum temperature of Pectinase would be between 30-40 degrees and the enzyme would have decreasing efficiency towards 0 degrees, but the results did not support my prediction that the enzyme would be completely useless at a higher temperature of 70 degrees, where I thought it would have denatured. The graph shows that at 70 degrees, juice is still being produced even at the higher temperature of 80 degrees; juice is still being produced, even though it was a small amount, this is obviously an anomaly.
At 70 degrees, I had expected the three -dimensional structure of the enzyme to be destabilized and Pectinase to be inactive, but this was not the case because juice was still being produced.
The graph below would have been a more desirable graph and was what I had initially expected.
The graph started at the origin (0, 0) because at a temperature of 0, Pectinase is inactive due to no kinetic energy, therefore the pectin remains intact and keeps the fruit cells glued together, and therefore no fruit juice is produced at this temperature.
Using the graph, I calculated the rates of juice production by calculating the two gradients. The first gradient was calculated to find the rate at which juice was produced up to the optimum temperature; this was found to be 3.9ml/10degrees which equals 0.39ml/degree, this value is the rate of fruit juice produced from 0-40 degrees. The second gradient was calculated to find the rate of juice produced after the optimum temperature, when the amount stated to decrease; this was found to be 1.7ml/-16degrees = -0.1063ml/degree, this is negative since there is a decrease. This signifies that the amount of juice produced changes as a higher rate after the optimum temperature, this is due to the Pectinase denaturing and therefore being unable to works as effectively as before.
I carried out the experiment twice to obtain more reliable results. I carried out a statistical test, namely a two-tailed T-Test on my results mainly for validity. I found the standard deviation to be 0.177 which I feel is reasonable and with the help of a statistical table (found in appendix) the test proved that my two sets of results were consistent with each other.
My experimental evidence can be seen to prove my prediction correct apart from the anomalous results I obtained towards the end which could have been caused by a number of reasons.
I can conclude with a high confidence level that my experimental evidence (results) confirm my prediction. From my experimental evidence and theory I can conclude that temperature does have an effect on the Pectinase. This effect is the higher the temperature the more efficiently Pectinase works until the optimum temperature, whish believe is between 35-40 degrees at which Pectinase works best. At higher temperatures past this optimum, Pectinase is less effective until a certain temperature at which it denatures and can no longer function, I still believe this temperature to be at 70 degrees.
EVALUATING EVIDENCE AND PROCEDURES
The experiment worked well overall, proving beyond reasonable doubt that the optimum temperature for Pectinase was between 35-40 degrees. I believe that the results I obtained were sufficiently accurate and the experimental procedure went to plan.
My experiment was suitable to investigate the effect of temperature on activity of Pectinase. I was able to easily change the temperature. The activity of the enzyme was measured using the amount of juice produced. The more efficient the enzyme worked, the more amount of juice was produced.
Despite all this, the results obtained were not 100% accurate. There was a clear trend as temperature increased as I had predicted but towards the temperatures of 70-80 degrees, I had anomalous results, this can be seen looking at the graph. At 70 degrees no juice was expected to be produced due to the denaturation of Pectinase, but despite this juice was still produced at this temperature and at the higher temperature of 80 degrees. I found a number of possible reasons for these anomalous results.
LIMITATIONS/ SOURCES OF ERROR
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WATERBATHS: I think the water baths were the main sources of error. Despite the fact that I was provided with the number of water baths I had asked for, they were all different. The temperature range for one of the water baths was 10-50 degrees while another was 20-100 degrees, they were all different sizes and makes. The water baths also had different volumes of water in them and this could have caused some errors in the results obtained.
Another major problem with the water baths was that they were not all at the exact temperature required; I checked this by placing thermometers in the water baths. The temperatures kept on fluctuating, I know this could be a factor for anomalous results because temperature has a great effect on Pectinase and could have affected the results obtained.
This source of error is very significant as it has a major effect on my measurement of enzyme activity, which is the dependant variable that I am investigating.
2. MEASURING CYLINDERS: These all measured the juice produced in whole numbers so I had to estimate the amount to 1.decimal place. This could reduce the accuracy of my results because my estimation might be wrong, but I tried to be as accurate as I could.
3. FRUIT PIECES: I cut the fruit into pieces with a knife and I tried as best as I could to make sure they were all the same size, but due to a time restriction I might not have been as precise as I would have liked to be. Errors could occur if they were not the same size because surface area also affects Pectinase. Larger Surface area means more area for Pectinase to work on and this would therefore affect the results.
4. PECTINASE: The Pectinase used in this experiment was a bacterial enzyme, and these do not necessarily behave in the same way as normal enzymes. It could therefore have been able to survive without denaturing at high temperatures, this would account for the anomalous results at the temperatures of 70-80 degrees.
5. PH: Unfortunately this was not controlled during the experiment and could have disrupted the results because pH also has an effect on the way enzymes works. PH alters the enzyme activity by
- Causing changes in protein structure.
-
Changing the binding of substrate to the enzyme.
-
Changing the properties of amino acids or co-factors involved in the catalytic activity of the enzyme.
-
Altering the ionization of the substrate.
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Temperature at 0 degrees: I had to obtain this temperature using ice water because the water baths couldn’t go to this temperature. The problem with using ice water was that, there was no way of maintaining the required temperature. This isn’t such a major source of error since only one temperature is affected.
IMPROVEMENTS
1. Better quality water baths (which stays at the required temperature) should be used which are all the same and time should be taken to ensure that the water baths all have the same amount of water in them.
2. Measuring cylinders with decimal places should be used to obtain a more accurate measurement of the juice produced. Using this will greatly reduce errors and increase accuracy.
3. To control the pH, buffers should be used. Buffers withstand small changes in pH by losing and gaining hydrogen ions so the pH should be kept constant.
4. The fruit pieces should be measured using a ruler, although this might be time-consuming, accuracy of results would be greatly improved because the surface area would be the same.
5. Pectinase extracted from fruits during the ripening stage would be a more natural source of the enzyme instead of bacterial enzymes. This enzyme would behave in a more predictable manner.
6. The experiment should be repeated more than twice to obtain more results and a better average. It should be repeated at least 3 times to obtain more reliable results, minimise errors and obtain more accurate average.
7. The juice should be left in the filter to be collected for a longer period, for example a couple of hours for greater amounts of fruit juice to be collected. This would give a better indication of the effect of temperature on Pectinase.
8. The experiment should be conducted over a longer temperature range and at more intervals, like 5 degrees instead of 10. This would greatly improve the accuracy of my results and more results would be obtained for a better graph.
CONCLUSION
Because of the several sources of errors and limitations of the techniques used, my data may not be very reliable. Therefore, the validity of my conclusion has been affected although I feel that the trend shown is correct apart from the anomalous results obtained towards the end.
Despite all the limitations I described before, I feel the investigation went very well, even better than I had originally anticipated. The accuracy of the results was certainly good enough to make a sensible conclusion. My conclusion is safe because even though the sources of error have a negative effect, the effect of temperature on Pectinase is still apparent.
In my opinion, if the experiment had been conducted under more strict conditions and with more advanced instruments, the conclusion would not have been so different. The only difference might have been the individual results, which might have been more accurate and the graph might look slightly different.
My Original Plan was altered in these aspects:
- I had originally planned to use apples instead of plums but due to the fact that another person was doing the exact same experiment with apples. I decided to be different and used plums instead of apples and I’m glad I did.
- I was initially going to use 1% Pectinase solution but, following the pilot study I found that 2% would be much more effective.
FUTHER INVESTIGATION
The enzyme Pectinase could be investigated even further in these aspects:
- Effect of different concentrations of Pectinase on the amount of juice produced.
- Comparison of juice produced from different fruits.
- Investigating how Pectinase works in clarifying juice.
- Effect of pH changes on Pectinase.
- Comparison of microbial Pectinase with natural plant Pectinase.
REFERENCES
BOOKS
1. MICROORGANISMS AND BIOTECHNOLGY
JOHN ADDS, ERICA LARKSON AND RUTH MILLER
2. MICROBIOLOGY AND BIOTECHNOLOGY
CAMBRIDGE SCIENCES
3. BIOLOGICAL SCIENCE 2
D.J Taylor, G.W Stout, N.P.O Green.
4. ADVANCED BIOLOGY
CJ Clegg, DG Mackean
5. ADVANCED BIOLOGY STATISTICS
Andrew Edmondson and David Bruce
- STATISTICS FOR BIOLOGISTS
Webb Blackmoore
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