We will need to keep the concentration the same for both experiments as doubling the concentration percentage means doubling the amount of enzyme in solution. For example, here is a beaker with a 10% & 50% solution of neutrase in it. I have shaded the neutrase orange to show how much of the solution is neutrase.
This will halve the reaction time and give anomalous results. Having a percentage higher than 2 would give a lower overall time and a percentage under 2 would give a lower overall time.
We will need an accurate judge of when the liquid has become decolorized. Although we can never be sure that we have got it right we will try to make it as accurate as we can by:
a) Using the same person to judge each time.
b) Constantly keeping an eye on the mixture from a top-down view while the reaction is in progress.
c) Taking an average of all three results we have gathered as an overall time.
We will need to repeat the experiments three times to be reasonably sure that our results are all
accurate. If we have any results that seem to be incongruent then they will be discounted in the final average.
Method
Preliminary Experiment Plan and Evaluation
Aim: To find out the optimum temperature of the enzyme “neutrase”
Equipment Needed
2 measuring cylinders.
Neutrase at three different temperatures.
2% marvel milk in water solution at three different temperatures.
100cm3 glass beaker.
Card with a marking on.
Stopwatch/clock.
Ice.
Heating Bath.
The three different temperatures that we are using are 10, 40 & 70oc we will need one measuring cylinder with 1cm3 markings, and one with 10/5cm3 markings.
Prediction
I think that the 40oc enzyme will work the best as this temperature is the closest one we are using to those found in the body in which the enzymes work. Any hotter than this and the enzymes will become denatured, any cooler and the enzyme will slow down as it has less energy to move and will therefore work slower. I predict that this will happen in the 10 & 70oc tests. I predict that the 10oc test will be slow and the 70oc test will not work as the enzymes will be denatured.
Method
We were given the enzymes at three different temperatures (cooled by ice or heated in heating baths). We were also given all of the equipment listed above. We took 2cm3 of neutrase in a 10cm3 measuring cylinder and 20cm3 of Marvel milk in a 50cm3 measuring cylinder. We then brought the two mixtures to our desks, poured them into the glass beaker and started the stopclock. We then placed the beaker on the marked card. We watched the beaker from the top down to see if the cross was visible. Once we could see the cross we stopped the timer and took a reading.
Results.
Above is a graph of how long it takes for the enzyme to work, relative to temperature. The graph assumes that 40oc is the enzyme’s optimum temperature.
Possible Errors.
- It is hard to judge when you can see the cross by eyesight alone.
- We only had 3 temperatures, more temperatures would have given a better idea of the optimum pH
- We did no repeats, one of the results could have been anomalous as we had nothing to check against.
-
It was hard to get 2cm3 into the measuring cylinder, too much went in and then we poured too much out, syringes would be better.
- We did not take into account that the reactants may have cooled between the bath and our desks.
Evaluation
The experiment went well (mostly) we kept to the plan, washed the beaker after every test, tried to cut down the time between the water bath and our desks, measured the reactants with the same measuring cylinder which was also washed after every experiment, used the same timer and used the same person to judge the decolorization. Because of this I think the test was as fair as it could have been. The only things that went wrong was that once we forgot to wash the beaker and had to start again and one time we started the stop clock late and had to add 6 seconds to the final time.
Improvements
If I were to do my experiment again I would:
- Repeat each temperature three times.
-
Have 5 different temperatures instead of three, 0, 20, 35, 60 & 80oc would be good temperatures to use.
-
I would use a graduated pipette / syringe to measure 2cm3.
- I would measure the temperature just before the reaction to get the most accurate results
- I would take the average of all three results to get the best set of results.
How Our Preliminary Experiment Altered Our Plan.
1) In our preliminary experiment we were using 10cm³ or Marvel milk and 1cm³ of neutrase. However, using these values you could see through the Marvel milk before you even added any neutrase. This would have made all of the results 0 seconds and impossible to draw any conclusion from.
2) We discovered that it is very hard to measure out 2cm³ in a measuring cylinder so we will use a graduated pipette in our final experiment.
3) We found that if you measured the liquid temperature at your desk it took about 20 seconds for the thermometer to stop going up and down before you could obtain a clear reading. We also found that to obtain 2 readings with 1 thermometer took quite a long time. So, by the time you had measured the second mixture, the first one had cooled down / warmed up. Therefore we decided it was best to measure the temperature in the water bath / ice tray.
We have discovered these problems and have changed our method accordingly.
Equipment Needed
To make sure that an accurate experiment is carried out I will need the following pieces of equipment:
1 Syringe / graduated pipette.
1 25cm³ measuring cylinder.
A 2% solution of neutrase in water and a 2% solution of Marvel milk, also in water, at five different temperatures.
1 Thermometer.
100cm³ glass beaker.
Laminated card with a marking on it.
1 Stop Clock.
Some ice cubes.
1 Tray.
1 Water Bath.
Goggles
Gloves
Outline of Experiment
First we will make sure we have all of the equipment listed above on our desk and laid out correctly so that we do not have to go and get something half way through the experiment. Nothing should be in the way or cluttering our desk (e.g. books, pens etc.) We will also make sure we have our goggles and gloves on.
Then we will start the experiment, we will repeat this procedure three times for each the experiments. The neutrase and Marvel milk will be at 10, 35, 40, 60 & 80oc.
We will first place the glass beaker on the marked card and go to collect some reactants. We will measure the temperature at the water bath or ice tray and then one of us will measure out 20cm³ of Marvel milk in a 25cm³ measuring cylinder. The other one of the pair will use the graduated pipette to accurately measure 2cm³ of neutrase. We found in our preliminary experiment that it is very hard to get some neutrase out of the conical flask as your fingers will not go in with the graduated pipette. The easiest way round this is to pour some neutrase into a glass beaker and fill up the graduated pipette from there. We also found that it is hard to get 2cm³ of neutrase when the scale is in the beaker so we will fill the syringe up to the top and then press the plunger down until there is only 2cm³ left in the syringe.
Once we have done this we will take the neutrase and Marvel milk back to our desk. Once it is at our desk we will pour in the neutrase and milk and start the stop clock. One of us will look down on the reaction and keep our head there until we can see the marking on the card below. Once we can see the marking we will stop the stop clock and take down the time.
We will then wash the glass beaker, syringe and measuring cylinder in preparation for the next experiment.
We will measure how fast the reaction works by timing the decolorization of the milk with a stop clock and then taking down the time displayed when we can first see the cross. Then we will eliminate any anomalous results and take an average of the remaining results.
We will record our results to the nearest hundredth of a second as this is the smallest amount of time that the stop clock will display.
To make sure the test is fair we will:
Use a buffer solution to make sure the pH is the same for all of the experiments.
Try and measure accurately and with the same equipment for each of the experiments.
The concentration of both will need to be the same but we cannot do anything to control this as we will be given the solution of milk and neutrase and will not be involved in any way in creating the 2% solution.
We will use the same person to judge when the mixture is decolorized, as a different person could have worse/better eyesight than the other and this would result in inaccurate results.
To keep the temperatures as near to the target temperature as possible, we will leave them in the water bath/ice tray for the same amount of time.
We will repeat all 5 experiments 3 times and then get rid of all obvious anomalous results. Only when we have results that seem consistent will we take the average figure and use this as a final result.
Make sure we wash all of the equipment before we start the next test.
We will change the variable so we readings at 10, 35, 40, 60 & 80oc. Each reading will be taken 3 times.
Some safety precautions I will need to take are:
Wear goggles to protect my eyes.
Wear gloves to protect my hands
Do not sit down.
Do not run with chemicals or glass.
Do not eat or drink whilst in the lab.
When we have collected the results in a table we will analyze them and then present them in a graph and a table.
Results
Table
With the 70o reading we left it for 5 minutes and there was no change. We saw little point in carrying on the experiment so assumed that all of the enzyme was denatured and that it would not work. We then carried on with the next experiment.
Graph
Analysis
At 0oc there is very little energy in the enzymes and the substrate. This means that they are traveling very slowly. If they are traveling slowly then the chances that the enzyme and substrate will meet up is very small. The lower the temperature, the smaller this chance. The enzymes need to collide with a substrate in order to create products from the substrate. Once they have met up, the substrate is adsorbed onto the active site of the enzyme and the substrate is turned into some products. At zero degrees all of this happens very slowly. This is reflected in the results. Zero degrees takes the longest to react out of al the values that we could collect. This is reflected in the experiments high average time of 73s. This is much higher that the 20o experiment, which had an overall time of 51s. The comparison of the two times shows that the 20o experiment had more energy than this one and that therefore this experiment had a small amount of energy.
At 20o the enzyme is gaining energy. It has a lot more energy than at 0oc this is reflected in its time. At 20o the enzyme possesses a little energy and will be speeding round quite fast. There will be a few collisions and products will be formed at a reasonable rate as the enzyme and substrate are traveling quite fast. When the enzyme and substrate meet up, the substrate will be adsorbed onto the active site of the enzyme and the enzyme will turn them into products. This is reflected in the average time of 51s. This is lower than the 0oc experiment where the conversion took 73 seconds but much higher than the 35 oc experiment, where the process took 4s. This shows that it has less energy than the 35o experiment, but more than the 0o experiment.
At around 35oc the enzyme is at its optimum temperature. This means that the 35o experiment should go the fastest. The enzymes have a lot of energy as they are quite hot, but they are not hot enough for the denaturisation to begin. This will result in fast moving enzymes and substrate. As the enzyme and substrate have a lot of energy and are moving fast they will collide a lot. Once they have collided the substrate will be adsorbed onto the surface of the enzyme and will be converted into products. This is reflected in the extremely low average time of 4s. This shows that out of all my temperatures, 35oc is the best temperature for converting the substrate into products.
At 60o the enzymes started to become denatured. This means that there are fewer enzymes that are capable of turning the substrate into the products. When an enzyme becomes denatured its active site is deformed. Once the active site is deformed the enzyme will stop accepting the substrate. So, once the enzyme is denatured it is incapable of turning its substrate into products. If a percentage of the enzymes become denatured then there will be less collisions between working enzymes and substrate and the products will form at a slower rate. This is proven in the results. The 60o experiment had an average time of 73s. This is much higher than the 35o experiments result of 4. As the temperature is higher there must be something else inhibiting the conversion of substrate to products and this is the increasingly denatured enzyme.
At 80o the enzymes have almost all been totally denatured. This means that the experiment will take a very long time to complete. With not many enzymes left in a working condition, the chances of a working enzyme and substrate meeting are lowered. The enzymes have a lot of energy but there are so little of them that the extra energy does little in comparison to the effect of the huge amounts of enzymes that have been denatured. Once a pair does meet up though, the substrate will be adsorbed onto the enzyme and the enzyme will convert the substrate into products. When I did this experiment, we added the enzyme and substrate to a beaker and then we waited for 5 minutes. After five minutes the milk had still not decolorized so we decided that it was not worth waiting longer and that we should start the next temperature. All I can say is that at 80o the enzyme takes over 300 seconds to denature which is a huge amount in comparison to the 60p experiment on which the result was 73s. I can conclude from this that the only reason tat the enzyme’s performance would be worse at 80, where it would have more energy) than at 60 is that the enzyme was denatured.
This conclusion does not fully support my hypothesis, though it does support it in part. I predicted that under 40oc the enzyme would not work very well. This has been disproved as we have shown that 35o is the optimum temperature and 40o is not. I did predict however that over 40o the enzyme would be increasingly denatured and this is what seems to have happened because the 60o and 80o tests have much larger times than the 35o one.
Conclusion
With my conclusion I stick to my hypothesis. I think that around 40oc is Neutrase’s optimum temperature. I think that increasing the temperature would have the effect of giving more energy, but at the same time denaturing the enzyme more and more depending on the temperature increase. I think that lowering it below 40oc would have the effect of taking away energy and so making the whole process slower as there would be fewer collisions at a lower speed. I now think though that the enzyme’s optimum temperature might be a bit below 40oc but we didn’t do enough experiments to determine what happens around 40oc. This could be a possible improvement for a future experiment.
Evaluation
My experiment went well in general. There were no major problems in my method but we did find ways to make it more accurate.
We got one anomalous result. On our 0o test we had a variation of 7 degrees, the biggest gap that we had in any of our experiment. The results for that test were 68, 72 and 79o. I think that we got this anomalous result because the temperature could have been more or less in that particular experiment as the temperature largely depended on how long it took for us to get the experiment from the bath to our desks. The 2ml3 of enzyme could have warmed/cooled very quickly and this would affect the reaction time.
Errors:
Temperature control
We had no accurate and fast way to check the temperature. We needed to check the experiments fast to prevent them from cooling down but we only had mercury thermometers. If the temperature was too high it would have given the enzyme and substrate more energy and therefore a higher chance of a collision. If they had collided more then the overall time would have been higher. If the temperature was raised extremely high the enzymes would denature, making a collision between an active enzyme and the substrate less likely, resulting in a smaller overall time. If we lowered the temperature then we would have been deceasing the energy in the enzyme and substrate, causing them to move slower and have fewer collisions. Once the enzyme and substrate collided the substrate would be adsorbed onto the active site of the enzyme and would break off as two products. A data logger would have made the whole process a lot faster and therefore we could have taken accurate readings in a small amount of time. This would make the whole experiment more accurate.
Decolorization Test
It was very hard to tell when the milk had decolorized. Using our eyes to judge was a very inaccurate way of judging and was probably often out by quite a few seconds. The decolorization is brought about when the enzymes collide and the substrate is adsorbed onto the active site. Once the products are locked onto the active site they change into two products. The products of our reaction are colorless. So we can tell when the reaction is complete when we can see the marked card underneath the beaker as if the reaction was still underway it would be obscured. Judging the decolorization would have been helped by a colorimeter. A colorimeter works by shining a known amount of light through a sample of the milk and neutrase. The colorimeter then picks up the light on the other side and works out how much light has got through and how much has been blocked and how much light has got through. This would make the point of decolorization indisputable as the colorimeter would be set up in the same way each time. This would make the experiment much more accurate.
PH
We could not fully control the PH. Once the enzyme and substrate have collided the enzyme needs to adsorb the substrate onto its active site. Altering the pH to extreme -/+ pH would have deformed the active site rendering the enzyme useless. However unlike extreme temperatures, once the pH is brought back too around the enzymes optimum pH the enzyme will work again, forming products from reactants or vice versa. Although all of the experiments contained exactly the same ingredients we had no assurance that the pH was constant throughout the experiments as the mixtures were pre-prepared and we had no involvement in there creation. However a buffer solution would have kept the solution’s pH constant. A buffer solution is a solution that will keep a mixture/solution at a constant pH even if acid/alkali is added. A buffer solution would have increased the probability of a constant pH.
Volume
We tried to keep the volume the same for all three experiments. We did this by using measuring cylinders and graduated pipettes. Having in equal volumes in the tests would have made the tests unfair and even time averages for single tests anomalous. If you increase the volume of the enzyme or substrate you are increasing the amount of enzyme or substrate in the experiment. If you have extra enzyme the reaction will go faster as there will be more enzyme to react with the proportionally less substrate. If you have more substrate then there will be proportionally fewer enzymes and more substrate to convert to products per enzyme. This will result in a slower reaction. The measuring techniques that we used were fairly accurate but to make them more precise we could have used A grade measuring equipment as A grade measuring equipment is quite expensive but made to be more accurate.
Concentration
We tried to keep the concentration the same but as the solutions were provided for us we had no direct control over the concentration. Increasing or decreasing the percentage concentration of enzyme or substrate would have exactly the same effect as increasing or decreasing the volume. Increasing the percentage concentration of enzyme would have increased the amount of enzyme in a set volume of water meaning that the reaction would go faster as there would be proportionally less substrate to each enzyme. This would mean that all the substrate would be converted quicker. Decreasing the percentage concentration of enzyme would have decreased the amount of enzyme in solution meaning proportionally more substrate to each enzyme. This means that there would have to be more collisions to get the same effect, thus taking more time. Increasing the percentage concentration of substrate would have the same effect as increasing the volume as there would be more substrate per enzyme so the reaction would take longer. Decreasing the percentage concentration of substrate would decrease the substrate per enzyme making the experiment shorter. If we had made up the solution we should have used a balance to at leas three decimal places to weigh reactants and also measured the water in the most accurate way possible.
Is My Evidence Enough To Support My Conclusion?
No. I do not think that there is enough evidence to support my conclusion in my experiment. This is because:
-
We set the temperature interval too high. I have absolutely no idea what happens at 40oc. For this reason I cannot say what the optimum temperature is. I cant even give it to the nearest 5oc
- I don’t know if my results are reliable. For the possible errors given above and also because we had no hand in creating the enzyme and milk solutions. This could mean that they are inaccurate and might not be 2% solutions at all.
- There was a period in which the enzyme and substrate could have cooled down (in-between the water baths and my desk)
- Sometimes we were late in starting the stopclock. We were often half a second out and once we forgot to start it for six seconds, then realized and had to start the experiment again.
Further Experiment
My preliminary experiment was clearly full of errors and anomalous results. It also had an extremely small set of experiments. We took three values on a scale of -273 - ∞. This is clearly not enough and for these reasons we did a second experiment. Taking five values and carrying it out in a way as to generate as little error as possible. We have now identified errors in this, second experiment and I am using that as the preliminary experiment for another experiment, for which I will write a method.
Method
First we will check that our working area is free from clutter and unneeded equipment. We will then make sure we have all of the equipment that we need for the experiment and we will make sure we have our goggles and possible gloves on because the neutrase is a biohazard.
We will then begin the experiment. We will repeat the experiment at 1 degree intervals from -10oc to 100oc we will do each 1o interval experiment 5 times. We will then discard any anomalous readings and take an average of the readings left.
We will place the cuvette into the colorimeter and put a funnel into the cuvette. We will then go to collect 1cm3 of Marvel Milk using a 1cm3 gradated pipette. We will then collect a 0.1cm3 solution of enzyme using a 1cm3 syringe with 0.1cm3 graduations. With a different syringe we will collect 0.1cm3 of a pH 7 buffer solution. We will use such small amounts as the cuvettes for the colorimeter are very small and cannot hold much liquid. We will then use a data logger with two probes to check the temperature of the enzyme and substrate before adding them together with the buffer solution into the cuvette. Once they are in the cuvette we will put the cuvette with the reactants into the colorimeter (which will already have a sample of the solvent, water, in it) and at the same time start a stopclock. We will then see how long it takes for the colorimeter to read 95%. Once it reads 95% we will stop the stopclock and take down a reading off of the stopclock. We will then put the cuvette in for washing and take out another cuvette. We will then wash out the graduated pipette and the syringe ready for the next experiment. We will use the same water in the colorimeter to make sure that that does not add another possible error.
To get our results we will be measuring how long it takes for the milk to decolorize at 1o intervals between -10 and 100oc. This will give us an idea of the enzymes reactivity.
I expect that the results would put the enzymes optimum temperature at around 37oc. Any higher or lower and the overall time will go up due to the enzymes being denatured or the energy being taken away.
