Biology Cell Biology

1. Design

1.1 Defining the problem

1.1.1 Focus Question-

What will happen to the rate of reaction of the amylase in the starch when the temperature is changed?

1.1.2 Hypothesis-

The rate of reaction of amylase in starch will change as the temperature is changed.

1.1.3 Theory-

A enzyme is a catalyst (increases the rate of reaction)(Malcolm P 2008). These enzymes are important within the body to reconstruct the nutrients we use into the compounds we need. Without these enzymes the reactions would occur far too slowly and we would slowly die. One of these enzymes is amylase. Amylase is an enzyme which breaks starch down into sugar (maltose). Amylase is commonly found in saliva. This is why if you hold a piece of starch rich bread in your mouth for long enough it begins to taste sweet. This is because the starch begins to break down into maltose. Amylase breaks down starch into sugar by the breaking of the disaccharide maltose (Two linked glucose molecules)(Wang S 2008). All enzymes are subject to denaturing. This means that the enzyme will become “denatured” and will not function as efficiently or at all. This can be caused by an effect such as adverse temperatures or PH in conjunction to the enzyme. Amylase optimal temperature is about 40 degrees Celsius. (Incognito 2008).

The reaction of starch with amylase is invisible to the naked eye. Iodine (or a similar indicator) needs to be added to the solution. This will react with the starch, colouring a dark brown or a purplish colour. Once the amylase is added to this solution the brown or purple colour will begin to change in to a bright yellow colour (the same colour as the iodine) as the starch is transformed into maltose. The faster this happens the more active the reaction. This transformation of brown to clear is known as hydrolysis. The time that it takes for the hydrolysis to fully complete the transferral conjunction with the temperature can be used to find the rate of reaction. From this is can be calculated at which temperature the enzyme amylase is most effective. Amylase is a very important component in the transferral of starch into sugar which can later be transferred into energy. All though too much can be a bad thing, too little will result in shortages of energy and tiredness, fatigue and possibly death. (Tray P 2008)

1.1.4 Investigating variables

Table 1- Practical variables

Type of variable

Identified variable

Independent

The temperature of the solutions

Dependent

How long it takes for hydrolysis to be completed

Controlled

Air temperature

Controlled

Container for solutions

Controlled

Apparatus used for measuring solutions

Controlled

Enzymes and chemicals used.

Controlled

Method of time keeping

Controlled

Experimental repeats

1.2 Controlling variables

1.2.1 Table 2 Controlling variables

Variables

Control treatment

Air temperature

The air temperature should initially be recorded with a thermometer. Preferably with use of an air con a temperature of 24 degrees should be achieved and the experiment should be kept out of direct sunlight to avoid degrading the integrity of the experiment.

Container for solutions

Test subjects will be placed in the sockets of a spot tile. Each tile is can hold 1ml to 0.7ml

Apparatus used for measuring solutions

One glass pipette socket (0.01ml uncertainty) with pump. 3 sockets to avoid contamination

Enzymes and chemicals used.

50mls of amylase, 50mls of starch and 25mls of iodine

Method of time keeping

The use of a stopwatch. Human error= 0.1, electronic error= 0.001

Experimental repeats

5 repeats for each temperature range, final result will be averaged.

1.2.2 Control experiment

The control experiment consists of the assumed optimal temperature of amylase. This is 40 degrees Celsius, a few degrees higher then body temperature. At this optimal temperature the speed of hydrolysis should occur faster than in other temperature ranges.

1.3.3 Method

Method inspired by (Teacher union 2007) and (Science project 2008)

1. 0.02mls of amylase was added to micro test tube.

2. Test tube was placed in water bath untill required temperature was obtained.

3. 0.1mls of iodine was added to starch

4. 0.2 mls of starch was added to the solution

5. Time for full hydrolysis to occur was timed and recorded.

6. 1,2,3,4 and 5 were repeated 4 more times (simultaneous reaction could not be performed but reaction would could not be set off simultaneously and it would be too difficult to time 5 moving reactions)

7. All steps were repeated for the temperature 30C, 35C, 40C, 45C and 50 C.

2.0 Data collection and processing

2.1.1 Raw data table- (table 5 raw data table)

Table showing the time for a full hydrolysis reaction to occur at differing temperatures at what colour they were at the end of five minutes. (0 means full hydrolysis never o0ccurred).

2.1.2 Qualitative data

Both the starch as the amylase was transparent clear in colour while the iodine presented a yellow colour. When the iodine was added to the amylase the solution turned yellow like the iodine, but when the starch was added then the concoction turned a heavy purple. This purple colour (in a successful reaction) slowly changed from a purple to a blue from a blue to a red and from a red to a yellow. The reaction also gave off a sweet aroma similar to caramel.

2.2 Processing raw data

2.2.1 Mathematical calculations (table 6 calculations)

Statistical Analysis

Formula used

Sample calculation

Mean (used for each temperature range, only use real values, do not use zeros)

Average= Sum of samples/number of samples

30C= 287+296/2

30C=292

Rate of reaction (used with each average temperature).

Temperature used/the average speed (seconds) of the reaction= Rate of reaction.

Rate of reaction= 30/292= .102 is the rate of amylase of transferring starch to sucrose.

Standard deviation

S=Standard deviation

X= real value- mean value squared/ degrees of freedom

30C= Standard deviation= 0.105-0.102 squared/2-

=0.00024

2.3 Presenting processed data

2.3.1 Overview

The raw data table was processed into two tables. The first displayed the average of time at each distinct temperature (Zero values were not counted in averaging). This was also displayed with the standard deviation and rate of reaction. This data could be visually interpreted to establish the differences between each of the temperature thresh hold and could be further extrapolated into graphs. The second table is a simplification of which temperatures actually reacted. While not all of the concoctions fully hydrolysed, many of them still did react and it is important to mention this, as to eliminate bias in the fact that all of one reaction may have reacted but not completely. This data was then interpreted into a two graphs. One displaying the rate of reaction verses the temperature and a small table summarising the total number of reactions. This data in turn can be further interpreted.

Graph 1 (Reaction rate vs. temperature)

The graph above gives an accurate coloration to the hypothesis (The Reaction rate will change at different temperatures). This can be seen as the rate of reaction increased as the temperate does. The rate of increase is also relatively uniform with an R value of 0.9584. This means that the graph is almost linier meaning there is little differentiation between each of the temperature thresh holds. This is with the exception of the change between 45C and 50C which was quite substantial compared to the other changes but was probably due to more activation in the formulas. There is a standard deviation. This deviation gets larger with each subsequent temperature. This is most probably due to the increased number of successful (but slightly differing) samples in each heightened temperature. Although even with the standard deviation does rise with each temperature thresh hold, its effect is negligible on the total scale of the graph.

Graph 2 (total activations)

This graph shows the total number of reactions for each temperature in the experiment. This is important to mention to show that in most of the solutions reaction did occur but just at different levels of intensity. These show that the largest majority of the reactions did not become completely denatured and still showed reaction but at slower rates. This shows that even at adverse temperatures reaction between amylase and starch shall still occur but at different rates.

3.1 Conclusion and evaluation

3.1.1 Conclusion

The hypothesis that as the temperature increases the rate of reaction will change was proven undeniably correct. By the data seen in graph one it can easily be seen that as the temperature rises the rate of reaction inversely raises as well. This can be seen at near constant rates except with a large gap between 45 degrees and 50 degrees. The rate for reaction increases at a rate of about 0.050 for each 5 degrees except for between 45 and 50 were this value effectively doubles. This is most likely due to the increased number of experiments which on average was inconsistent with the 4 other degree levels. But apart from this, the data was relatively consistent in its rise which could be represented by the linier R value of 0.9584. This rise probably would have continued as the temperature rises. However eventually there would have been a point where the enzyme amylase simply becomes denatured by the incredible heat.

At this point the reaction rate would most likely begin to drop. As the temperature drops the rate of reaction would also decrease untill the enzyme would have become completely denatured. The body temperature is 37 degrees but this is obviously not the optimal temperature for amylase to exist at. The body would process starch in to sucrose far more efficiently at a higher temperature. The reason for this lower then optimal temperature in the body is probably due to the fact that the body does not want to process to much sucrose. Sucrose is a very potent form of sugar which in excess can cause several complications in humans. These can include insomnia and shock. With the large amount of starch humans absorb it may cause complications if we did transfer large amounts of it into sucrose. The amount of starch transferred into sucrose is capped due to the physical limitations of the human body.

This is turn is actually a good aspect because we do not absorb excess amount of sucrose from the starch we eat but it is also an inhibitor because we cannot take full advantage of the quick energy boost from starch products. Another reason for this lower then optimal temperature in the human body may be the fact that amylase is not the only enzyme in the human body. The human body holds a multitude of chemicals and enzymes, all of which denature at different temperature. It is highly possible if the body temperature was increased to improve the affect of amylase, another enzyme would become denatured because the temperature has risen too much. The body temperature is constantly (except for fevers) in perfect sync. This sync is performed to make sure that all enzymes and chemicals are acting at a safe level. This shows a symbiotic relationship between bodily temperature and the enzymes of the human body. If just a single enzyme is removed from the body then all systems would slowly begin to fail. So balance between the bodily temperature and the denaturisation rates of the body’s enzymes are crucial to survival.

3.1.2 Limitations of experimental design

The reaction rate between 45C and 50C was most alarming in relation to the other sets of data. It was at least twice as large as the changes between previous temperatures thresh holds. This is most likely due the increased amount of reactions finishing in a decreased period of time. This could be solved by simply increasing the number of replications to achieve a more plausible average. But this in itself creates an issue. As seen in graph 1 the standard deviation increases as the number of reactions that fully hydrolyse increases. This means that if to man replication are done the standard deviation will begin to have a significant effect on the reliability of the results. The results of time were given in seconds. Unfortunately on the day of the experiment there was a lack of stop watches so I had to use a standard clock. This affected the precision of my results. It would have been more accurate if I had waited for a stop watch or used a better alternative then a standard clock.

The time of the reacting was stopped when the colour was perceived to be yellow. Unfortunately the human eyes have very distinct limitations and it is difficult to perceive between two similar colours. This could possibly affect the overall results because the time on each sample may have been stopped early or later than others due to the perception of when the solution turned yellow. It would have been far more efficient to have used a colour spectrometer which can convert colour into a numerical number. This could then be used to set a standard for yellow and time would only be stopped when subsequent solutions reached this number or the time on the experiment had expired. This could also have been used in a different way. Instead of recording the time at which hydrolysis occurred, with a colour spectrometer the intensity of the reaction could have been recorded. This would have been a more viable alternative to time because those reaction which did not fully complete would still be included in the results therefore supplying more reliable data. It also would have been more efficient to use a better heating apparatus.

A water bath was used and temperature had to be maintained at constant levels. This required a lot of fiddling with the output of the hotplate and probably spawned some temperatures which were not constant and accurate. Alternatively an air space heater could have been used which can control the rate of temperature in the substance trough the use of computers. This error is random in the effect that temperature was controlled to an extent but not perfectly, but also systematic due to the heating apparatus used. It also would have been more efficient to use a data studio thermometer over a regular mercury thermometer because the regular thermometer had to constantly be removed to make sure a correct temperature was been recorded.

3.1.3 Improvements to experimental design

Error indentified

Type of error

Factor for control

The number of solutions

Systematic

This should be increased but not to a rate were standard deviation becomes too large.

Timing apertures

Systematic

A stop watch or similar timing device should be used

Colour regeneration

Systematic

A colour spectrometer should have been used over the eyes. This could also be used to record the intensity of the reaction

Independent variable

Systematic

The variable recorded should have been intensity of the reaction. By using a larger majority of the sample would have been able to record. This would have improved the validity of the experiment.

Heating apparatus

Systematic and random

A more accurate device such as an air space heater should have been used.

Apparatus for measuring temperatures

Systematic

A more accurate heating apparatus such as a data studio measurer over a regular mercury thermometer.

Bibliography

Genetics 2008, Genetic crosses, Ryan P, View 12/02/09, http://images.google.com.au/imgres?imgurl=http://ib.berkeley.edu/courses/ib162/Week2a_files/image006.jpg&imgrefurl=http://ib.berkeley.edu/courses/ib162/Week2a.htm&usg=__p0P5TrzbIybnEVp-6dzAf3IFGuo=&h=362&w=394&sz=16&hl=en&start=1&um=1&tbnid=Lur7Zz3OJ17OHM:&tbnh=114&tbnw=124&prev=/images%3Fq%3Dmonohybrid%2Bcross%26hl%3Den%26sa%3DN%26um%3D1

Amylase 2008, Starch hydrolysis in amylase, Wang S, Viewed 12/02/09 http://www.engr.umd.edu/~nsw/ench485/lab5.htm

Amylase, 2008, Optimal temperature of amylase, Incognito 2008, viewed 12/02/09, http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T4D-4BT8GYV-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b4a12e24117a3e29477b8cfbf4092ee4