Investigation into the effect of enzyme concentration of catalysers of sucrose by sucrase.

James Clothier

Investigation into the effect of enzyme concentration of catalysers of sucrose by sucrase

Planning

Aim: I aim to find out the effect of enzyme concentration on the substrate. I will try to find out how fast the substrate sucrose is broken down using different concentrations of sucrase, to see if there is a link between enzyme concentration and the rate at which the substrate is broken down.

An enzyme is made of protein, they are protein molecules which are called a biological catalyst. These molecules speed up chemical reactions and remain unchanged when the reaction is finished. Enzymes have an active site, this is a place where another molecule or molecules can bind to. These molecules are the substrate of the enzyme. The shape of the enzymes active site will allow the substrate to fit onto it. The substrate is held in place by temporary bond that form between the enzyme and the substrate, the bonds form between the R-groups of the enzymes amino acids and the substrate.

Only one type of enzyme will work on one type of substrate molecule. This is because the shape of the enzymes active site will only fit the molecule which will fit into its active site.

The enzymes catalyse reactions where substrate molecules are split into two or more molecules. In this case

Sucrose glucose + Fructose

Sucrose is broken down into two molecules by sucrase into glucose and fructose which are reducing sugars.

When the reaction is finished, the products leave the active site and the enzyme is unchanged, then it is ready to receive another substrate molecule.

Factors that can affect the reaction rate are the temperature, enzyme concentration and pH. As the temperature increases, the enzymes and substrate are given more energy so they move faster and collide more, this means the substrate will enter the enzymes active site more with more energy allowing bonds to be broken. As the temperature increases the reaction speed increases, when the reaction works at its fastest it has reached its optimum temperature (38 ˚C), but over a certain temperature the enzymes structure vibrates energetically and some of the hydrogen bonds that holds the enzyme molecule together in its shape begin to break. This means the active sites shape changes and the substrate molecule cant fit onto the active site, the enzyme is said to be denatured. 1

The pH can effect the reaction rate, most enzymes work best at 7, pH is measured in hydrogen ions, these hydrogen ions can interact with amino acids R groups, affecting the way in which they bond which effects the 3D shape of the enzyme and denatures it. 2

If the enzyme concentration is increased the reaction rate will increase because there are more active sites for the substrate to fit into, which means the substrate will be broken down into two or more products quicker. This will only happen if the is enough substrate available. 3

Hypothesis: I predict that as the enzyme concentration is increased, the substrate sucrose will be broken down quicker. I think this because as the enzyme concentration is increased, there are more active sites for the substrate to fit into, so the substrate sucrose will be broken down quicker, as explained in the theory.

Null hypothesis: however, increasing the enzyme concentration could mean that there is no change at all in the rate of reaction.

Variables:

Control variables-My control variables will be sucrose and the temperature. I will control the temperature because I want the conditions to be the same for each concentration. I will keep the concentration and amount of sucrose the same because so it is fair and so I will get reliable results

Independent Variables-The independent variable will be sucrase because this is what is being tested, the concentration will be changed to find out if enzyme concentration has an effect ...

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Null hypothesis: however, increasing the enzyme concentration could mean that there is no change at all in the rate of reaction.

Variables:

Independent Variables-The independent variable will be sucrase because this is what is being tested, the concentration will be changed to find out if enzyme concentration has an effect on substrate breakdown.

Dependent variable-The dependent variable will be the time, the time will be different according to the concentration of sucrase, because as the enzyme concentration increases the reaction speed increases.

Extraneous variable- The extraneous variable is the room temperature because it cant be controlled.

Method: For my preliminary experiment this is the method I used

I will get 3 test tubes and place them in a test tube rack.
I will clean the test tubes out with distilled water so it isn’t contaminated with any other chemicals.
Then I will place 5cm3 of 2% sucrose solution into one of the test tubes using a 5cm3 syringe. I will use a syringe because it is more accurate than using a measuring cylinder.
Then I will place 5cm3 of 1% sucrase solution into one of the test tubes using a 5cm3 syringe. I will use separate syringes so the solutions don’t get contaminated which will effect the results.
Then I will place 1cm3 of benedict’s solution into the last test tube using a 1cm3 syringe.
Then I will label each test tube with its name with a permanent marker pen, so the test tubes don’t get mixed up.
Then I will place the sucrose and sucrase solution into a water bath at 38 ˚C. This is because 38 ˚C is the optimum temperature for a reaction to take place which is explained in the theory. I will leave the test tubes in the bath for 5 minutes to allow the solutions to reach this temperature. I will time them with a stop watch. I will check with a thermometer that they have reached this temperature after 5 minutes. 3
Then simultaneously I will pour the sucrase solution into the sucrose solution and start the stop watch. I will start the stop watch now because the solutions will start reacting from this point.
I will then swirl the contents of the test tube and quickly put it back into the 38 ˚C water bath for 30 seconds. I will put it back into the 38 ˚C water bath because it is the optimum temperature for the chemicals to react.
After 30 seconds, I will remove 1cm3 of the mixture using a 1cm3 syringe and put it into the Benedict’s solution in the test tube rack. I will put it in the benedicts because the reaction produces glucose which is a reducing sugar, so the glucose will reduce the copper in the benedict’s which will turn the solution green then red.
Then I will put the test with the mixture into a water bath at 50 ˚C. I will put it in a water bath at 50 ˚C because it is a good temperature for the benedict’s to react with glucose produced from the reaction.
I will do a sample before this so that I can use it to help me tell when I want to record the end point. I will do this so that all the reactions end points will be recorded in the same place so the test is fair.
When the mixture begins to change colour I will compare it to the sample, and when I think they are the same colour I will stop the clock and record the time that it took to change colour.
I repeated this twice and got the results as shown below

This is the method I used for my preliminary experiment. I will use the same method for my final experiment, but I will use the concentrations 1%, 0.8%, 0.6%, 0.4% and 0.2%. I will repeat the experiment three times for each concentration, which will give me 15 results. I will do this so that I can notice any patterns in the data, and it will allow me to do an average of the results so I can plot graphs with them.

I will then choose to concentrations which I think don’t have a very big difference in time, I will then repeat the experiment ten times on these two concentrations, I will do this so that I can do a statistical test on them. The statistical test will tell me if there is a difference between the two concentrations or if they are the same.

I chose to do this method because it worked well when I done my preliminary experiment, so I will use it to do my final experiment.

To do different concentrations of sucrase I will have to dilute them.

I will dilute the concentration of sucrase by using distilled water. I will use this because it doesn’t contain any chemicals.
I will use a 5cm3 syringe to put the correct amount of sucrase into the test tube.
Then I will use a different syringe to put in the right amount of water to get the right dilution.
To get 1% enzyme concentration I will use 5cm3 of sucrase.
To get 0.8% enzyme concentration I will mix 4cm3 of sucrase and 1cm3 of distilled water.
To get 0.6% enzyme concentration I will mix 3cm3 of sucrase and 2cm3 of distilled water.
To get 0.4% enzyme concentration I will mix 2cm3 of sucrase and 3cm3 of distilled water.
To get 0.2% enzyme concentration I will mix 1cm3 of sucrase and 4cm3 of distilled water.

Apparatus:

Risk Assessment: I will were goggles for eye protection because the chemicals could splash into my eyes. I will be careful not to spill any solutions on my skin because they could irritate it.

Results

Analysis

Conclusion- I have proved that my experimental hypothesis is correct. I have done a statistical test which proved that there was a difference between 0.6% and 0.8% concentration of sucrase.

I think that my data is a good set of results. The data for the 5 different concentrations of sucrase shows a difference, I plotted a graph for the averages of these results. The graph shows an increase in time for the reaction to end as the concentration decreases. The trend is that as the concentration of sucrase increases the time for the reaction to end decreases. This is because as the enzyme concentration increases, there are more active sites for the enzyme to collide with, so the substrate is broken down quicker.

Evaluation

When doing 0.8% concentration, I got the time 4.59 minutes. This is an anomalous result. 0.8% has an average of 3.24 minutes, which shows that it is anomalous.

I think that the accuracy of the measurements could have been improved, the measurements were not that accurate because the syringe didn’t have decimals on it. I could have used a burette with is measured in decimals which would have give me much more accurate results.

There could have been anomalous results because there could have been slightly more sucrose in than 5cm3 which would have taken the sucrase longer to breakdown. The sucrase could have been diluted too much by accident, which would have taken the sucrose longer to be broken down. The solution could have got contaminated, by the syringes getting mixed up and some sucrase could have been in the sucrose. The variation in temperature could have caused an anomalous result. The variations in the amount of benedict’s solution used could have cause reaction to take longer to end.

I think the reliability of the evidence is good. Every different concentration was put under the same conditions and each solution was measured in the same way.

The main source of error was the way that I measured the end point. I used a sample to measure the end point, and I judged the colours with my eyes. The colours could have been slightly different but I couldn’t tell. By judging the end point this way, I might have stopped each reaction in different places, meaning each reaction had different end points, this would make my results not very reliable. The sample would also still be reacting, so it would get darker, so near the end of the experiment I would be stopping the reactions at a longer point. I could improved the way I judged the end point by using a colorimeter. I would tell me the exact colour of the solution, so I could stop each reaction at the same place, this would make my experiment much more accurate and reliable.

The strong correlation from the data sown in the graphs and in the tables shows that the limitations that have been described has not had a great effect on my result, and my conclusion therefore have a high level of validity.

I drew a graph with error bars, which shows that there is no overlap with different concentrations of sucrase. This shows that as the concentration of sucrase increases the reaction time is faster.

Jones, M. (ed).(2000).Biology 1. University press: Cambridge. ISBN: 0 521 78719 X
Introduction to Enzymes. (2003). Worthington biochem. URL:
Green, N.P., Taylor, D.J. Biological Science, Cambridge University Press, 1984.

Investigation into the effect of enzyme concentration of catalysers of sucrose by sucrase.

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