Immobilized Enzymes (Yeast)

 Table 1 showing some common application of immobilized enzymes.

Catalase is an enzyme that is found in most living tissues.  It breaks down peroxides, which are generated in these tissues and are harmful.  Yeasts, which contain catalase, are therefore used in this investigation.  They are immobilized on the surface of sodium alginate, i.e. microencapsulation.  The decomposition of hydrogen peroxide is sped up as the enzyme catalase breaks down hydrogen peroxide into water and oxygen, which escapes as gas bubbles and is trapped in the burette: -

2H2O2(l) → O2(g) + 2H2O(l)

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Method

Preparing immobilized yeast

3g of calcium chloride will be weighed out using a 2 decimal place top pan balance.  The calcium chloride will be dissolved in 200cm3 of distilled water measured from the 100cm3 measuring cylinder using a glass rod.  The 1.5% calcium chloride solution will be then made up and will be contained in the 250ml beaker.  2g of yeast will also be weighed out using the balance and will be dissolved in 10cm3 of distilled water measured by a 10ml plastic syringe to make 50% yeast suspension, and will be mixed well by syringing the suspension a couple of times.  0.4g of sodium alginate will be weighed out using the balance and will be dissolved in 50cm3 of distilled water and will be kept in the autoclave to prevent solidification.  A 5ml plastic syringe will be used to transfer 2ml of yeast suspension and 8ml of sodium alginate solution to a 100ml beaker and will be mixed well be syringing the mixture several times.  5ml of the alginate/yeast mixture will be sucked up and will be added to the calcium chloride solution one drop at a time.  The rest of the mixture is covered by a cling film to prevent contamination of the enzymes.  The beads will then be allowed to set for a few minutes.  A plastic tea strainer will be used to separate the beads from the calcium chloride solution.  

Investigating the rate of reaction on hydrogen peroxide solution

The apparatus are then set up as shown below to measure the rate of reaction on hydrogen peroxide solution:-

5 alginate beads will be placed in the conical flask and will be stoppered well with a stopper connected with a delivery tube.  The burette will be filled with water and will be inverted over the delivery tube to collect gas bubbles that will be produced when the reaction starts.  The pinch valve will be used to adjust the level of water and will be recorded.  70cm3 of hydrogen peroxide solution will be measured by a 100cm3 measuring cylinder and will be poured gradually to the flask through the Thistle funnel.  The solution will be in excess to provide excess substrate molecules and to make sure the end of the funnel will be covered by the solution that the gas produced in the reaction will not leave the system through the end of the funnel.  The bubbles produced while pouring the hydrogen peroxide solution in will not be collected as there is air in the flask, which is not oxygen gas produced in the reaction.  The burette will then be placed at the end of the delivery tube to make sure no gas bubbles will be left out.  The reaction will be timed when the first bubble appears.  The readings on the burette will be taken after 3, 5, 7, and 9 minutes.  The experiment will be repeated using different number of beads: 5, 10, 15, 20, 30, 40, 50, 60, 70 and 80.  

Apparatus List

Choice of Apparatus

Yeast will be used as the enzyme for the investigation since there is no ethical problem.  The enzymes will be attached on the sodium alginate beads, as it is non-poisonous.  50cm3 burette will be chosen to collect oxygen bubbles because it has a narrow bore, and small graduations from which readings can be read off accurately.  

Predictions

Increasing the number of alginate beads, i.e. yeast concentration with excess substrate molecules, the volume of oxygen collected should increases in direct proportion.  The rate of reaction will also be directly proportional to the enzyme concentration.  The reasons of both predictions are because there are more active sites available for the substrate to fit in which will be able to form more products: -

Modification

While investigating the rate of reaction on hydrogen peroxide solution, 5 alginate beads were first used.  However no gas bubbles were produced in 5 minutes.  Ten more beads were the added to start the experiment with 15 beads.  Gas bubbles were produced very slowly.  Thus the minimum number of beads for the investigation was set to be 15 and the experiment was repeated using the following number of beads: 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 and 80.  

Risk Assessment

Variables:

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Data Analysis

During the investigation, the following data were recorded and graphs were plotted.  

Figure 1 showing the burette readings after different times using a various number of alginate beads.

Figure 2 showing the volume of oxygen gas generated after each time interval and is plotted as figure 4.  

Figure 3 showing the gradient (or rate) of oxygen gas produced under different number of beads.

Figure 5 is then drawn to show the rate of reaction against the number of beads.

Calculating the equation of the regression line

Let the equation be y = a + bx

Σx = 665  ;  Σy = 237.18  ;  Σx2 = 37275  ;  Σy2 = 4686.3604  ;  Σxy = 13130.95  ;  n = 14

b        = n∙Σxy – Σx∙Σy

        n∙Σx2 – (Σx)2

        = (14)(13130.95) – (237.18)(665)

                (14)(37275) – (665)2

        = 0.327894505…

a        = Σy – b∙Σx

        n

        = 237.18 – (0.327894505…)(665)

                        14

        = 1.366439561…

Equation is y = 1.37 + 0.328x      (correct to 3 sig. fig.)

Finding the product-moment correlation coefficient, r

r        =         n∙Σxy – Σx∙Σy        

          √ [n∙Σx2 – (Σx)2] [n∙Σy 2 – (Σy)2]

        =                 (14)(13130.95) – (237.18)(665)                

          √ [(14)(37275) – (665)2] [(14)(4686.3604) – (237.18)2]

        = 0.956630201…

        = 0.96      (correct to 2 d.p.)

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Discussion and Evaluation

Conclusion

From figure 5, increasing the concentration of enzymes would increase the rate of reaction, i.e. the rate of reaction was directly proportional to the amount of enzymes which was roughly the same as the original hypothesis, except the regression line did not pass through the origin.  It was because the hydrogen peroxide might decompose to form oxygen gas without any catalase.  Further details will be discussed in the ‘improvements’ section.

From figure 4, the best-fit line among the 14 coloured lines was the purple line with 55 alginate beads.  New beads were used for the experiment, which means the oxygen gas collected was all produced by an enzyme concentration of 55 beads.  Whereas for the others, the beads were used to carry on the next experiment which the oxygen gas might not be all produced from the respective number of beads. Further details will be discussed in the ‘limitations’ section.

Limitations

1. The beads were not identical.  There were an uneven number of yeast cells in each bead, which made it difficult to control the exact amount of enzymes added to the hydrogen peroxide.  If a bead containing a larger amount of yeast cells was used, the reaction rate would increase and would affect the reliability of the results.
2. During the experiment was carried out, it was observed that one bubble decreased about 0.2cm3 of the burette reading.  This increased the time interval between each bubble.  Thus some of the gas could not be collected at the time the reading was taken, which had affected the reliability of the data obtained.  
3. The yeast cells were not as efficient as expected.  They were thought to be reused for each experiment.  However the beads floated onto the surface of the hydrogen peroxide solution after they were added in for a few seconds, since oxygen gas was formed on the active sites of the enzyme but they did not fall out completely.  This had blocked the hydrogen peroxide substrate to fit in the active site, i.e. acting as an inhibitor.  The values of oxygen gas produced were less than expected, and the oxygen produced for the previous experiment might carry along to the next one in which the number of beads had changed.  There was a significant error and had seriously affected the reliability of the results.

Percentage uncertainty

Uncertainty value of the 50cm3 Grade B burette = 0.05cm3

Average volume

Largest percentage uncertainty in volume of oxygen gas collected = 0.05/21 = 0.00238%  (3 s.f.)

The value is small enough to be ignored, which means the data obtained are precise enough.  

Improvements

1. A control experiment should be done when investigating the rate of reaction.  The set-up should be the same but without adding any immobilized yeast.  There might be a change of the burette reading after each time interval, since hydrogen peroxide decomposes easily into water and oxygen gas.  The change of the reading should also be included in the table of result and should also be plotted on figures 4 and 5.  
2. Under observation during the experiment was carried out, 1 bubble decreased about 0.2cm3 of the burette reading.  This increased the time interval between each bubble.  Thus some of the gas could not be collected at the time the reading was taken.  A smaller internal diameter of delivery tube should be used to improve accuracy of the volume of oxygen gas produced.  
3. New beads should be used each time as oxygen produced on the active sites of the enzymes did not fall off, which blocked the hydrogen peroxide substrate to fit in, and evidence was obtained from the purple line with 55 alginate beads which had proved the reliability of using new beads for each experiment.
4. Since the rate of reaction of catalase in the yeast cell is concerned in the experiment, only catalase should be immobilized instead of a whole yeast cell which will be more precise.  

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