Investigating the effect of concentration change in Immobilised enzymes

Aim

I will investigate how change in concentration of immobilised enzymes affects the rate of reaction.

Hypothesis

I predict that the rate of reaction will increase, as the concentration of enzyme increases. Immobilising the enzyme may change the rate of reaction to using free enzyme.

(see prediction for details)

Theory

I will test my hypothesis by keeping the concentration of enzyme the variable in my experiment. I will use Amyloglucosidase (AMG) as my enzyme and Starch solution as my substrate.

AMG hydrolyses α 1, 4 and α 1, 6 linkages (bonds) in Starch. During hydrolysis, Glucose units are removed in a stepwise manner from the non-reducing end of the Starch molecule.

The rate of hydrolysis depends on the type of linkages in the substrate as well as its chain length. α 1,4 linkages are more readily hydrolysed than α 1,6 linkages. For example Maltose is hydrolysed at a low rate because it contains 1, 6 linkages.

Starch is made from glucose units linked to form either a linear polymer called Amylose or a branched polymer called Amylopectin. Glucose units in both polymers are linked by α 1, 4 bonds, the sides that branch into Amylopectin are linked by α 1, 6 bonds. Both Amylose and Amylopectin are broken down into extra cellular amylases that are produced by many kinds of organisms, including bacteria and fungi. Apart from Amyloglucosidase, many other enzymes act on Starch in different ways such as α-Amylase, β-Amylase, Pullulandase and Glucose Isomerase.

Saccharification is the process by which Starch is made sweeter by treatment with AMG. The AMG can theoretically hydrolyse Starch completely to Glucose. In practise a little Maltose and Isomaltose are produced too. This is a catabolic reaction by which the polysaccharide, Starch is broken down into its monosaccharide, Glucose as it reacts with water. The enzyme AMG will speed up the reaction by reducing the activation energy needed for the reaction to take place, AMG catalyses the reaction.

Enzyme + Substrate → Enzyme Substrate Complex → Enzyme + Product
AMG + Starch → AMG + Starch Complex → AMG + Glucose

An immobilised enzyme is an enzyme that is bound to a surface so that they are not able to dissolve. They take form of small beads. There are many techniques of immobilisation. I will use gel entrapment, the enzyme is mixed with gel-forming ingredients and when the gel forms the enzyme remains trapped in the gel matrix. The pores are large enough to let the substrate in but not let the enzyme out.

I will use this method because it is the quickest and most effective way of immobilising AMG in a school laboratory environment. I will use Sodium Alginate solution as my gel forming ingredient.

The first commercial use in the 1970’s, was the development of immobilised Glucose Isomerase for the preparation of high-fructose syrups from Starch. Other uses of immobilised enzymes include:

Furnase to turn Furnatic acid into Malic acid for the food use.
Use in the pharmaceutical industry of Pericillin Amidase to prepare 6-APA from negative Penicillin.
Use of Nitrile Hydratase to prepare Acryamide from Acyloritrile.

The advantages of immobilisation are:

Prevention of losses due to flushing away of enzyme
A more stable enzyme produced
The possibility to produce an enzyme with altered properties
The enzyme can be removed and reused over and over again
The enzyme may be more stable at extreme pH or temperatures and it may therefore last longer
The enzyme does not denature
The product is not mixed in with the enzyme and doesn’t need separation which reduces costs.
It allows continuous processing rather than a batch system. This allows for a much smaller set-up and reduces capital costs.

Immobilising enzymes also has disadvantages:

Losses of enzyme activity can occur during the making of the beads
Diffusion of substrates and products may take place by separating the enzyme into an immobilised layer.
The enzyme may have a more constrained conformation in the immobilised state, giving it a lower catalytic activity.
May be a high initial investment for the immobilisation, compared to the free enzyme

Immobilisation is still used widely in industry especially for the production of sweeteners. Many people think that the advantages out weight the disadvantages.

Prediction

As stated in my hypothesis, I predict that the rate of reaction will increase at a steady rate, as the concentration of enzyme increases. Immobilisation of the enzyme will decrease the rate of reaction. So on the whole, reactions between immobilised AMG and Starch will be slower than using free enzyme in solution.

At low AMG (enzyme) concentrations there are more Starch (substrate) molecules than available active sites, so the reactions will be slow. By increasing the concentration of AMG, the number of active sites will increase and therefore the rate of reaction will increase. Eventually when it reaches optimum point, increasing the AMG concentration will have no effect on the rate of reaction. This is because the number of Starch molecules will now become the limited factor because there are now more enzyme molecules than substrate molecules.

Immobilising the enzyme will decrease the rate of reaction because the enzymes have a constrained conformation. This causes a lower catalytic activity as there is less chance of a collision between the enzyme and substrate. Free enzymes are constantly moving in solution and therefore have a greater chance of colliding with the substrate. I will find out if my prediction of immobilisation is correct because I am not investigating the effects of immobilisation.

I predict the graph showing the effect of enzyme concentration against rate of reaction will look as follows (only if the substrate is in excess):

Preliminary Work

I will carry out some preliminary experiments for one concentration of AMG to test my method and to make any corrections or modifications if necessary. It will also give me some practise of doing my experiment so I do not make any mistakes and can collect accurate results.

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Method 1: I will use 1cm³ of AMG enzyme and mix it with 10cm³ of Sodium Alginate solution. Then I will draw up the mixture into a syringe and drop it into 100cm³ of Calcium Chloride solution to form my immobilised enzyme beads. I will then separate the beads and pour them into a syringe to form a column. Then I will pour 50cm³ of Starch solution into the column. When my experiment is set up, I will open the tap at 30 second intervals to test the Starch solution.

I can find the effects of the enzyme by testing the Starch solution. There are two ways of testing my Starch solution; I can either test for the amount of Glucose produced or the amount of Starch still to be hydrolysed.

Test 1: I can test for the presence of Starch by using the Iodine test. After dropping Iodine solution onto the sample, if it turns blue/black this indicates that there is Starch present, but if it does not this means there is no Starch present.

Test 2: I can test for the presence of Glucose by using the Diabur-test 5000. This will tell me the amount of Glucose present for up to 2%. The Diabur strip will change colour depending on the percentage of Glucose produced. The Diabur test will give me a range of greens and browns and I will need to use the chart to match the colour of the strip to find out what percentage of glucose has been produced.

For my preliminary experiment I will use both tests to find out which test works better and which to use for my main experiment. I will stop the experiment when the Iodine test gives a negative result showing that all Starch has been hydrolysed.

Results

I have found out that the concentration of Glucose increases with time. This shows that Starch is being broken down into Glucose by the enzyme. More than 2% of Glucose was produced by 150 seconds. My results show that it takes 26 minutes (1560 seconds) for 1 cm³ for the AMG enzyme to completely hydrolyse 50 cm³ of Starch solution into Glucose.

I stopped the experiment at 26 minutes because iodine solution remained its original orange/red colour which showed that there is no Starch present. This experiment was very time consuming and therefore I may not use Iodine test in my main experiment.

The Diabur test only shows the amounts of Glucose present up to 2%, and therefore does not show the percentage of Glucose produced if it is over 2%, but only tells me that it is over 2% by turning a dark shade of brown. The reaction using 1cm³ of enzyme was much faster than I expected and it may be difficult to analyse the rate of reaction.

My preliminary work has helped me to refine my method. I will make changes to my procedure to make it more accurate and effective for my main experiment. It has also reassured me that my method works and can give me reliable results to analyse. Carrying out my preliminary experiment has given me practise of doing the standard immobilised enzyme experiment.

My preliminary work has helped me make these changes:

I have found out that using the Iodine test is to time consuming for my experiment because the rate of hydrolysis is very slow. I will need to do three repeats for my main experiment and therefore spending 30 minutes (approx) on each reaction is to long. I will not have enough time to complete my repeats. The experiments using Diabur test are faster than I expected, but as the concentration of the enzyme decreases, I predict that the time taken will increase. Therefore I will use 1cm³ as my highest concentration so this shouldn’t be a problem. Also the Diabur test is much more useful than the Iodine test because the Diabur test tells me the amount of Glucose produced. The Diabur will give me a reading of 0%, 0.1%, 0.25%, 0.5%, 1.0% and 2% whereas the Iodine test tells me if Starch is present or not, in a Yes/No answer. It will be easier to produce a graph from the range of results of the Diabur test and therefore easier to analyse. An advantage of the Diabur test only going up to 2% is that my experiments will not take very long, so I can take my time to carry out each experiment, checking for health and safety as I go. More time will help me carry out my experiments more safely and accurately.

Ideally I should test the Starch solution at much more regular intervals, for example every 15 seconds would be more appropriate because it is a fast reaction. I can not increase my time intervals because I only have a limited amount of Diabur test strips as they are very expensive. Also taking a reading every 15 seconds will be rushed and I may not get an accurate reading. I have decided to have a 30 second time interval to test the Starch solution. I have already used this time interval for my preliminary experiment and found that it works well and is easy to carry out.

Also from my preliminary work I have found that I only need to use 25cm³ of Starch solution instead of 50cm³. This is because I am using a 20cm³ syringe barrel to form my column of beads and only 25cm³ of the solution fits into the syringe if I fill the barrel to the top.

I have found that when adding the Sodium Alginate solution to the enzyme, bubbles start to form by stirring the mixture because of this bubbles are trapped in the beads. This may lead to inaccurate results therefore I need to be careful to avoid the formation of bubbles; I can do this by stirring slowly.

Now I ready to start my main experiment and I will make the modifications stated above.

Main experiment

I will change the concentration of the enzyme by using the same volume of solution each time but I will change the amount of enzyme used each time. I will add distilled water to dilute it and make up 1cm³ of solution each time.

The table shows the concentrations of enzyme solution I will use. I am using 5 different concentrations.

Method

Equipment and materials:

Amyloglucosidase (AMG) 1cm³
5% Sodium Alginate solution 10cm³
Calcium Chloride solution 100cm³
1% Starch solution 25cm³
Semi-quantative Glucose test strips - Diabur test 5000

10 cm³ plastic syringe
Glass rod
2 × 100cm³ small beaker
Small sieve/ tea strainer
Small nylon gauze
20cm³ Syringe barrel with small tube attached to nozzle
Tubing clip (Hoffman clip)
Clamp stand
Stop clock
Spotting tiles

Method

Add 1cm³ of enzyme solution to 10cm³ of 5% Sodium Alginate. Carefully stir the enzyme into the Alginate solution to ensure a thorough mix. Try not to stir bubbles into the mixture.

Using a 10cm³ syringe draw up the enzyme alginate mixture, again avoiding the formation of air bubbles.

Drop the enzyme alginate mixture drop by drop into the Calcium Chloride solution so that it forms small regular beads

To ensure the beads are fully set, leave them in the Calcium Chloride for 10 minutes.

Separate the beads from the Calcium chloride solution using a small sieve.

Cut a disc of nylon gauze and fit it into the bottom of the syringe barrel. This will prevent the column becoming blocked by one of the beads. Hold the syringe using a retort stand.

Pour the beads into the syringe barrel to form a column.

Test the Starch solution for the presence of Glucose with a Glucose test strip (Diabur test 5000)

Pour the Starch solution into the column so that it runs slowly over the enzyme beads, (make sure the clip is closed when pouring) controlling the rate of flow with the tubing clip.

Test a few drops of the processed Starch for the presence of Glucose using the Diabur test strips. Take this test every 30 seconds until there is 2% glucose present, shown by the Diabur strip turning dark brown, use the colour chart to help identify the percentage. The apparatus should be set up exactly as in the diagram below:

Repeat the experiment using all the different concentrations of enzymes shown in the table.
Repeat the same experiment for all the steps from 1-11 two times.

Risk Assessment

When AMG (liquid and granulate) is stored at a temperature of 25 °C, the declared activity is maintained for at least six months. When stored at 5 °C the product will maintain the declared activity for at least one year.

AMG is non-flammable, completely miscible with water and safe when used according to directions (on container).

The granulate form of AMG is more hazardous. Enzyme dust may cause irritation when inhaled. Avoid direct contact with the product or inhalation of dust from the dried product. In case of accidental spillage and contact with skin or eyes, rinse promptly with water, may cause allergic reaction or minor irritation.

Liquid enzyme preparations are dust free preparations. However inappropriate handling may cause formation of dust or aerosols.

Spilled preparations should be removed immediately to avoid formation of dust from dried preparations. Take up by mechanical means, preferably by a vacuum cleaner equipped with a highly efficient filter. Flush remainder carefully with plenty of water.

Starch solution, Sodium alginate, and Calcium chloride solution are not harmful but avoid contact with eyes and skin, and in case of accident rise thoroughly with water.

To protect myself and avoid accidents I will do the following:

Wear protective glasses- goggles
Wear impermeable gloves
Wear suitable protective clothing- Lab coat

Enzymes are used in industry but they can cause harm to the environment. This risk is low because they are specific in their action and are therefore less likely to produce unwanted by-products. They are biodegradable and so cause less environmental pollution. They work in mild conditions i.e. low temperatures, neutral pH and normal atmospheric pressure and therefore save energy.

Fair test

I need to keep my experiment a fair test to achieve accurate results. I will do this by keeping all variables controlled. The only independent variable in my experiment will be the concentration of enzyme. The only dependent variable will be the time taken

I will have to use a fresh amount of Sodium Alginate solution and Calcium Chloride for each experiment, but I can keep them constant by using them from the same preparations each time. I will keep the amount of Sodium Alginate solution and Calcium Chloride solution constant.
I will use AMG enzyme and distilled water from the same containers each time
I will use Diabur strip from the same container each time (same batch number)
I will do all my experiments at room temperature and pressure
I will keep the size of the enzyme beads constant, I will use 0.05 cm³ of solution for each bead.
I will keep the concentration and amount of Starch substrate constant
I will use the same apparatus each time e.g. beaker, syringe, stop watch etc.

Results

0.2cm³ of enzyme, 0.8cm³ of distilled water:

* shows any anomalous results

0.4 cm³ of enzyme and 0.6 cm³ of distilled water:

* shows any anomalous results

0.6 cm³ of enzyme. 0.4 cm³ of distilled water:

* shows any anomalous results

0.8 cm³ of enzyme, 0.2 cm³ of distilled water:

* shows any anomalous results

1.0 cm³ of enzyme, 0.0 cm³ of distilled water:

* shows any anomalous results

I have carried out all my experiments for all the concentrations including 3 repeats. My experiments were successful. The 3 repeats of all the experiments gave very similar results. This shows that my results are reliable and I can now analyse them. I have also worked out the average of the 3 repeats shown in my results tables, but I have left out any anomalous results.

I have worked out the average time taken by adding the 3 repeats together and dividing by 3:- E.g. for 1.0cm³ of enzyme, >2% glucose:-

(180 + 150 + 180) = 170 seconds

Analysis

I will now analyse my results in comparison to each other. I will use them to identify any trends and patterns found. I will construct some graphs and tables to help me come to a conclusion.

Firstly I will produce a graph from the results of all concentrations of enzymes showing the amount of glucose produced against the average time taken.

A graph showing the percentage of Glucose present against time taken, for different concentrations of enzyme:

The graph shows that the percentage of Glucose increases as the time increases. This is the simple trend for all concentrations. All the graphs have strong positive correlation. The graphs become steeper (the gradient increases) as the concentration of enzyme decreases, this shows that as the concentration of enzyme increases as the time taken decreases. Similarly, the time taken for 2% Glucose to be produced decreases at a steady rate as the concentration of enzyme increases for example, at 0.2 cm³ it takes 1250 seconds and at 1.0 cm³ it takes170 seconds.

I will now use the time taken to convert it into rate of reaction, this will help me compare the relationship between rate of reaction and the percentage of Glucose produced which will help me come to a conclusion. The rate of reaction is calculated by the reciprocal of the time taken (1÷ Time taken).

E.g.: for 1.0cm³ of enzyme concentration the time taken to reach 0.5% Glucose was 40 seconds, so the rate of reaction at 0.5% Glucose is: 1 ÷ 40 = 0.025

E.g.2.: for 0.8cm³ of enzyme concentration the time taken to reach 0.5% Glucose was 220 seconds, so the rate of reaction at 0.5% Glucose is: 1÷ 220 = 0.0045 …etc

I have drawn a graph to show the rate of reaction against percentage Glucose produced relationship:-

The graph shows that the rate of reaction increases as the concentration of enzyme increases. At a low percentage of Glucose there is a higher rate of reaction and at a high percentage of glucose there is low rate of reaction. For example for the concentration of 0.2 cm³, at 2% glucose the rate of reaction is 0.008 and at 0.1% glucose the rate of reaction is 0.0019.There is a steady increase in rate of reaction until 0.1% Glucose for all concentrations, this is the optimum rate of reaction (fastest) and there is a steady decrease after this point. The rate of increase is higher than the rate of decrease, showing that the rate of reaction is faster during the beginning of the experiment and slows down near the end.

Conclusion

I predicted that the rate of reaction will increase at a steady rate, as the concentration increases and that immobilisation of the enzyme will also decrease the rate of reaction and the reactions between AMG and Starch should be slow. My prediction was correct, but to find out the effects of immobilisation I will need to do further experiments using free enzymes. The time taken for the reaction varied from 170 seconds to 1250 second (21 minutes) showing the dramatic effect of changing the concentration of enzyme. The more enzymes there are, the faster the reaction will take place. Rate of reaction and time taken are proportional to each other therefore the rate of reaction increases as the time taken decreases. This concludes to the statement that when there are fewer enzymes, the reaction will be slow and when there are more enzymes, the reaction will be fast.

This is because at low AMG enzyme concentration (0.2cm³) the reaction will be slow because there is not enough active sites on the enzyme to occupy all of the Starch substrate molecules so less collisions can take place between enzyme and substrates. As the enzyme concentration increases (0.6cm³) the rate of reaction increases because now more enzyme substrate complexes can be formed. At the point when 0.1% Glucose is produced by the enzyme, increasing the enzyme concentration will not increase the rate of reaction because now all the active sites are being used. This is the optimum point which has the fastest rate of reaction. After this point the rate of reaction gradually decreases producing a curve because the number of Starch substrate molecules is now the limited factor. The active site on the enzyme has to collide with the non-reducing end of the starch to break the α 1, 4 and α 1, 6 linkages (bonds) between the Carbons. The decrease in rate of reaction could be due to the immobilisation of the enzyme, causing a constrained conformation making it difficult for the Starch to get into the pores of the beads. Immobilisation may have caused the AMG to lose it activity as the experiment progressed. These factors can cause a decrease in rate of reaction after the optimum point.

Evaluation

I think my results are reliable because they are repeatable. I have repeated my experiments 3 times and have only found slight changes but they all still contain the same pattern and trends.

Therefore I can say my conclusions from this experiment are reliable and accurate for the reaction between immobilised AMG and Starch solution.

I think this my method was very suitable for this investigation as it was simple to carry out. It had no complications and gave accurate results. But my method had many limitations. The Diabur test was a limitation because it only gave percentage of glucose produced up to 2%, for percentage above 2% the values were not stated, which lead to my experiments being short which was an advantage as I had to carry out 15 experiments.

Also the Diabur test did not change colour straight away, it took about 30 second for the Diabur strips to change colour, and sometimes I had more than one reading for >2% glucose because I did not know I had reached 2% until the Diabur changed colour . Also I did not have very long to carry out my experiments, if I had more time I would have investigated more concentrations to make sure my results were reliable.

There were many errors in my procedure which may have caused my results to be inaccurate. It was easy to test using Diabur strips but because my time interval was every 30 seconds, I had to be very quick and attentive to make sure I place the strips in the right order. Also I had to be careful not to touch the top of the strip which reacted with the starch because I would contaminate the strip and so my results would not accurate, which was quite fiddly to do in 30 seconds. Also it was difficult to test exactly at 30 second so my results may be 1 or 2 second out, this reduces the accuracy of my results. This experiment is very time consuming as I can not keep the beads for more than a day. I had to make new bead for each reaction, making the beads and setting up the experiment took approximately 30 minutes, so I had to spend 30 minutes at the begging of every lesson to do this which considerably reduced my time to actually carry out the experiments. This made me to rush my experiments causing more chance of mistakes and accidents. I also had to use lots of Diabur strips, I only had a limited supply as they are very expensive to use so I had to cut each strip into two to reduce costs, this was very time consuming and fiddly. I was difficult to produce beads of exactly the same size by using a syringe, different sizes of beads could have reduced the accuracy and reliability of my results as bead size is also a factor affecting the rate of reaction of enzymes. To keep my experiment a fair test I decided to use all my solution from the same preparations but in practise there was purposely not enough made because the solutions would have lost there affectivity if left for to long so I had to use different preparations of solutions such as Sodium Alginate, Calcium chloride, and Starch solution. This may have affected the reliability of my results.

There were no errors in the procedure except for the measuring equipment used, e.g. beakers and syringes are not 100% accurate and there may have been slight errors caused by this. When pouring a solution into a beaker, you have to be at eye level to get an accurate reading.

I could improve my method to reduce the main sources of error. I would make sure the beads are already made for me before the lesson and make sure they are of accurate sizes and have no bubbles in by using industrial machinery. I would increase my time intervals to 1 minute instead of 30 seconds, which will give me more time to organise my Diabur strips. It will be ideal to work in pairs, or groups for more organisation. I would explore other ways of testing for Glucose which are easier and cheaper than using Diabur strips. I would make a larger preparation of each experiment enough to last for all the experiments and I would do all my experiments in one day. This would be easier to do as the immobilised beads will only have to be made once, because they do not get used up in the experiment and are active for about one day. This would also reduce the chances of error as I have more dependent variables.

I had a few anomalous results, when testing the solution with the Diabur strips. At zero all the strips gave a reading of 0% which was accurate. After that the Diabur should turn light green and graduate to dark green and light and dark shades of brown. On a few occasions after zero usually at 30 second or 1 minute the Diabur test turned black and then the rest the readings were normal. This may be because some of the previous Starch solution was left on the tap because I may not have rinsed it out thoroughly causing errors, so when I opened the tap to test the starch solution at 30 second I was actually testing the starch solution from the previous experiments which was over 2% glucose produced. I think it turned black because it may have been a very strong concentration of Glucose, a lot more than 2% so the percentage was off the scale of the Diabur test.

I could gather further evidence to support my conclusion. I would do this by carrying out further investigations and experiments. To test the reliability of my results I would repeat my experiment still using immobilised enzymes but using a different enzyme and substrate. I would use Lactose as my enzyme and immobilise it using the same procedure as before. I would mix the lactose with sodium alginate and form beads in calcium chloride solution. Then I would set the bead up in a column as before and use pasteurised milk as my substrate. I would pour the milk over the beads as I did with the starch and use the Diabur test and investigate other ways of testing which are more effective. I would also do the same test using AMG as my enzyme and Starch solution as my substrate but I would not immobilise the enzyme and use it free. This will help me compare the free enzyme to immobilised enzymes and I can come to a conclusion for the effect of immobilising enzymes.

Investigating the effect of concentration change in Immobilised enzymes

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