"An investigation to find out the optimum temperature for the activity of Lipase".

Step 6:        We then time how long it took for the pink colour change to a white mixture indicating the fat had been hydrolysed.

Step 7:         The beaker of water was then allowed to cool to 60°C and steps 4 to 6 were repeated.

Step 8:         The water was further cooled to 40°C and steps 4 to 6 were repeated.

Step 9:         Then the water was cooled to 20°C and steps 4 to 6 were repeated.  

Step 10:        Finally a control experiment was preformed using tap water at room temperature following steps 4 to 6.

Safety procedures

Lipase is an irritant substance so I took care not to get it on my skin by wearing surgical gloves and goggles to prevent contact with my eyes.

Phenolphthalein is flammable liquid so I made sure I kept it away from any naked flames such as Bunsen burners.

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• Results

Below is a table of the times it took for lipase to convert enough fats into fatty acids and glycerol to lower the pH below 8.3 so that the indicator would show this.

If the results are put into a scatter graph (below) and a polynomial trend line added you can see the rate of reactions in comparison.

At 20°C we can see the lowest ROR at 2.9 g/s, next is the control experiment which was carried out at room temperature 21°C and the ROR was slightly higher at 3.2 g/s.

At 40°C the rate of reaction increase noticeably up to 5.2 g/s as the time taken was nearly halved.

At 60°C there was a slight increase in ROR than previously with the time taken only slightly quicker.

When tested at 80°C the reaction surpassed the allocated 30min barrier set out and there was no change in the colour of the mixture.

From the graph we can see the general trend that if you raise the temperature you will get an increased rate of reaction (ROR) until a certain temperature, when the reaction slows and stops.

• Discussion

At 20°C the ROR was slowest due to the kinetic energy being applied. Heat provides kinetic energy to the molecules in the mixture, which increases the speed at which they travel about. The chemical reactions in this experiment rely on random collisions between the Lipase and the fat in the milk. At 20°C the kinetic energy is low so the particles will only be travelling in the mixture at a slow speed, which results in a slower ROR because fewer enzymes and substrates collide less frequently.

(Boyle, Indge, Senior, 1999)

For the control experiment, at 21°C the ROR was slightly higher due to a small increase in kinetic energy. At this slightly higher temperature there is more a little kinetic energy so the molecules are moving faster resulting in more collisions therefore we see a higher ROR.

At 40°C we saw a steep increase in the rate of reaction from 2.9g/s at 20°C to 5.2g/s at 40°C. At 40°C there is a large increase kinetic energy from 20°C so the molecules are moving around much faster, resulting in many more collisions than previously so there is more successful enzyme reactions taking place faster which increases the ROR.

At the temperature of 60°C the ROR was only slightly higher than for 40°C. the increase in kinetic energy has increased the ROR but by only seeing a slight increase it suggests that denaturing has began to occur. As the Kinetic energy increases, as well as the molecules moving faster the lipase molecules vibrate more violently. At a certain point this vibrating becomes too great and the weak hydrogen bonds that hold the proteins into its specific protein structure begin to break. Each enzyme is specific and each work to catalyse one reaction. E.g. Lipase hydrolyses Lipids, or Amylase breaks down Starch. (Roberts, 1986)

These breaks result in the lipase’s structure shape changes, which changes the shape of the active site. With a different shaped active site the substrate will be not fit into it and will no longer be able to form complexes with it. This specificity is due to shape of their active site due to the precise protein structure each enzyme has. (Green, Stout, Taylor, 1991) How the active site makes each enzyme specific can be understood more clearly by looking at the Lock and Key hypothesis put forward to how enzymes react. It suggests that for each enzyme there is a “groove” called an active site (seen as the lock) and there is one specific substrate (the key) that can fits into this groove in order for them to react. (Simpkins, Williams, 1987) (See diagram 1 below)

If denaturing occurs then the precise structure of the enzyme is alters, altering the shape of the active site. Denaturing is irreversible and once it has occurred renders the enzyme useless as the substrate can no longer ft into the active site. (Fullick, 1994) This in turn lowers the ROR because fewer enzymes are now available to form complexes with the fats. (Toole, Toole, 1995) (See diagram 2 below)

Even though at 60°C denaturing has began to take place it still proved to be the optimum temperature for the activity of lipase in this investigation.

At the top temperature of 80°C due to the large amount of kinetic energy subjected to the lipase it had become denatured so much that would have never of saw a visual change in the colour of the phenolphthalein.

• Conclusion

From the results I obtained from this investigation I found that the optimum temperature for the activity of Lipase was 60°C even though the Lipase had begun to denature. These results therefore weaken my initial hypothesis that I predicted the optimum temperature to be 40°C. I found did found that as the temperature increased the rate of reaction would increase and then at a high temperature would be slower due to denaturing of the enzyme.

However, even though the optimum temperature was found to be 60°C I put forward a new hypothesis based on the polynomial trend line shown on the graph which suggests that a temperature around 45-50°C would prove give a higher ROR.

• Evaluation

From experience of this investigation if it was to be repeated then certain changes would have to be made in order to obtain more accurate results. Certain procedures and pieces of equipment could be altered for more accuracy.

Equipment/ Procedures that caused problems with inaccuracy:

1. Changing temperatures
2. Use of Phenolphthalein
3. Temperatures of Milk, Sodium Carbonate and Lipase

(a) If you look at the table of results there is a temperature for the start of each individual test and one for the end.

At the start of each experiment the temperature was correct however when the temp was taken after the colour had turned clear it had dropped. Ideally these temperatures should have been the same, but the method of using beakers of hot water didn’t account for any cooling.  With the temperature falling throughout the test it meant the Kinetic was falling, resulting in inaccuracy for the time taken because it doesn’t represent a true figure for teach temperature.

Solution – To overcome this Water baths could have been used which would keep the water at a constant pre-set temperature to stop any changes.

(b) The decision for when the lipase has hydrolysed the fats enough to lower the pH below 8.3 was decided when we thought the solution had turned clear. This method proved to have flaws, as we couldn’t be completely sure if the solution was the same colour that the previous test had been stopped at.

Solution – By using an electric pH meter a more accurate reading could have been taken. Rather than relying on visual colour change a pH value could have been accurately given so that once the pH dropped below a pH value, such as 8.3 the test could have been stopped.

3. The method of heating the milk, sodium carbonate and phenolphthalein mixture and the lipase solution prevented accurate results being obtained as the temperatures for each weren’t tested and we assumed they would have reached the required temperature after 5 min in a beaker of water of the required temperature. This would result in temps of the solution being lower then what we tested for which would give results that were untrue.

E.g. For 60°C this was the fastest rate of reaction, but if the solution was only at say 50°C when we thought it was 60°C then denaturing wouldn’t be occurring as fast as it should at 60°C and therefore would give a faster ROR which would be an untrue indication for activity at this temperature.

Solution – the solutions should be left in the water bath for longer until when tested and when they had reached the required temperature.

Other changes I would make to my procedure if I were to repeat it would be the intervals of the temperatures being tested. From the results I gained the optimum temperature was found to be 60°C. Since the temperatures tested went up each time by 20°C we can’t say that 60°C is the true optimum temperature for the activity of lipase just the optimum temperature found in this investigation. From previous knowledge and suggestion from the trend line on the graph in fact it lies somewhere between 40°C and 60°C. Knowing this next time I would test temperatures at 5°C intervals maybe between 40°C and 70°C so a more accurate optimum temperature could be put forward.

Another change would be the number of times each temperature would be tested. Due to time constraints each temperature was only tested once which meant any anomalous results might not have been noticeable. Repeating each temperature twice or even three times would eliminate the chance of this, and then maybe the mean ROR for each temperature could be found.

From the results obtained a proper conclusion cannot be drawn up. The methodology taken of this investigation contains flaws that might only be small but overall add up significantly and in effect produce inaccurate results.

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BIBLIOGRAPHY

• Boyle, Ingham, Senior, 1999, Human Biology, USA, McGraw Hill
• Fullick, 1994, Advanced Human Biology, Oxford, Heinemann
• Green, Stout, Taylor, 1993, Biological Science 1- Organisms, Energy and Environment, Cambridge
• Roberts, 1986, Biology - A functional approach 4th edition, London, Nelson
• Simpkins, Williams, 1995, Advanced human Biology, Harper Collins
• Toole and Toole, 1995, Understanding Biology for A-Level 3rd edition, London, Stanley Thornes
• Vander, Sherman, Luciano, 1994, Human Physiology, London, Collins