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Comparing the effect of different temperatures on free lipase and immobilised lipase

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Introduction

Comparing the effect of different temperatures on free lipase and immobilised lipase Plan Lipase is an enzyme which hydrolyses fats to fatty acids and glycerol. Glycerol has three hydroxyl groups. Fatty acid molecules are much larger than glycerol molecules and consist of long hydrocarbon chains. Lipase works by hydrolysis, adding three molecules of water across the ester link. Hypothesis My hypothesis is that immobilised lipase will work more efficiently at high temperatures than free lipase. Free lipase is when lipase is prepared free in solution. It is used to digest fats as, for e.g., in some biological washing powders. It is also used in the food industry. Free lipase works best at temperatures of 30-50�C. However, when an enzyme is immobilised some of its physical properties are changed. There are three ways to immobilise enzymes. Entrapment is the gentlest method of immobilisation and does not damage enzymes. The enzymes are also more stable due to entrapment. The chief effect of immobilising an enzyme is to slow the rate at which denaturation occurs at high temperatures, enabling the enzyme to be used in industrial processes that are performed at 40�C, or more. Immobilised enzymes are much cheaper as they can be recovered at the end and reused, they also help avoid contamination. I suggest that due to the effect of immobilisation, it is likely that immobilised lipase will be more resistant to denaturation at higher temperatures. ...read more.

Middle

I will check the temperature of the solutions with a thermometer. As soon as the 5 minutes has completed I will mix them together and start a stopwatch. I will then record the time when the solutions turn white (like the untreated milk) I will repeat this experiment at different temperatures (30, 40, 50, 60, 70�C). I will repeat each reading. Risk Assessment Lipase enzymes This is in powdered form. All enzymes have biological activity and need to be treated with care. Avoid inhaling powder. Sodium Carbonate and Phenolphthalein Very low risk Keep in clearly labelled container Results Temp �C Time to reach end point (s) Free Lipase Repeats for Free Lipase Time to reach end point (s) Immobilised Lipase Repeats for Immobilised Lipase 20 17.06 16.53 49.36 50.13 30 3.46 3.55 25.27 24.51 40 2.27 2.16 11.07 11.33 50 4.00 4.52 13.21 17.19 60 Denatured 0 56.17 59.09 70 Denatured 0 0 0 To enable me to compare the effects of temperature on each enzyme I calculated the averages of the times and then the overall rate of reaction for each using the formula: 1 time taken for colour change (s) I then expressed the rate of each reaction as a percentage of the maximum rate. The results are shown in the following summary table. Summary results table Temp �C Time to reach end point (s) ...read more.

Conclusion

The colour of the phenolphthalein changes slowly in some cases. In the slower reactions this was particularly difficult. Whilst I made an effort to distinguish between the untreated milk and the reactions, and those where the solution remained pink by counting the rate of reaction as zero where there was no sign of change this remained quite an inaccurate method. The other major limitation involved temperature treatment especially at higher temperatures it could have easily made a difference in the results. The solution itself took longer to reach these high temperatures so the time of treatment did vary. The overall reliability of the investigation was also dependent on the time constraints. To cover the full range of temperatures there was only enough time to carry out one set of repeats. It is obvious from some of the variability of the results that this was an important limitation. Overall it appeared that this investigation did provide some evidence to support my hypothesis that immobilised lipase would work better over a wider temperature range than free lipase. Clearly repeating these tests at least twice would be the most important type of further work, which would be needed. If the trends shown in the first set of data were consistent it would also be important to investigate smaller increases in temperature between 40 and 60�C. Also seeing how the length of each temperature treatment affects the enzymes. If time were available it would also be profitable to investigate a wider range of enzymes when immobilised by entrapment in a sodium alginate gel. ...read more.

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