Investigating the effect of temperature on enzyme activityInvestigation into the effect of temperature on enzyme activity is part of the Component 1 specification(Biological reactions are regulated by enzymes). You should read over the theory of enzyme structureas well asthe effect of temperature and pH on enzyme activity from the drop down menu above. Introduction Phenolphthalein is an indicator that is pink in alkaline solutions of about pH10, but turns colourless in pH conditions less than 8.3. In this investigation, an alkaline solution of milk, plus lipase enzyme and also phenolphthalein indicator will be used. The milk contains lipids (review their structure from Component 1 > Biological molecules from the drop down menu above). Over time this milk solution willchange from pink to colourless as the fat/lipid in milk is broken down by the lipase enzyme to form fatty acids (and glycerol) thus reducing the pH to below 8.3. The time taken for this reaction to occur is affected by temperature (review this from the Component 1> Enzymes > Enzyme properties section from the drop-down menu above). Sodium carbonate Milk, full-fat or semi-skimmed 1. Place atest tube containing 2cm3 of lipase solution in the 25 oC water bath Hazard > • Use a pH probe or data logger to give quantitative results Practical Techniques • Use appropriate apparatus to record a range of quantitative measurements (mass, time, volume, temperature, length and pH)
Phenolphthalein in a dropper bottle
lipase solution (5g/ 100cm3)
Sodium carbonate solution (0.05 mol dm–3)
5x Test tubes and rack
2 x 10cm3 syringes/measuring cylinders
2cm3 syringe
Stirring rod Thermometer
Water baths set to 15oC, 25 oC, 35oC, 45oC and 55oC. Ice
2. Place 5 cm3 of milk into a separate test tube (contains lipids whichwill bethe substrate for the lipase enzyme)
3. Add 5 drops of phenolphthalein indicator to the same test tube as the milk
4. Add 7 cm3 of sodium carbonate solution to the same test tube as the milk
5. Place this test tube in the 25 oC water bath for 10 minutes to equilibrate to this temperature
6. Use a syringe to add 1 cm3 of lipase into the milk solution test tube in the water bath and start the stop clock
7. Stir the contents of the test tube until the solution loses its pink colour, record the time taken
8. The time you have recorded is the time it has taken for the lipase to hydrolyse enough of the lipid (and therefore release enough fatty acids) to neutralise the Sodium carbonate to just below pH 8.3
9. Repeat steps 1 – 7 for four other temperatures 15 oC, 35 oC, 45 oC and 55 oC
Risk Assessment
Sodium carbonate is irritant at high concentrations
May splash or transfer into eye when placing into test tube
Use low concentrations and wear safety glasses
Phenolphthalein indicator contains ethanol which is flammable
If using Bunsen burners for water baths, could ignite the ethanol
Do not use ethanol near Bunsen burners
Further work
• Itis possible to vary the lipase concentration and so look at the effect of enzyme concentration on breakdown of milk fat
•Also, different types of milk could be used to explore the effect of changing substrate (fat) concentration on the reaction :
E.g. Full cream, semi-skimmed and skimmed milk
• Use laboratory glassware apparatus for a variety of experimental techniques to include serial dilutions
• Use ICT such as computer modelling, or data logger to collect data, or use software to process data
See Also
A milkman adds a very small amount of baking soda to fresh milk. (a) Why does he shift the pH of fresh milk from 6 to slightly alkaline?(b) What do you expect to observe when the milk comes to boil?(c) Why does this milk take a long time to set as curd?[Solved] A milkman adds a small amount of baking soda to fresh milk: