Experiments

• Temperature: using a water bath
• Substrate concentration: same volumes of starch solution to each test tube.
• Enzyme concentration: same volume of amylase solution to each test tube.

• pH: by using different pH buffers (4,7,9)
• (A buffer contains certain pH)

• 11 steps.
• 1. Set up water bath (37*)
• 2. Put starch and iodine solution in 3 tubes.
• 3. Put amylase and buffer (4,7,9) into 3 others.
• 4. All tubes in water bath and leave
• 5. Pour an amylase tube into a starch tube. Shake and repeat for each pH
• 6. Put back and measure time for colour fade
• 7. Graph
• 8. Test for sugar (steps1+2, Benedict’s into each and heat)
• 9. Brick red colour means there’s reducing sugar
• 10. Repeat experiment with boiled amylase (control)
• 11. Expected result: enzyme works best at pH7, blue/black faded and Benedict’s was positive.

• Buffer solution of pH4 stayed blue/black after 15 mins.
• Buffer solution of pH7 stayed, after 15 mins, blue/black then 5-6 mins later turns red/yellow.
• Buffer solution of pH9 stayed blue/black after 15 mins.

37* C

Starch solution and iodine solution

Amylase solution and a pH buffer (either 4,7 or9)

• Pour one of the amylase tubes (amylase+pH buffer) into one of the starch tubes (starch+iodine)
• Gently shake
• Repeat for each pH.

Measure time taken for blue/black colour to disappear (for amylase to work on starch).

• Test for sugar- repeat steps 1 and 2. Add Benedict’s solution to each boiling tube and heat for 5 min.
• A brick red colour indicates a reducing sugar is present.

Repeat the whole experiment with boiled amylase as a control.

Expected result: the enzyme works best at pH7, the blue/black colour fades and Benedict’s test results as positive

• Water bath
• Starch solution
• Boiling tube
• Amylase solution
• PH buffers (4,7,9)
• Thermometer
• Iodine solution
• Watch (measure time)
• Benedict’s solution
• Boiled amylase (control)

• 10 steps.
• 1. Set up 5 water baths (0,20,35,55,80*C)
• 2. Put starch and iodine in 5 boiling tubes
• 3. Put amylase into 5 more boiling tubes.
• 4. Pair of tubes into each bath and leave 5min.
• 5. Pour amylase tube into starch tube, shake, repeat for each temperature.
• 6. Put back into bath, measure time for colour fade.
• 7. Sugar test- (steps1+2) Benedict’s into each and heat.
• 8. Brick red colour indicates reducing sugar
• 9. Repeat whole experiment with boiled amylase
• 10. Expected result: enzyme works best at 37*C, blue/black colour faded, Benedict’s test positive.

• Expected result: the enzyme works best at 37*C,
• the blue/black colour fades and
• Benedict’s resulted in a positive test.

Five water baths set up at 0, 20, 35, 55, 80 *C.

Put starch solution and drops of iodine solution into each of 5 boiling tubes.

Put amylase solution into five more boiling tubes.

• Pour the amylase into the tube of starch solution and gently shake.
• Repeat for each temperature.

• Measure the time taken for blue/black colour to disappear.
• ( measures the rate of enzyme activity, time for starch to be converted to maltose. Done by taking some of the solution out every 1 minute and testing for the presence of starch with iodine)

• Steps1+2, Benedict’s solution to each boiling tube and heat for 5 min
• A brick red colour indicates a reducing sugar is present.

The whole experiment repeated with boiled amylase

• Water baths of different temperatures
• Boiling tubes
• Starch solution
• Iodine solution
• Amylase solution
• Watch (measure time)
• Benedict’s solution
• Boiled amylase (control)

• 11 steps.
• 1. Put starch and iodine into 2 boiling tubes. Label 1+2.
• 2. Put amylase into 2 other boiling tubes. Label 3+4.
• 3. Place tube4 into water bath at 100*C for 10min.
• 4. Place all 4 tubes in water bath at 37*C
• 5. Pour tube1 into tube3 (unboiled). Shake
• 6. Pour tube2 into tube4 (boiled). Shake and note any colour changes.
• 7. Put tubes back into water bath(37)
• 8. Expected results: unboiled amylase blue/black colour fades.
• 9. Reducing sugar test by adding Benedict’s and heating
• 10. Brick red colour indicates presence of reducing sugar.
• 11. Unboiled amylase will turn red on heating with Benedict’s solution and blue will not disappear in boiled amylase

Expected result: the unboiled amylase blue/black colour fades.

After testing for reducing sugar- unboiled amylase results as positive (red) and the boiled amylase will negative (remain blue).

Starch solution and drops of iodine into two boiling tubes, labelled 1 and 2.

Amylase into two other boiling tubes and label 3 and 4.

Place tube4 into water bath at 100*C for 10 mins.

All four tubes are placed in water bath at 37* for 5 mins.

Pour tube1 into tube3 (unboiled amylase). Gently shake.

• Pour tube2 into tube4 (boiled amylase)
• Shake.
• Note any colour changes

• Starch solution
• Iodine solution
• Boiling tubes
• Water bath at 100*C and 37*C
• Thermometer
• Benedict’s solution