1st Method (SL) using white tile and stop watch
How changing Temp/Salt conc affect the hydrolysis of starch by enzyme amylase?
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- Place 50ul iodine onto white tile
- Add 10ul 0.1% enzyme into 1ml 1% starch
- Pipette 50ul of enzyme/starch mixture onto white tile containing iodine
- Repeat for every 30s interval until all starch is hydrolysed (iodine remains yellow)
- Rate enzyme activity = 1/ Time taken to hydrolyse 1% starch
- Assume conc of starch remains the same
Starch + iodine will form blue black colouration (abs at 576nm )
Absorbance drop as starch digested, blue black turn colourless
Measure absorbance change over 120s, digestion of starch by amylase.
Research Question
How changing Temp/Salt conc affect the rate of amylase digestion of starch ( by monitoring the absorbance change over time)
Research Question
How changing Temp/Salt conc affect the rate of amylase digestion of starch ( by monitoring the absorbance change over time)
Set up
1. 1ml 0.01% starch in cuvette
2. 40ul iodine (adjust vol) for a blue black
3. Set Abs at 576nm
4. Place cuvette in spec and set to Abs vs time
5. Add 10ul 1% amylase into cuvette
6. Start data collection immediately
7. Rate of digestion = Abs change over 120s
Video, Starch hydrolysis by amylase (Abs decrease over time)
Video, Starch hydrolysis by amylase (Abs decrease over time)
Treatment of amylase at different salt Concentration
8. Incubate enzyme at different salt conc for 10mins
9. After 10min repeat step 1 to 4, adding 20ul treated amylase and measure Abs over time.
Tips to make it work.
Set up
1. 1ml 0.01% starch in cuvette
2. 40ul iodine (adjust vol) for a blue black
3. Set Abs at 576nm
4. Place cuvette in spec, set to Abs vs time
5. Add 10ul water to 10ul 1% amylase, (20ul total) and transfer to cuvette containing 1ml starch + 40ul iodine
6. Start data collection immediately
7. Rate of digestion = Abs change over 120s
Treatment of amylase at different Salt Concentration
9. After 10min repeat step 1 to 4, adding 20ul treated amylase and measure Abs over time.
Tips to make it work.
- Starch must be freshly prepared and accurate
- Volume iodine adjusted accordingly to get blue black colouration
- Blue black colouration must be dilute so Beer's Lambert Law is obeyed
- Trial and error needed to get the right starch/iodine solution for this study
- Prepare a large stock of 0.01%starch/iodine and add 1 ml to cuvette instead of adding 1ml starch and 40ul iodine to cuvette ( pipetting error)
- Make sure use Lugol's Iodine (I2+KI) as iodine will not work.