Saturday, June 4, 2011

IB Biology, IB Chemistry, enzyme kinetics amylase for IA and EE

IB Biology IA, EE on enzymes kinetics amylase using Visible spectrophotometer
EE and IA on enzyme study is safe, easy and doable for IB students
Starch digestion using Amylase
Source for amylase from bacteria, malt or salivary amylase.
Click here (amylase) or Click here to purchase ( pancreatin amylase) from Carolina.
Introduction
2 methods to quantify rate of starch digestion ( Abs vs time and Abs vs conc )
1st Method--(Abs vs Time)
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 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 absorbance 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 = Absorbance change over 120s




Video, starch hydrolysis by amylase (Abs decrease over time)

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Treatment of amylase at diff Temp


8. Incubate 0.5ml 1%amylase at different temp for 10mins
9. Starch sol must also be at the same temp as amylase.
9. After 10min repeat step 1 to 5 by adding treated amylase and measure absorbance over time




Treatment of amylase at diff salt Conc
Student data on effect of salt conc on amylase activity
Set up
1. 1ml 0.01% starch in cuvette
2. 40ul iodine (adjust vol) for a blue black
3. Set absorbance 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 diff Salt Conc


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.






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2nd Method ( Abs vs Conc ) suitable for EE ( 2 parts Expt )
How changing Temp affect amylase digestion of starch ( Change in Starch Conc over time )
Part 1. Standard Calibration Curve for Starch Conc
Starch with iodine solution forms blue black colouration.
1. Prepare 0.01% starch solution ( 0.01g in 100ml )
2. Perform a 2 fold dilution ( 0.01, 0.005, 0.00250, 0.00125, 0.000625%)
3. Add 2.5ml 0.01% starch to cuvette
3. Add 40ul 1% iodine solution ( adjust volume accordingly to obtain appropriate blue black)
4. Set visible spec at 576nm
5. Prepare a blank ( 1ml water + 1ml iodine ) and calibrate it (zeroize it)
6. Prepare cuvette (2.5ml 0.01%starch + 40ul iodine) and measure absorbance at 576nm
7. Repeat step 6 with different starch conc and plot std calibration curve.
Hints and tips 
  • Starch iodine must be dilute as Beer's Law, works for diluted solution.
  • Prepare fresh starch solution
  • Volume of 1% Lugol Iodine must be adjusted accordingly so a blue black is formed.



Part 2
Expt set up
1. 1ml 0.01% starch in cuvette
2. 40ul iodine ( adjust vol) for a blue black
3. Set absorbance 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 over 120s = (Initial -Final) Abs = (Initial -Final) Starch Conc
8. From Std curve of Absorbance and Starch Conc




9. Initial Abs (0.6) = Initial Starch Conc (0.0084%)














10. At 120s, measure the Abs
11. Final Abs (0.54) = Final Starch Conc (0.0075%)
12. Enz activity = Change in Starch Conc 
13. (0.0084-0.0075)/120s = (7.5x10^-6)%/s








Treatment of amylase at diff Temp 



14. Incubate 0.5ml 1%amylase at different temp for 10mins
15. Starch sol must also be at the same temp as amylase.
16. After 10min repeat step 1 to 5 by adding treated amylase and measure absorbance after 120s
17. Find Ini/Final Starch Conc shown above

Source of picture
http://www.helixxtec.com/Helixx/CoolPrep/coolprep.htm
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Video, Starch/Iodine Standard Calibration Curve

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.

EE topics on Enzymatic study
Interesting to find out the efficiency of our own salivary amylase

  • Investigation on the effects of pH, Salt, Temp and heavy metal inhibitors on Human salivary Amylase using Visible Spec for quantification.
  • Comparative study on effectiveness of amylase from diff organs (salivary vs pancreatic ) or diff source (salivary, malt, fungi (Amyloglucosidase)
  • Effect of calcium ions on amylase activity ( Amylase is calcium dependent enzyme )

Diff amylase be purchased from HERE(Carolina) or HERE( NCBE)
Source for info, Click HERE
Hope you find joy and passion on doing Science EE Research.........................Lawrence Kok

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