Tuesday, June 7, 2011

IB Biology IA, enzyme amylase experiment on carbohydrates hydrolysis

IB Biology EE, enzymes kinetics amylase experiment using colorimeter
Simplified version for SL Biology. For  HL Biology, Click HERE to view HL version
Introduction
2 methods  ( Abs vs time or Abs vs conc )
1st Method--(Abs vs Time)
Starch + iodine form blue black colouration (abs at 576nm )
Absorbance drops as starch digested, blue black turn colourless
Measure absorbance change over 120s, digestion of starch by amylase.

Set up
1. 50ul 1% starch in cuvette
2. 20ul iodine (adjust vol) for a blue black
3. Add 2ml water ( dilute it for Beer's Law)
4. Set absorbance at 576nm
5. Place cuvette into spec, set Abs vs time
6. Measure Intial Abs = 0.364





7. Add 20ul 1% amylase into cuvette
8. Start data collection immediately
9. Rate of digestion = Abs change over 120s
8. Final Abs = 0.207
9. Rate of enzyme activity = Abs change/120s
.....................................................................................................................................................
2nd Method ( Abs vs Conc ) 
Click HERE to view how to prepare Std Calibration Curve ( HL students )

Expt set up
1. 50ul 1% starch in cuvette
2. 20ul iodine ( adjust vol) for a blue black
3. Add 2ml water ( dilute it for Beer's Law)
4. Set absorbance at 576nm
5. Blank it with (20ul iodine + 2ml water)
6. Place cuvette in spec, measure Abs
7. Initial Abs = 0.377




8. Remove cuvette out, add 20ul 1% amylase 
9. After 120s, place cuvette into spec and measure final Abs
10. Final Abs = 0.226
11. Rate of enzyme activity
= (Initial - Final) Abs 
= (Initial - Final) Starch Conc
= Decreased in Starch Conc




12. From Std curve of Absorbance vs Starch Conc

Initial Abs = 0.377, Ini Starch Conc = 0.0051
Final Abs = 0.226,  Final Conc = 0.0029
Rate enzyme activity 
= Change in Starch Conc/time
= (0.0051- 0.0029)/120s
= 1.83 x 10^-5M/s








Video, Starch Hydrolysis by amylase using Abs/Time or Abs/Conc


....................................................................................................................................................
For HL or EE students
Using DNS method to quantify glucose produced by starch hydrolysis
Click HERE to view glucose quantification with DNS
Steps to follow
  • DNS solution = yellow
  • Glucose (all reducing sugar ) + DNS = red brown colouration
  • Prepare Glucose Std
  • Digest Starch/Sucrose/Cellulose with enzymes produce glucose
  • React with DNS = red brown 




Glucose Std Calibration Curve


3 Ways to quantify starch hydrolysis
  • Abs vs Time ( Easy ) for SL
  • Abs vs Conc ( Hard ) for HL
  • DNS method ( Challenging )  for EE








Trials Result done ( More trials needed to perfect this method)
Hydrolysis of starch, sucrose and cellulose by enzymes


DNS = yellow ( no glucose )
DNS + sucrose = yellow ( no glucose )
DNS +sucrose+enzyme = red brown (glucose)


DNS + starch = yellow ( no glucose )
DNS +starch+enzyme =red brown(glucose)


DNS + cellulose = yellow ( no glucose )
DNS+cellulose+enzyme=red brown(glucose)




Limitation and problems encounter in DNS Method
Hydrolysis of starch by amylase produce glucose /maltose
DNS = yellow ( no glucose )
DNS + starch = yellow ( no glucose )
DNS + starch + enzyme = red brown ( glucose )
DNS + enzyme = red brown !!!!!!!!!!! ( glucose )


Means enzyme reacts with DNS producing a colour change or might due to heat or other chemical rxn.






Hydrolysis of Cellulose with enzyme ( cellulase )


DNS = yellow ( no glucose )
DNS + cellulose = yellow ( no glucose )
DNS+cellulose + enzyme = red brown(glucose)
DNS + enzyme = red brown!!!!!! ( glucose )


Limitation is enzyme reacts with DNS producing colour.












DNS method, will have to make a few assumptions and modifications
  • Absorbance is due to glucose + fructose + amt enzyme added
  • Blank it with (enzyme + DNS)
  • Absorbance of (starch+enzyme +DNS) -  Absorbance of ( enzyme + DNS)
Video, Hydrolysis of Starch/Sucrose/Cellulose with DNS

EE topics 
Using this quantification technique on glucose for investigation on
  • Hydrolysis of starch by diff amylases (sources) producing glucose
  • Factors (pH, ionic salts, temp, inhibitors) affecting enzymatic hydrolysis of starch/sucrose/cellulose using DNS/Visible Spec as quantification method
  • Hydrolysis of cellulose with enzyme cellulase  producing glucose.
  • Optimization of this technique, more sensitive, easy for students to use ( instead of Benedict's and Sugar dip stick) ( My current EE 2011 )
  • Colour stabilisation using potassium sodium tartrate and phenol solution
  • Modification of this method, to prevent interaction between enzyme and DNS
  • Immobilisation of enzyme on alginate/agar beads, enable us to reuse enzyme and prevent enzyme reacting with DNS ( My current EE 2011 )
Click HERE on glucose quantification using DNS
Click HERE on sucrose quantification using DNS
Click HERE for EE Research Questions on enzyme sucrase using DNS
Click HERE to purchase DNS kit
Click HERE to view MSDS toxicity of DNS.
Most welcome to collaborate with students from TCIS (Korea)
Click HERE to connect
Demands great analytical skills. Great failures make great mens and nothing worthwhile in life is ever achieve without great failures/struggles. It is OK to try and fail...........Lawrence Kok

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