IA on effect of pH on the rate of hydrolysis of starch by amylase enzyme (fungal extract), measured using visible spectrophotometer.

1. Effect of pH on the rate of hydrolysis of starch by amylase enzyme (fungal extract), measured using visible spectrophotometer.
Starch react with I2 to form a blue black product.
Blue black colorization can be monitored using visible spec.
Enzyme amylase will breakdown starch to maltose.
As starch is broken down, I2 wont be able to bind with starch.
Resulting in colorless product.
Rate of starch hydrolysis – measured by change in blue black over time
Starch + I2 + enzyme → maltose + simple sugars
Reaction mechanism
Procedure:
1% (w/v) starch – 1g in 100ml water
1% (w/v) iodine – 1 g in 100ml water
1% (v/v) Enzyme amylase obtained from fungal extract
pH buffer (1, 4, 5, 7, 9) were used.
1ml starch + 2ml water + 50ul I2 was added into a cuvette. (Blue black)
100ul enzyme added to 100ul pH buffer. Incubate for 5 mins, at RT.
100ul of enzyme + pH buffer mix was then transfer to cuvette containing starch.
Mix and abs over time taken.
Rate of hydrolysis – decrease in abs over time.
Starch + I2 (Blue black) I2 (yellow)
pH buffer (1, 4, 5, 7, 9) were used.
Negative control - starch + I2 + water without any enzyme
was done, to confirm that breakdown of blue black colorization
is due to enzyme, and not due to other factors

2. Go to expt – press calibrate Insert cuvette (blue black) and press collect
Insert a blank containing water
Press stop, and click on rainbow icon. Select abs vs time. λ max at 594nm will
be automatically chosen
Abs vs time
Effect of pH on the rate of hydrolysis of starch by amylase enzyme (fungal extract), measured using visible spectrophotometer.

3. Change of abs over time were plotted.
Rate of hydrolysis – decrease in Abs over time
Slope/gradient taken over 40s
pH Rate
Abss-1
1 0.0002505
4 0.001047
5 0.001082
7 0.004221
9 0.006004
-ve
(control)
0.0001442
Data collected.
Effect of pH on the rate of hydrolysis of starch by amylase enzyme (fungal extract), measured using visible spectrophotometer.
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0 2 4 6 8 10
rate
of
hydrolysis
pH
rate of hydrolysis vs pH
-ve control compared to pH 1
very little hydrolysis at pH 1
Fungal amylase seems to work in alkaline medium
Pancreatic amylase (animal) can be investigate.
Liver and yeast amylase can be compared if they work well at pH 9