ENZYMOLOGY- LAB Bch 206

LAB MANUAL
Dr. Muhammad Ansar

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PRACTICAL # 1 CALCULATIONS

Percentage Solutions: Fraction of the weight or volume of solute related to the total weight or
volume of solution
 Weight by volume
 Weight by weight
 Volume by volume
Q. Make 10% weight by volume solution of NaCl.
Q. Make 40 ml of 10% weight by volume solution of SDS.
Q. make 70% volume by volume solution of ethanol.
Molarity:
No. of moles of solute per liter of solution.
Molarity= moles of solute/ liters of solution
Normality:
Different than molarity. Normality never less than molarity.
Number of mole equivalents of solute per liter of solution
N=nM
n= integer (no. of replaceable H+ or OH- ions in acid or base)
1M NaOH solution 1N
1M H2SO4 solution is 2N
Dilutions:
Concentrated Solution to Diluted solution
C1V1=C2V2
C1=Concentration of Initial/Stock Solution
C2= Desired final Concentration
V1= Volume of Stock solution to be use for Dilution
V2=Desired final volume
You have Stock Solution of 1000ug/ml BSA, make 200ul of 20ug/ml.
Dilution Factor: Total volume of solution per aliquot or sample volume (DF=Vf/Vi)
1X dilution

2
2X dilution
Serial Dilutions:
Any dilution where the concentration decreases by the same quantity in each successive step.
Suppose prepare stock solution of 100mg/ml
From Stock Solution make 5, 10, 15, 20, 25, 30 mg/ml respectively

Sr. no. Dilution Concentrations Volume of BSA to be added (ml) Volume of distilled
mg/ml water to be added (ml)
1. 5

2. 10

3. 15

4. 20

5. 25

6. 30

Double dilution:
Series of 1/2 dilutions. Each successive tube will be 1/2 the amount of original solution.
Starting solution 500ug/ml, next 1/2 dilution will 250ug/ml, next 1/2 dilution will be 125ug/ml, next 1/2
dilution will be 62.5, next 31.5.

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PRACTICAL # 2
STARCH STANDARD CURVE
Standard Curve:

It is also known as calibration curve, a type of graph which represents relationship between two
quantities. . They are obtained by relating a measured quantity to the concentration of the
substance of interest in "known" samples, i.e. Standards of known concentration. These
standards provide a reference to determine unknown concentrations. Thus amounts chosen of
standards need to span the range of concentrations expected to be found in the "unknown"
sample concentration.

Spectrophotometer:

Spectrophotometer is an instrument which measures the amount of light of a specified

wavelength which passes through a medium. It allows selection of a wavelength (corresponding
to maximum absorption of solute) to pass through the solution. It works on the principle of Beer-
Lambert Law: Amount of light absorbed by medium is directly proportional to concentration of
solute present.

A=Ԑ l c

Where Ԑ= molar absorptivity, l = path length, c = concentration

Absorbance A is negative log of transmittance T. A= -log T

Application:

Quantitative analysis

Qualitative analysis

Enzyme Assays

Physiochemical studies

Dilution conc. (mg/ml) Vol. of starch Vol. of distilled A610

solution (ml) water (ml)
1. Blank
2. 10
3. 20
4. 30
5. 40
6. 50
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 7. 60
8. Unknown

Practical # 3

Determination of catalytic activity of amylase by using starch solution

THEORY
Carbohydrates
Monosaccharides (Glucose, Fructose, Glactose)
Disaccharides (Sucrose, Maltose, Lactose)
Polysaccharide (Cellulose, Glycogen, Starch)

Starch is a polysaccharide which odorless, tasteless, insoluble in cold water and white in color. It
gives blue color with Iodine. Composed of two types of molecules:
Amylose: 20-25%, it has  1-4 glycosidic bond and is straight chained.
Amylopectin: 80%, it has  1-4 and  1-6 glycosidic bond and is branched polymer.

Starch reaction with Iodine

Iodine - KI Reagent: Iodine is not very soluble in water, therefore the iodine reagent is made by
dissolving iodine in water in the presence of potassium iodide. This makes a linear triiodide ion
complex with is soluble. The triiodide ion slips into the amylose coil of the starch causing an
intense blue-black color.
I2 + I - I 3-
Amylase:
It is a glycoside hydrolase which catalyzes the hydrolysis of starch into sugar.

Starch Amylase Maltose Glucose

Enzyme Unit:
Amount of enzyme required to convert 1umol or 1mg of substrate into product under certain
working conditions.
Enzymatic activity:
Amount of product formed (µmoles) or substrate reduced per ml of enzyme in one minute is
called enzymatic activity. SI units: katal
Enzyme activity in units
Large unit U = µmol / min / ml
Small unit µ = nmol / min / ml

Procedure

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 Stock solution of Starch: 50mg/ ml
 Make 2ml dilutions of 5, 10, 15, 20, 25, 30, and 35mg/ml respectively and one of
unknown starch concentration (should make by instructor to give to students) from stock
solution through C1V1 = C2V2 equation.
 Take absorbance at 580 nm of all dilutions including unknown sample
(spectrophotometer should be zeroed through distill water before taking absorbance of
dilutions) and plot a graph of absorbance v/s concentration. (This is the standard graph).
 Now keep one tube as blank (2ml distill water+ 2µl amylase + 2µl iodine reagent).
 Add 50ul of 0.4% bacterial amylase solution (ph 6.6 Phosphate buffer) to 2ml of each
dilution including unknown sample.
 Incubate for 5 min at room temperature and add 2 ul of iodine reagent to each dilution
and unknown sample.
 Observe the blue color produce in each sample tube. The intensity of color is directly
proportional to the unhydrolyzed starch present in the sample. This will give the rough
estimate of enzymatic activity of amylase enzyme.
 Add drop of concentrated HCl in each tube (including blank) to stop the enzymatic
reaction (This is an optional step).
 Measure absorbance at 580 nm of each sample tube (spectrophotometer should be zeroed
by blank). Absorbance value is directly proportional to the unhydrolyzed starch present in
the sample after catalysis.
 If you are not interesting in optional step then immediately take absorbance at 580nm of
each sample (spectrophotometer should be zeroed by blank) after adding iodine.
 Record the values.
 Plot the graph of absorbance v/s concentration on the standard graph and compare them
both.
 Comparing the values of absorbance will give you the value of remaining or
unhydrolyzed starch concentration (X2) after enzymatic reaction for each dilution and
unknown sample.
 Determine the concentration of unknown sample from standard curve/graph.
 Measure the enzyme activity.
 Enzyme Activity: X1 –X2 / incubation time (mg/ml min)
 Where X1 is the initial Starch concentration [e.g. 5, 10, 15, 20, 25, 30, 35 and of
unknown sample (18) mg/ml]
 X2 is the starch concentration after enzymatic reaction [derive through graph of
absorbance v/s concentration of unknown sample, it should be less than the initial
concentration of unknown sample because now the starch is hydrolyzed through enzyme
( when no standard graph was plotted)].
 X2 can also be measured through standard graph as discussed earlier.
 Here X1 – X2 = product produced after starch hydrolysis
 Product produced after starch hydrolysis / incubation time = Enzymatic activity

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 3

2.5 2.44 2.42 2.42 2.42

2.3
2.2
2
Absorbance 580nm

1.9

1.5
1.3
1

0.6
0.5

0
0 10 20 30 40 50 60
Starch concentration (mg/ml)

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Practical # 4
Effect of Temperature on the catalytic activity of alpha-amylase

Theory
Enzyme require optimum conditions for maximum activity. Different factors affect the enzyme
activity
o Substrate Concentration
o Enzyme Concentration
o pH
o Temperature
o Inhibitors (Assignment on Amylase Inhibitors)
Amylase inhibitors are also called starch blockers and are used in diet supplements,
Diabetes drugs. As amylase catalyze starch hydrolysis yielding glucose units.
Optimum Conditions: Optimum conditions refers conditions like pH and temperature where the
enzyme activity is highly favorable (maximum activity)
Effect of Temperature
As the temperature increases kinetic energy of the colliding molecules increases, so does the rate
of reaction but very high temperature breaks the hydrogen bonds causing the enzyme to denature
and become nonfunctional.
Low temperature conditions slows the rate of reaction and enzyme activity is reduced.

Procedure:
 Effect of Temperature
 Make a solution of starch 10 mg/ml.
 Take absorbance A1 at 580 nm of simple starch solution (spectrophotometer should be
zeroed through distill water before taking absorbance of starch solution)
 Transfer 1ml of starch solution in five tubes.
 Add 1ml of distill water in a tube (Blank tube).
 Add 50ul of alpha-amylase solution in each tube including blank tube and incubate
sample tubes for 5 min at five different temperatures.
 Add 60ul of Iodine reagent to each tube and observe the blue color produce in each
sample tube. The intensity of color is directly proportional to the unhydrolyzed starch
present in the sample. This will give the rough estimate of enzymatic activity of amylase
enzyme at 5 different temperatures.
 Add drop of concentrated HCl in each tube (including blank) to stop the enzymatic
reaction (This is an optional step).
 Measure absorbance at 580 nm of each sample tube (spectrophotometer should be zeroed
by blank).
 If you are not interesting in optional step then immediately take absorbance at 580nm of
each sample (spectrophotometer should be zeroed by blank) after adding iodine.
Absorbance value is directly proportional to the unhydrolyzed starch present in the
sample after catalysis. So higher the absorbance; less will be the enzymatic activity.
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 Record the values. (All values must be smaller than or equal to that of starch solution not
greater than starch solution because here the enzyme has worked on starch).
 Plot the graph of absorbance v/s temperature (inverted curve).
 Calculate the enzymatic activity through absorbance at each temperature.
 Plot the graph of enzymatic activity v/s temperature (bell shaped curve).
 Find the optimum temperature where enzymatic activity is maximum and absorbance is
minimum for starch-iodine complex because at optimum temperature, enzyme works at
its best and therefore it will hydrolyze maximum starch to its product, remaining
minimum starch in the sample.
1
0.9 0.9
0.8 0.8
0.7
0.6 0.6
Absorbance at 0.5 0.5
580nm
0.4 0.4
0.3
0.2
0.1
0
0 10 20 30 40 50 60 70 80 90
Temperature in celsius

70

60

50

Enzymatic activ- 40
ity in µmol/
min/ml 30

20

10

0
0 10 20 30 40 50 60 70 80 90
Temperature in celsius

Enzymatic activity in µmol/min/ml

Temperature Absorbance
1. 4 (ice cold water)
2. 25 (Room Temp)

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 3. 37 incubator
4. 65 incubator
5 >80

 Calculate Enzymatic Activity

First of all take difference of all absorbance by subtracting the absorbance at each temperature
from the absorbance of simple starch solution. This will give the absorbance of product which is
produced during the hydrolysis of starch i.e. glucose.
For example, simple starch solution has the absorbance value of 1.029 and at 37˚C the solution
after reaction has 0.45 absorbance. A1 – A2 = ΔA
1.029 – 0.45 = 0.579
Now ΔA / incubation time (This will give the absorbance of product produced per minute) = A
0.579 / 5 minutes = 0.1158 A
Now apply Beer-Lambert Law
A= εlc
Where A = absorbance, ε = extinction coefficient or molar absorptivity of product in L/moles
cm, l = cuvette path length and c = concentration of product.
Mostly l = 1cm or 10mm
And ε for glucose = 0.0136 L/mole cm (not confirm)
Calculate concentration by above mentioned equation
So c= A / εl
C= 0.1158 / 0.0136 × 1 = 8.514705882 moles / min / L
Calculate enzyme activity in IU
From above, if cuvette contained 1L, then 8.5147 moles of product would be made every minute
by the 50µl of enzyme.
But in our experiment, cuvette contained only 1ml
So, 50 µl of enzyme made only 8.5147 × 10-3moles/min
1ml of enzyme would make 1 / 50 × 10 -3 × 8.5147 × 10-3moles/min = 2×10-5 × 8.5147 × 10-
3
moles/min = 1.70294 × 10-7 moles/min/ml of enzyme or 0.170294 µmoles/min/ml
1L of enzyme would make 1 / 50 × 10 -6 × 8.5147 × 10-3moles/min = 2×10-8 × 8.5147 × 10-
3
moles/min = 1.70294 × 10-10 moles/min/L of enzyme or 0.000170294 µmoles/min/L
Hence the enzyme activity in this hypothetical example is 0.170294 µmoles/min/ml of enzyme.

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 1
0.9
0.8
0.7
0.6
Absorbance at 0.5
580nm
0.4
0.3
0.2
0.1
0
0 2 4 6 8 10 12 14
pH

Absorbance at 580nm
60

50

40
Enzymatic activ-
ity in µmol/ 30
min/ml
20

10

0
0 2 4 6 8 10 12 14
pH

Enzymatic Activity

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