1

Effect of substrate concentration on α -Amylase

Aim: To determine the effect of sub state concentration on α –amylase by Shoffer-Somogyi method.

Principle: With a fixed concentration of enzyme an increase of substrate will result at first in a very rapid rise
in velocity or reaction sate. As the substrate concentration continues to increase, however the increase in sate of
reaction begins to slow down until, with a large substrate concentration, no further change in velocity is
observed.
Michealis and others reasoned correctly that an enzyme catalyzed reaction at varying sub state concentrations is
diphasic i.e. at low substrate concentration the active sites on molecules (enzyme) are not saturated by substrate
and the enzyme rate varies with substrate molecules concentration (phase1). As the number of substrate
molecules increases, the sites are covered to a greater degree until at saturation no more rites are available, the
enzyme is working at full capacity and now the rate is independent of substrate concentration. (Phase II).

Procedure: Pipette out the Enzyme and substrate as shown in tabular column. Incubate exactly for 3 minutes
and follow the Shoffer – Somogyi method. Plot the graph of substrate concentration against Enzyme activity.

Conclusion: As the substrate concentration increases, the activity increases. However after certain
concentration of substrate, the activity remains same because of saturation of Active site on Enzyme with
substrate Molecules.

Substrate Enzyme Time DNS H2O O.D. Mg of Activity

Sl.No.
ml ml (minutes) ml ml 540nm Maltose mol/ml/min
Heat in Boiling water
bath for 5min & cool

1
2
3
4
5
6
7
 2

EFFECT OF TIME ON α -AMYLASE

Principle:

Procedure:

Pipette out 0.5ml of enzyme & 0.5ml of substrate at room temperature into different test tubes. Incubate all test
tubes in room temperature exactly for 3 minutes stop the reaction at end of 30sec., 1, 1½, 2, 21/2, 3 minutes by
adding 1ml of DNS reagent. Then keep the tubes in boiling water bath exactly for 5 minutes and coll. Add 8ml
of water O.D. is read at 540nm after adjusting calorimeter with blank. Calculate the activity at different time
intervals. Plot the curve taking time on the absicca and activity on ordinate. Comment on your results.

Reagents:

1) DNS Reagent: -
1 gm of 3,5 dinitro salicylic acid in 200ml of NaOH. Add to 50ml of water. Add the suspension carefully to
50ml of water containing 30gms of sodium potassium tartarate and made upto 100ml. Filter if necessary.

2) Phosphate Buffer of pH 6.9 (0.02M).

Prepare 0.2M NaOH and 0.2m sodium dihydrogen phosphate 24ml of NaOH to 50ml of 0.2M Sodium
dihydrogen phosphates and dilute it to 100ml and standardize it to pH 6.9.

3) Enzyme: 25mg in 10ml Buffer.

4) Substrate: 1.1 Starch in Buffer solution.

Substrate Enzyme Time DNS H2O O.D. Mg of Activity

Sl.No.
ml ml (minutes) ml ml 540nm Maltose mol/ml/min
Heat in Boiling water
bath for 5min & cool

1
2
3
4
5
6
7

Result:
 3

DETERMINATION OF KM OF α -AMYLASE

BENFELD & TITRATION METHOD.

Aim: To determine the Km & Vmax of α -amylase by Benfeld and titration method.

Principle: the concentration of substrate affects the rate of enzyme-catalyzed reaction. After a certain
concentration, a point is reached finally where the Enzyme is saturated with the substrate. When the substrate
concentration has no influence on the rate of formation of products. Km is the Michealis Menten constant is it
is the substrate concentration that gives the half of Maximum velocity. Which is calculated from graph by
plotting 1/V agains 1/S.

There fore

Large Km = Low Enzyme – substrate affinity.

Small Km = High Enzyme – substrate affinity.

The kinetic constants Km & V are must conveniently determined from a linear transformation of the Michealis
equation, obtained by taking reciprocals.

½ = 1/V + Km/V × 1/S

A plot of 1/v against 1/s therefore gives a strait line of slope Km/V. The reciprocals of the kinetic constants can
then be determined from the intercepts in the axis, since when

1/s = 0, 1/U = 1/V

& When 1/V = 0, 1/S = 1/Km

The graph of 1/V against 1/S is known as line weaver-Burk plot and is the method frequently used to calculate
Km.

Procedure:

A) Determination of Km by DNS method.

Pipette out Enzyme, Substrates, Buffer as shown in tabular column. Incubate exactly for 3 minutes are cease
the reaction by adding 1ml of DNS.
Heat for 5 min. in boiling water bath, cool and add 8ml of water. Read the absorbance at 540 nm and calculate
km & Vmax from graph.

B) Determination of Km by Shaffer Somogyi method.

Pipette out enzyme, substrate, Buffer as shown on tabular column. Incubate for 3 minutes and cease the
reaction by adding 5ml of SSR reagent. Heat in boiling water bath for 15 minutes and cool. Add 1ml of
NH2So4 and titrate against sodium thio sulphate solution. Note down the reading. Calculate the activity and
Km from graph.
 4

Reagents:

1) DNS Reagent:
1 gm of 3,5 dinitro salicylic acid in 200ml of NaOH. Add to 50ml of water. Add the suspension carefully to
50ml of water containing 30gms sodium potassium tartarate and made upto 1000ml. Filter if necessary.

2) Phosphate Buffer of pH 6.9 (0.02m)

Prepare 0.2m NaOH and 0.2m sodium dihydrogen phosphate. Add 24ml of NaOH to 50ml if 0.2M Sodium
dihydrogen phosphate and dilute it to 100ml and standardize it to pH 6.9.

3) Enzyme: 25mg on 10ml of Buffer.

4) Substrate: 1% starch in 100ml Buffer.

LAB-REPORT:

Determinat
ion of Km Subs- O.D. µ g Km
Buffer Enzyme DNS H2O V 1/V
Incubate for 3 minutes minutuuuru

by DNS trate 540 of S 1/S from

Heat in BWB for 5 min. & cool

ml ml ml ml M/L M/L
Method ml nm Maltose graph
No.
1 0 1 - 1 8 -
2 0.1 0.4 0.5 1 8 1000
3 0.15 0.35 0.5 1 8 1400
4 0.2 0.3 0.5 1 8 1400
5 0.25 0.25 0.5 1 8 1560
6 0.3 0.2 0.5 1 8 1680
7 0.35 0.15 0.5 1 8 1760
8 0.4 0.1 0.5 1 8 1760
9 0.45 0.05 0.5 1 8 1840
10 0.5 0 0.5 1 8 1760

Calculation of Activity:

Enzyme unit: 1 Enzyme unit is defined as µ g of Maltose liberated per ml per minute.

Ex. 1. V = µ g of Maltose x 2 x 1000

342 x 3

= 1000 x 2 x 1000

342x3

V = 1949.31.
 5

Results:

1. Km from graph = 33.33.

2. Vmax from graph = 1.42.
3. Slope = 23.47.

LAB – REPORT

Determination of Km by Shoffer – Somogyi: Method.

µ g
Sub- 0.1N Burette Reading
Sl. Buffer Enzyme SSR of V 1/V S 1/s
strate H2So4 Differ-
No ml ml ml mal- M/L M/L M/L M/L
ml ml I F ence tose
ml ml ml
1 0 1 - 5 1 0
Heat in BWB for 15 min. & cool

2 0.1 0.4 0.5 5 1 800

3 0.15 0.35 0.5 5 1 1250
Incubate for 3 minutes

4 0.2 0.2 0.5 5 1 1500

5 0.25 0.25 0.5 5 1 2000
6 0.3 0.2 0.5 5 1 2225
7 0.35 0.15 0.5 5 1 2500
8 0.4 0.1 0.5 5 1 2600
9 0.45 0.05 0.5 5 1 2650
10 0.5 0 0.5 5 1 2975

Result:

1. Km from intercept = 33.33

2. Vmax from intercept = 1.42
3. Slope = Km = 23.47.

Vmax

Calculation of V & 1/V: -

0.5ml of enzyme in 3minutes liberate say 140 g of Maltose. Therefore 1ml of enzyme in 3minutes liberates
140x2 = 280 g Maltose.
 6
∴ 1ml enzyme in 3 minute liberates 280.
∴ 1ml enzyme in 1minute = 280 = 93.3µ g.
3

1 litre of enzyme in 1 minute liberates = 93x1000

= 93000 µ g of Maltose.

∴ Activity in 1moles = 93000

342

V = 27:19 moles.

∴ 1/v = 0.036 mol/min/ml.

Calculation of S & 1/s: -

1% of starch is used.
For test tube No.2.
We have 0.1ml of substrate used.
∴ 1gm = 100ml of phosphate buffer.
1000mg = 100ml
∴ 10mg = 1ml
1mg = 0.1ml
We want for 0.5ml.

∴ 1mg – 1ml
100ml

1000x1 = 1000mg = 1gm.

Since Molecular weight of starch is 2x105 D.

2x105 mg of starch in 1 litre = 1 mole of solute.
-5
1gm of starch = one litre = 1 = 0.5x10 mole/litre
5
2x10

∴ 1/s = 1 = 2x105 moles/litre.

0.5x10-5

DETERMINATION OF KI OF α -AMYLASE

Aim: To determine the Ki of –amylase by DNS and Shoffer-Somogyi method.

Principle: In the presence of an inhibitor, the Michealis & Menton equation is altered as shown below, where
‘i’ is the concentration of inhibitor and Ki the inhibition constant.
 7
No Inhibitor V = V
1+(km/s)

Competitive inhibitor = V=v

1 + Km

S (1 + i/Ki)

Non-competitive inhibitor V = V

(1 + Km/s) (1 + i/Ki)

taking reciprocals of the above equation we have

No inhibitor 1 = 1 =Km 1
V V V S

Competitive inhibitor = 1/V = 1/V + Km/V (1 + i/Ki) 1/S

Non-competitive inhibitor 1/V = 1/V (1+ i/Ki) + Km/V (1 + i/Ki) 1/S

A Graph of (1/V) against (1/S) can be used to determine Ki and the type of inhibition. A competitive inhibitor
alters km and how competitive inhibitor cases change in V.

i.e. Kx = Km (1 + i/Ki)

Vx = V
(1 + i/Ki)

V and Km are determined in the absence of an inhibitor and I is known. Therefore Ki can be reading
calculated.

Procedure: -

A) Preparation of standard graph by DNS method.

Pipette out the enzyme, substrate, and buffer as shown in tabular column. Incubate exactly for 3 minutes and
cease the reaction by addition of 1ml of DNS reagent. Heat in boiling water bath for 5 minutes and cool. Add
l8ml of water and read the absorbance at 540nm. Calculate the amount of Maltose liberated and its activity.
Plot the graph of 1/V against (1/S) which gives Km & Vmax. This is standard graph.

B) Preparation of standard graph by Shoffer-Somogyi method.

Pipette out Substrate, Enzyme, and Buffer as shown in tabular column. Incubate for 3 minutes
exactly and cease the reaction by adding 5ml of SSR reagent. Heat the tubes in boiling water bath
for 15 minutes and cool. Add 1ml of H2SO4 (0.1N) and titrate against standard sodium thio
sulphate. Note down the reading. Calculate 1g of Maltose liberated, activity (V), 1/V, S, 1/S. Plot
the graph of 1/V against 1/S and calculate the Km & Vmax from graph. This is the standard graph.

C) Effect of Inhibitors: -
 8
Repeat the above experiment, but this time incorporate 0.1ml of the inhibitor into the reaction
mixture, again adjusting the value of buffer to give a final reaction volume of 10ml. Repeat the
experiment with 0.4 & 0.6ml of inhibitor by adjusting the volume of buffer. Calculate 1/V & 1/S.
Plot the graph of 1/V against concentration of salicylic acid. Calculate the Ki from graph.

D) Determination of Ki from Dixon plot: -

Plot the graph of 1/v against concentration of Salicylic acid. Determine the value of Ki.

Reagents: -

1) DNS Reagent: -
1 gm of 3,5 dinitro salicylic acid in 200ml of NaOH. Add to 50ml of water. Add the suspension
carefully to 50ml of water containing 30gms of sodium potassium tartarate and made upto 100ml.
Filter of necessary.
2) Phosphate buffer of pH 6.9.
3) Enzyme: 25mg in 10ml of buffer.
4) Substrate: 1% Starch in 100ml Buffer.
5) Salicylic acid: Stock – 10mM.
Above solution is diluted to 0.1mM. 0.4mM, 0.6mM.

Lab-Report

Standard Graph by DNS method.

Km
Sl. Substrate Buffer Enzyme DNS Water O.D. Mg of V 1/v
S 1/S From
No. ml ml ml ml ml 540nm Maltose M/L M/L
graph
Incubate for 3 minutes

1 0 1 - 1 8 -
2 0.1 0.4 0.5 1 8 520
3 0.15 0.35 0.5 1 8 1060
4 0.2 0.3 0.5 1 8 1300
5 0.25 0.25 0.5 1 8 1580
6 0.3 0.2 0.5 1 8 2080
7 0.35 0.15 0.5 1 8 2480
8 0.4 0.1 0.5 1 8 2800
9 0.45 0.05 0.5 1 8 3160
10 0.5 0 0.5 1 8 3580

Lab-Report.
 9

Burette Reading µ g
Sub- Enzy- 0.1N V
Sl. Buffer SSR of 1/V S 1/s
strate me H2So4 Diffe- M/
No. ml ml mal- M/L M/L M/L
ml ml ml I F rence L
tose
ml ml ml

coolHeat in BWB for 15 min. &

1 0 1 5 1 -
2 0.1 0.4 0.5 5 1 275
0.5

Incubate for 3 minutes

3 0.15 0.35 5 1 1150
4 0.2 0.2 0.5 5 1 1150
5 0.25 0.25 0.5 5 1 1100
6 0.3 0.2 0.5 5 1 1400
7 0.35 0.15 0.5 5 1 1450
8 0.4 0.1 0.5 5 1 1825
9 0.45 0.05 0.5 5 1 1925
10 0.5 0 0.5 5 1 2000
0.5

Km = 90.9 from graph.

Vmax = 2

Lab-Report

Inhibition by 0.1mM Inhibitor

0.1 O.D.
Subs Enzy µ g
Sl. Buffer mM DNS Water 540nm V 1/V S 1/s
trate -me of
No. ml I ml ml M/L M/L M/L M/L
ml ml maltose
ml
1 0 0.5 0.1 0.5 1 7.9 0
2 0.1 0.4 0.1 0.5 1 7.9 740
Heat in BWB for 5 min. & cool

3 0.15 0.35 0.1 0.5 1 7.9 940

Incubate for 3 minutes

4 0.2 0.3 0.1 0.5 1 7.9 1180

5 0.25 0.25 0.1 0.5 1 7.9 1220
6 0.3 0.2 0.1 0.5 1 7.9 1500
7 0.35 0.15 0.1 0.5 1 7.9 1530
8 0.4 0.1 0.1 0.5 1 7.9 1980
9 0.45 0.05 0.1 0.5 1 7.9 2010
10 0.5 0 0.1 0.5 1 7.9 2280
 10

Lab-Report:

Inhibition by 0.4 mM salicylic acid

0.4 O.D.
Subs Enzy- µ g
Sl. Buffer mM DNS Water 540nm V 1/V S 1/s
trate me of
No. ml I ml ml M/L M/L M/L M/L
ml ml maltose
ml
1 0 0.5 0.4 0.5 1 7.6
-

Heat in BWB for 5 min. & cool

2 0.1 0.4 0.4 0.5 1 7.6

Incubate exactly for 3 minutes

710
3 0.15 0.35 0.4 0.5 1 7.6
800
4 0.2 0.3 0.4 0.5 1 7.6
1100
5 0.25 0.25 0.4 0.5 1 7.6
1260
6 0.3 0.2 0.4 0.5 1 7.6
1420
7 0.35 0.15 0.4 0.5 1 7.6
1610
8 0.4 0.1 0.4 0.5 1 7.6
1610
9 0.45 0.05 0.4 0.5 1 7.6
1910
10 0.5 0 0.4 0.5 1 7.6
1910

Lab-Report:

Inhibition by 0.6mM salicylic acid

0.6 O.D.
Subs Enzy- µ g
Sl. Buffer mM DNS Water 540nm V 1/V S 1/s
trate me of
No. ml I ml ml M/L M/L M/L M/L
ml ml maltose
ml
1 0 0.5 0.6 0.5 1 7.4 -
Heat in BWB for 5 min. & cool

2 0.1 0.4 0.6 0.5 1 7.4 400

Incubate exactly for 3 minutes

3 0.15 0.35 0.6 0.5 1 7.4 480

4 0.2 0.3 0.6 0.5 1 7.4 630
5 0.25 0.25 0.6 0.5 1 7.4 830
6 0.3 0.2 0.6 0.5 1 7.4 750
7 0.35 0.15 0.6 0.5 1 7.4 1100
8 0.4 0.1 0.6 0.5 1 7.4 900
9 0.45 0.05 0.6 0.5 1 7.4 1020
10 0.5 0 0.6 0.5 1 7.4 1060

Results: -
 11
1. From nature of graph salicylic acid is Non-competitive Inhibitor.
2. Ki from intercept = 0.33.

Effect of Temperature on solubility of α -Amylase.

Principle:
Molecules must posses certain energy of activation (E) before they can react, and enzymes function as
catalysis by lowering this energy of activation, there by enabling the reaction to proceed more rapidly.
The overall change in the free energy (DG) is un affected by the enzyme. Indeed enzymes are very
sensitive to elevated temperature because of protein nature of enzyme. Thermal Denaturation of the
enzyme protein with increasing temperatures will decrease the effective concentration of an enzyme and
consequently decrease the reaction rate. Above 45°c an opposing factor, namely thermal Denaturation
will become. However until at 55°c rapid Denaturation will destroy the catalytic function of the enzyme
protein.

Procedure:
Pipette out 0.5ml of substrate and 0.5ml of enzyme at different temperatures as shown in tabular
column. Incubate exactly for 3 minutes; cease the reaction by addition of 1ml of DNS reagent. Read
the absorbance at 540nm and calculate the activity. Plot the graph of activity against temperature.

Connects:
1. The enzyme shows maximum activity at 40c.
2. Thermal Denaturation of enzyme takes place after 40c and hence decreases in activity.

Reagents:

1) DNS Reagent:
1gm of 3,5 dinitro salicylic acid in 200ml of NaoH. Add to 50ml water. Add the suspension carefully
to 50ml water containing 30gms of sodium potassium tartarate and make upto 100ml. filter if necessary.

2) Phosphate Buffer of pH 6.9:

Prepare 0.2M NaoH and 0.2M sodium dihydrogen phosphates. Add 24ml of NaoH to 50ml of 0.2M
sodium dihydrogen phosphates and dilute it to 100ml and standardize it to pH 6.9.

3) Enzyme: 25mg in 10ml Buffer.

4) Substrate: 1% starch in 100ml Buffer.

Lab Report:

Activity
Sl. Different Substrate Enzyme DNS Water O.D. µ g of
µ Moles/m
No. Temperature ml ml ml ml 540nm Maltose
l
 12
1 1
Control 0.5 0.5 8 0

Incubate exactly for 3

2 1
0 0.5 0.5 8 480
3 1
10 0.5 0.5 8 680
4 1

minutes
20 0.5 0.5 8 960
5 1
30 (RT) 0.5 0.5 8 1120
6 1
40 0.5 0.5 8 1240
7 1
50 0.5 0.5 8 1200
8 1
60 0.5 0.5 8 1100

Calculation of Activity:

A= µ g of Maltose x ml

342 x 3

A= 480 x 2

1026

= 0.93 µ Moles/ml/minute.

Effect of pH on α -Amylase:
Principle:
 13
Since enzymes are proteins, pH changes will profoundly affect the ionic character of the amino and
carboxylic acid groups on the protein and will therefore markedly affect the catalytic site and
confirmation of an enzyme. Enzymes are active over a limited pH range only and a plot of activity
against pH usually gives a bell shaped curve. The pH value of Maximum activity s known as the
optimum pH and this is a characteristic of the enzyme, provided that the enzyme is stable under the
conditions studied. The variation of activity with pH is due to change in the state of ionization of the
enzyme protein and other components of reaction mixture.

Procedure:
Pipette out 0.5ml of substrate, 0.5ml of enzyme at different pH range as shown in tabular column.
Incubate exactly for 3 minutes and cease the reaction by DNS reagent. Read the absorbance at 540 nm.
Calculate the Activity plot the graph of pH against activity.

Comments:

1. The Enzyme shows Maximum Activity at pH 6.5.

2. The Activity is lost at pH 7.5, which refers to decrease in activity.

Reagents:

1) DNS reagent:

1gm of 3,5 dinitro salicylic acid in 200ml of NaoH. Add to 50ml water. Add the suspension carefully
to 50ml of water containing 30gms of sodium potassium tartarate and made upto 100ml. Filter of
necessary.

2) Phosphate Buffer:
Prepare phosphate buffer of pH range 4.5, 5,6,6,5,7,7.5 by mixing 0.2m NaoH and 0.2m sodium
dihydrogen phosphate. Standardize the pH to of required.

3) Enzyme: Pancreatic Amylase. 25mg in 10ml Buffer.

5) Substrate: 1.1starch in 100ml Buffer.

Activity
Sl. PH Substrate Buffer Enzyme DNS Water O.D. µ g of
µ moles/ml/mi
No. Range ml ml ml ml ml 540nm Maltose
n
 14
1 1 -
0.5 - 1 7 0.0 -
C 4.5 1 360
0.5 0.5 1 7 0.09 0.70
T
2

Heat in BWB for 5 minutes & cool

0.5 1 - 1 7 0.0 - -
C 5

Incubate exactly for 3 minutes

0.5 1 0.5 1 7 0.0 - -
T
3
0.5 1 - 1 7 0.0 - -
C 6
0.5 1 0.5 1 7 0.0 - -
T
4
0.5 1 - 1 7 0.0 - -
C 6.5
0.5 1 0.5 1 7 0.12 460 0.89
T
5
0.5 1 - 1 7 0.0 - -
C 7
0.5 1 0.5 1 7 0.12 460 0.89
T
6 -
C 0.5 1 - 1 7 0.0 - 0.62
7.5
T 0.5 1 0.5 1 7 0.08 320
 15
Effect of NaCl on α -Amylase
Principle: Enzymes require co-factors and co-enzymes for their activity. It is a co-factor. Which
increases the enzyme activity. At high concentration of salt the protein (enzyme) is precipitated. This is
known as salting out.

Procedure: Pipette out 0.5ml of substrate, 0.5ml of Buffer, 0.5ml of enzyme. Introduce varying
concentration of NaCl (1mM, 2mM, 4mM, 8mM, 10mM) into the reaction mixture. Incubate exactly for
3 minutes and cease the reaction by addition of 1ml of DNS reagent. Measure the absorbance at 540nm.
Calculate the activity. Plot the graph of activity versus concentration of NaCl.

Comments:
1) The activity of enzyme decreases as the concentration of NaCl is increased due to
precipitation of protein.

Reagents:

1) DNS reagent:
1gm of 3,5 dinitro salicylic acid in 200ml of NaOH. Add to 50ml water. Carefully trans for the
suspension to 50ml water containing 30gms of sodium potassium tartarate and make up the volume to
1000ml. Filter if necessary.

2) Phosphate buffer of pH 6.9.

Prepare 0.2M NaoH and 0.2M sodium dihydrogen phosphates. Add 24ml of NaOH to 50ml of Sodium
dihydrogen phosphate and make up the volume to 100ml with water. Standardize the solution to pH 6.9.

3) Enzyme: 25mg in 10ml Buffer.

4) Substrate: 1% starch is 100ml buffer.

5) NaCl: Stock 50mM.

Lab-Report

Sl. Substrate Buffer NaCl Enzyme DNS H2O O.D. µ G of Activity

 16
µ moles/
NO. ml ml ml ml ml ml 540nm Maltose
ml/min
-
1
0.5 0.5 0.5 10.5 -

Heat in BWB for 5 min & cool

Incubate exactly for 3 minutes
0.24
2 (1mM)
0.5 0.5 0.5 10.26 500
0.48
3 (2mM)
0.5 0.5 0.5 10.62 490
0.96
4 (4mM)
0.5 0.5 0.5 9.54 400
1.92
5 (8mM)
0.5 0.5 0.5 8.58 300
2.4
6 (10mM
0.5 0.5 0.5 8.1 280
)

Effect of Urea on α –Amylase

Principle: Urea is denaturing agent. At high concentration of urea, the protein is denatured and the
activity of enzyme is reduced and the activity is completely lost.

Procedure: Pipette out 0.5ml substrate, 0.5ml enzyme and incorporate the varying concentration of
urea into the reaction mixture as shown in tabular column. Cease the reaction after incubation for
exactly 3minutes and read the absorbance at 540nm. Measure the O>D. at 540nm. Calculate activity.
Plot the activity against concentration of urea.

Comments:
1) The protein is denatured at high concentration of urea and the activity is lost.

Reagents:
1) DNS reagent:
1gm of 3,5 dinitro salicylic acid is dissolved in 200ml of NaOH. 50ml of water is added. The
suspension is carefully transformed into 50ml water containing 30gms of sodium potassium tartarate.
Make up the volume to 1000ml. Filter if necessary.

2) Phosphate Buffer:
Prepare 0.2M Sodium hydroxide & 0.2M Sodium dihydrogen phosphates. Add 24ml of 0.2M NaOH to
50ml of sodium dihydrogen phosphate and make up the volume to 100ml. Standardize the solution to
pH 6.9.

3) Enzyme: 25mg in 10ml Buffer.

4) Substrate: 1% starch in 100ml buffer.

5) Urea: 0.08M stock.

Lab-Report:
 17
Sl. Substrate Enzyme Urea DNS Water O>D. G of V
NO. ml ml ml ml ml 540nm Maltose Moles/ml/min
0
1 0
0.5 - 1 10 0 0
1.5

Heat in BWB for 5 minutes & cool

2 300
0.5 0.5 (0.01) 1 8.5 0.073 0.58
3
3 40

Incubate for 3minutes

0.5 0.5 (0.02) 1 7 0.01 0.07
4.5
4 4
0.5 0.5 (0.03) 1 5.5 0.001 0.007
6
5 0
0.5 0.5 (0.04) 1 4 0 0
7.5
6 0
0.5 0.5 (0.05) 1 2.5 0 0
9
7 0
0.5 0.5 (0.06) 1 1 0 0
10.5
8 0
0.5 0.5 (0.07) 1 0 0 0

Determination of activity of proteolytic enzyme by kuznitz method

Aim: To determine the activity of proteolytic enzyme by Kuznitz method.

Procedure:

A) Tyrosine Calibration Curve:

Prepare the enzyme extract from young tendrils of papain using phosphate buffer. Pipette out 0.2, 0.3,
0.5ml of enzyme. Make the volume to 1ml with phosphate buffer and Incubate at 37°C for 5minutes.
Add 1mlof 1% casein, shake and incubate for 20minutes. Add 3ml of 5% TCA solution. After 1 hour
centrifuge and test for supernatant for Tyrosine. This is unknown and carryout FCR method for 5 test
tubes.

B) Standard Tyrosine Curve:

Pipette out standard Tyrosine of 0.1m 0.2, 0.5, 0.4, 0.5, 0.6 & 0.7ml of Tyrosine and make up the
volume upto 1ml with water. Add 5ml of alkaline copper reagent and incubate for 20minutes. Add
0.5ml of FCR reagent and read the absorbance at 660nm. Calculate the concentration and plot the
concentration against absorbance.

C) Assay of Enzyme activity:

Take 0.5ml of enzyme extract; make up the volume to 1ml with water. Add 5ml alkaline copper reagent
and incubate for 20minutes. Add 0.5ml of FCR reagent and read the absorbance at 660nm. Calculate
the amount of Tyrosine liberated and calculate the enzyme activity.

Definition of Enzyme Activity: (Papain)

1 unit of Enzyme activity is defined as amount, which releases 10µ g of Tyrosine equivalent per hour
under the conditions of assay.
 18
Reference:

1) Arman CR. 1970, Methods in Enzymology, Ed. Pelman Landel. Academic press, P-228-229

2) Kakade Ln.C., Cereal chemistry 1969, 46, P-518-526.

Reagents:

1) 1% Casein:
1gm of casein was suspended in 80ml of phosphate Buffer (0.1M) pH 7.6 and completely dissolved by
heating in boiling water bath for 15minutes. This brings about complete solution of casein. This
solution was cooled and made to 100ml with buffer and stored in fridge when not in use. Prior to use
casein solution was kept in hot water bath for atleast 5 minutes.

2) Phosphate Buffer: (pH 7.6).

Prepare 43ml of 0.1M NaOH to 50ml of 0.1M sodium dihydrogen phosphates and dilute to 100ml.
Standardize the solution by adjusting to pH 7.6.

3) 5% TCA solution.

4) Enzyme extract:

Some 4-5 tendril papain leaves are crashed using water and extract with phosphate buffer of pH 7.6.
Filtered and used as papain solution.

5) Tyrosine: 10mg/50ml (0.2M)

Lab-Repot:

PART – A

Sl. Standard Phosphate 1% 5% TCA

No. Enzyme ml Buffer ml Casein Solution
5minutesIncubate at 37C for

Shake and Incubate for

1 0 1 1 3
After 1 hour
Centrifuge
20minutes

2 0.2 0.8 1 3
And test for
supernatant
3 0.3 0.7 1 3
for Tyrosine
4 0.5 0.5 1 3

PART – B

Tyrosine Calibration Curve

Sl. Tyrosine Water Alkaline FCR µ g of Tyrosine O.D. at

No. ml ml Cu-reagent ml 660nm
 19
ml
1 1

Incubate for 20 minutes

0 5 0.5 - -
2 0.9
0.1 5 0.5 20 0.2
3 0.8
0.2 5 0.5 40 0.39
4 0.7
0.3 5 0.5 60 0.51
5 0.6
0.4 5 0.5 80 0.7
6 0.5
0.5 5 0.5 100 0.84
7 0.4
0.6 5 0.5 130 1.0
8 0.3
0.7 5 0.5 140 1.2

Lab-Report:

PART - C

Assay of Enzyme Activity

Alkaline Concentration
Enzyme O.D. V
Sl. Water Cu FCR Mean Of ((g)
Extract at µ g/ml
No. ml Reagent ml OD. Unknown
ml 660nm /hour
ml 1year
minutesIncubate Fro 20

minutesIncubate for 20

- 1 5 0.5 -
I - -
0.5 0.5 5 0.5 0.225
II 25 375
0.5 0.5 5 0.5 -
0.5 0.5 5 0.5 0.275
III 28 277.2
0.5 0.5 5 0.5
0.5 0.5 5 0.5 0.405
IV 46 276.0
0.5 0.5 5 0.5

Calculation of Activity

1) Enzyme extract = 5ml.

Activity = µ g of Tyrosine x ml x hour

10 x Incubation time
= 250 x 5 x 60
10 x20
 20
= 375 µ mol/ml/hour.

2) 0.5 ml liberates 28 g of Tyrosine

∴ 5ml liberates 280 g of Tyrosine

V = 280 x 5 x 60
10 x 20

= 277.2 µ mol/ml/hour.
21
 22

Assay of Alkaline Phosphatase

Principle: Alkaline Phosphatases (E.C. No. 3:1:3:1) is found in bone, kidney, liver and intestine and
acts on phosphoric esters with the liberation of inorganic phosphate.

When p-nitryl phenyl is used as substrate, p-nitrophenol and inorganic phosphates are liberated. The p-
nitrophenol is yellow in colour in alkaline condition and with absorbance at 405nm.

Units:

1 unit of enzyme activity is defined as moles of p-nitro phenol liberated per ml per minutes.

Procedure:

A) Standard PNP Curve:

In a series of test tubes pipette out standard PNP and water as shown in tabular column. Add 5ml of
NaOH to each tube and read the O.D at 405nm. Plot the graph of concentration against absorbance at
405nm.

B) Assay of Enzyme Activity:

To a clean dry test tube add substrate and enzyme as shown in tabular column. Keep the tubes in water
bath at 37°C for 3-5 minutes. Then add enzyme and incubate exactly for 10minutes. Stop the reaction
by adding 5ml of NaOH. Shake well and read the absorbance at 405nm. Find the amount of PNP
liberated from standard graph and calculate activity.

Reagent:

1) Sodium carbonate bicarbonate buffer:

0.02M of pH 9.5.

2) Substrate:
P-nitro phenyl phosphate – 5mM/litre in Buffer.

3) P-Nitro Phenol: 69.9 mg of PNP in 100ml 0.02M NaOH. Dilute 1ml of above solution in Buffer.

4) 0.02N NaOH.

5) Enzyme:

Buffer extract of germinated seeds is used as source of enzyme.

 23

Lab-Report:

1) Standard PNP Curve:

Sl. PNP 0.2M NaOH Concentration Of

O.D. at 405 nm
No. ml ml PNP µ moles
1 0.0 6 0 0.0
2 0.1 5.9 10 0.04
3 0.2 5.8 20 0.09
4 0.3 5.7 30 0.15
5 0.4 5.6 40 0.2
6 0.5 5.5 50 0.23
7 0.6 5.4 60 0.27
8 0.7 5.3 70 0.3
9 0.8 5.2 80 0.35
10 0.9 5.1 90 0.38
11 1.0 5 100 0.41

Sl Substrate Buffer Enzyme NaOH O.D. at Mol of V

No. ml ml ml ml 405min substrate Mol/ml/minute
0.0
Incubate at 37°C
for 10 minutes

1 0.1 0.9 5
0.2
2 0.1 0.7 5
57 142.5
0.2
3 0.1 0.7 5

Calculation of Activity:

Enzyme extract = 10ml

0.2ml liberates 57 µ mol of substrate.
∴ 10ml liberates 2850 µ mol of substrate.
∴ Enzyme Activity per minutes = 2850 = 285 µ mol/min/2gm of wheat

10
∴ Enzyme activity per minute = 285 = 142.5 µ mol/gm wheat.

Result:

1) The activity of alkaline Phosphatase is 142.5µ mol/gm of wheat.

Reagent:

1) David T. Plummer. (III edition) P-236.

 24

Assay of lipase activity

Aim: To assay the Lipase activity.

Principle:

Lipase hydrolyses the fats into Mono glycerides, diglycerides and free fatty acids and glycerol. After
complete hydrolysis all fatty acids are liberated. The free fatty acid liberated are measured either by
titration is decreasing pH. Fats are emulsified before they are acted upon by Lipase.

Procedure:

Titrometric Assay of Lipase:

Pipette out exactly 2.5ml of buffer into a conical flask containing 5ml of substrate. Keep the flask in
thermostat at 35°c for 5minutes. Add 2ml of enzyme solution. Shake well and incubate for 1Hcur at
37°C. After incubation add 5ml of Ethyl alcohol and ether 5ml to stop the reaction. Add a drop of
indicator and titrate against 0.1N NaOH.

Reagents:

1) 0.1N NaOH.
2) Phenophalean Indicator.
3) 0.1N Acetate Buffer of pH 5:

Place 32ml of 0.1N mol/litre acetic acid in a 100ml vol. flask and make upto the mark with 0.1N
mol/litre sodium acetate.

4) Substrate:

Dissolve 2gm of Gum acasia in 80ml of water. Ad 100mg of cholate or tauro cholate. Stirr well. Add
10ml of groundnut oil. Homogenies for 2minutes. Make the volume to 100ml with distill water.

5) Enzyme Extract:

10gms of castor seeds are homogenized with 20ml of ice cold Acetone. The suspension is filtered
quickly and residue is washed with acetone, acetone ether (1:1) and ether respectively. The air-dried
powder now called the acetone dry powder is used as source of enzyme. Suspend 2gm of acetone
powder in 20ml buffer for 120-15min and centrifuge. Supernatant is used source of enzyme. In
similion way enzyme extract is prepared for flesh castor seed, green almond, dry almond.
 25

Lab-Report:

Observations

a) Substrate A = oil + water + Acasia.

b) Substrate B = oil + water + Acasia + 100mg of B.Salt
c) Substrate C = oil + water + Acasia + 300mg of B.Salt
d) Substrate D = oil + water + Acasia z = 500mg of Bile salt.

Enzyme:

a) Enzyme I – Green castor.

b) Enzyme II – Dry castor.

c) Enzyme III – Green almond.

d) Enzyme IV – Fruit almond.

T.T. Sub- Differ- Activity V

Buffer Enzyme Ethanol Ether NaOH
No. strate ence (Unites) µ mol/
ml ml ml ml ml
ml ml ml/min
Incubate 5min. 37°C

1litre inoculation

1 27 5 - 5 5 4.2
0.6 300
2 26 5 1 5 5 4.2
26