Effect of substrate concentration on -Amylase

Aim: To determine the effect of sub state concentration on –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.

Sl.No.Substrate

mlEnzyme

mlTime

(minutes)DNSml

H2Oml

O.D.540nm

Mg of Maltose

Activitymol/ml/min

1234567

Heat in B

oiling water

bath for 5min &

cool

1

EFFECT OF TIME ON -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.

Sl.No.Substrate

mlEnzyme

mlTime

(minutes)DNSml

H2Oml

O.D.540nm

Mg of Maltose

Activitymol/ml/min

1234567

Heat in B

oiling water

bath for 5min &

cool

Result:

2

DETERMINATION OF KM OF -AMYLASE

BENFELD & TITRATION METHOD.

Aim: To determine the Km & Vmax of -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.

3

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:

Determination of Km by DNS Method

No.

Subs-trateml

Bufferml

Enzymeml

Incu

bate

for

3 m

inut

es m

inut

uuur

u DNSml

Hea

t in

BW

B f

or 5

min

. & c

ool H2O

ml

O.D. 540nm

g of

Maltose

VM/L

1/VM/L

S 1/SKm fromgraph

12345678910

00.10.150.20.250.30.350.40.450.5

10.40.350.30.250.20.150.10.05

0

-0.50.50.50.50.50.50.50.50.5

1111111111

8888888888

-100014001400156016801760176018401760

Calculation of Activity:

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

Ex. 1. V = g of Maltose x 2 x 1000 342 x 3

= 1000 x 2 x 1000 342x3

V = 1949.31.

4

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.

Sl.No

Sub-strate

ml

Bufferml

Enzymeml

SSRml

0.1NH2So4

ml

Burette Readinggof

mal-tose

VM/L

1/VM/L

SM/L

1/sM/L

Iml

Fml

Differ-enceml

12345678910

00.10.150.20.250.30.350.40.450.5

10.40.350.20.250.20.150.10.050

-0.50.50.50.50.50.50.50.50.5

Incu

bate

for

3 m

inut

es

5555555555

Hea

t in

BW

B f

or 1

5 m

in. &

coo

l 1111111111

080012501500200022252500260026502975

Result:

1. Km from intercept = 33.332. Vmax from intercept = 1.423. 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.

5

1ml enzyme in 3 minute liberates 280. 1ml enzyme in 1minute = 280 = 93.3g. 3

1 litre of enzyme in 1 minute liberates = 93x1000 = 93000 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 = 0.1mlWe want for 0.5ml.

1mg – 1ml 100ml

1000x1 = 1000mg = 1gm.

1

Since Molecular weight of starch is 2x105 D.2x105 mg of starch in 1 litre = 1 mole of solute.

1gm of starch = one litre = 1 = 0.5x10-5

mole/litre

2x105

1/s = 1 = 2x105 moles/litre.

0.5x10-5

DETERMINATION OF KI OF -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.

6

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 : -

7

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.

Sl.No.

Substrateml

Buffer ml

Enzymeml

DNSml

Waterml

O.D.540nm

Mg of Maltose

V M/L

1/vM/L

S 1/SKm

Fromgraph

12345678910

00.10.150.20.250.30.350.40.450.5

10.40.350.30.250.20.150.10.050

-0.50.50.50.50.50.50.50.50.5 In

cuba

te f

or 3

min

utes

1111111111

8888888888

-52010601300158020802480280031603580

Lab-Report.

8

Sl.No.

Sub-strate

ml

Bufferml

Enzy-meml

SSRml

0.1NH2So4

ml

Burette Reading gof

mal-tose

VM/L

1/VM/L

SM/L

1/sM/L

Iml

Fml

Diffe-rence

ml

12345678910

00.10.150.20.250.30.350.40.450.5

10.40.350.20.250.20.150.10.050

0.50.50.50.50.50.50.50.50.50.5

Incu

bate

for

3 m

inut

es

5555555555

Hea

t in

BW

B f

or 1

5 m

in. &

co

ol

1111111111

-27511501150110014001450182519252000

Km = 90.9 from graph.Vmax = 2

Lab-Report

Inhibition by 0.1mM Inhibitor

Sl.No.

Substrateml

Bufferml

0.1mM

Iml

Enzy-meml

DNSml

Waterml

O.D.540nm

gof

maltose

VM/L

1/VM/L

SM/L

1/sM/L

12345678910

00.10.150.20.250.30.350.40.450.5

0.50.40.350.30.250.20.150.10.050

0.10.10.10.10.10.10.10.10.10.1

0.50.50.50.50.50.50.50.50.50.5

Incu

bate

for

3 m

inut

es

1111111111

Hea

t in

BW

B f

or 5

min

. & c

ool 7.9

7.97.97.97.97.97.97.97.97.9

07409401180122015001530198020102280

9

Lab-Report:

Inhibition by 0.4 mM salicylic acid

Sl.No.

Substrateml

Bufferml

0.4mM

Iml

Enzy-meml

DNSml

Waterml

O.D.540nm

gof

maltose

VM/L

1/VM/L

SM/L

1/sM/L

12345678910

00.10.150.20.250.30.350.40.450.5

0.50.40.350.30.250.20.150.10.050

0.40.40.40.40.40.40.40.40.40.4

0.50.50.50.50.50.50.50.50.50.5

Incu

bate

exa

ctly

for

3 m

inut

es

1111111111

Hea

t in

BW

B f

or 5

min

. & c

ool 7.6

7.67.67.67.67.67.67.6

7.67.6

-7108001100126014201610161019101910

Lab-Report:

Inhibition by 0.6mM salicylic acid

Sl.No.

Substrateml

Bufferml

0.6mM

Iml

Enzy-meml

DNSml

Waterml

O.D.540nm

gof

maltose

VM/L

1/VM/L

SM/L

1/sM/L

12345678910

00.10.150.20.250.30.350.40.450.5

0.50.40.350.30.250.20.150.10.050

0.60.60.60.60.60.60.60.60.60.6

0.50.50.50.50.50.50.50.50.50.5

Incu

bate

exa

ctly

for

3 m

inut

es

1111111111

Hea

t in

BW

B f

or 5

min

. & c

ool 7.4

7.47.47.47.47.47.47.4

7.47.4

-400480630830750110090010201060

Results: -

10

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

Effect of Temperature on solubility of -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 45c an opposing factor, namely thermal Denaturation will become. However until at 55c 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:

Sl.No.

DifferentTemperature

Substrateml

Enzymeml

DNSml

Waterml

O.D.540nm

g of Maltose

ActivityMoles/ml

11

12345678

Control01020

30 (RT)405060

0.50.50.50.50.50.50.50.5

0.50.50.50.50.50.50.50.5 In

cuba

te e

xact

ly f

or 3

m

inut

es

11111111

88888888

04806809601120124012001100

Calculation of Activity:

A= g of Maltose x ml

342 x 3

A= 480 x 2 1026

= 0.93 Moles/ml/minute.

Effect of pH on -Amylase:

Principle:

12

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.

Sl.No.

PHRange

Substrateml

Bufferml

Enzymeml

DNSml

Waterml

O.D.540nm

g of Maltose

Activitymoles/ml/min

13

1CT

4.50.50.5

11

-0.5

Incu

bate

exa

ctly

for

3 m

inut

es

11

Hea

t in

BW

B f

or 5

min

utes

& c

ool

77

0.00.09

-360

-0.70

2CT

50.50.5

11

-0.5

11

77

0.00.0

--

--

3CT

60.50.5

11

-0.5

11

77

0.00.0

--

--

4CT

6.50.50.5

11

-0.5

11

77

0.00.12

-460

-0.89

5CT

70.50.5

11

-0.5

11

77

0.00.12

-460

-0.89

6CT

7.50.50.5

11

-0.5

11

77

0.00.08

-320

-0.62

14

Effect of NaCl on -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. G of Activity

15

NO. ml ml ml ml ml ml 540nm Maltosemoles/ml/min

1

2

3

4

5

6

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

-

0.24(1mM)

0.48(2mM)

0.96(4mM)

1.92(8mM)

2.4(10mM)

0.5

0.5

0.5

0.5

0.5

0.5 Incu

bate

exa

ctly

for

3 m

inut

es

Hea

t in

BW

B f

or 5

min

& c

ool 10.5

10.26

10.62

9.54

8.58

8.1

-

500

490

400

300

280

Effect of Urea on –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:

Sl. Substrate Enzyme Urea DNS Water O>D. G of V

16

NO. ml ml ml ml ml 540nm Maltose Moles/ml/min

1

2

3

4

5

6

7

8

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

-

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0

1.5(0.01)

3(0.02)

4.5(0.03)

6(0.04)

7.5(0.05)

9(0.06)10.5

(0.07)

Incu

bate

for

3m

inut

es

1

1

1

1

1

1

1

1

Hea

t in

BW

B f

or 5

min

utes

& c

ool

10

8.5

7

5.5

4

2.5

1

0

0

0.073

0.01

0.001

0

0

0

0

0

300

40

4

0

0

0

0

0

0.58

0.07

0.007

0

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 10g of Tyrosine equivalent per hour under the conditions of assay.

Reference:

17

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.No.

StandardEnzyme ml

PhosphateBuffer ml

1%Casein

5% TCASolution

1

2

3

4

0

0.2

0.3

0.5

1

0.8

0.7

0.5

Incu

bate

at 3

7C

for

5min

utes

1

1

1

1

Sha

ke a

nd

Incu

bate

for

20

min

utes

3

3

3

3

After 1 hourCentrifugeAnd test for supernatant for Tyrosine

PART – B

Tyrosine Calibration Curve

Sl.No.

Tyrosineml

Waterml

AlkalineCu-reagent

ml

FCRml

g of TyrosineO.D. at660nm

18

12345678

00.10.20.30.40.50.60.7

10.90.80.70.60.50.40.3

55555555

Incu

bate

for

20

min

utes

0.50.50.50.50.50.50.50.5

-20406080100130140

-0.20.390.510.70.841.01.2

Lab-Report:

PART - C

Assay of Enzyme Activity

Sl.No.

EnzymeExtract

ml

Waterml

AlkalineCu

Reagent ml

FCRml

O.D. at

660nm

Mean OD.

ConcentrationOf ((g)

Unknown 1year

Vg/ml/hour

I

II

III

IV

-

0.50.50.50.50.50.5

1

0.50.50.50.50.50.5

5

555555

Incu

bate

Fro

20

min

utes

0.5

0.50.50.50.50.50.5

Incu

bate

for

20

min

utes -

-

0.225

0.275

0.405

-

25

28

46

-

375

277.2

276.0

Calculation of Activity

1) Enzyme extract = 5ml.

Activity = g of Tyrosine x ml x hour 10 x Incubation time = 250 x 5 x 60 10 x20 = 375 mol/ml/hour.

19

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 mol/ml/hour.

20

21

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 37C 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.

22

Lab-Report:

1) Standard PNP Curve:

Sl.No.

PNPml

0.2M NaOHml

Concentration OfPNP moles

O.D. at 405 nm

1234567891011

0.00.10.20.30.40.50.60.70.80.91.0

65.95.85.75.65.55.45.35.25.15

0102030405060708090100

0.00.040.090.150.20.230.270.30.350.380.41

Calculation of Activity:

Enzyme extract = 10ml0.2ml liberates 57 mol of substrate.10ml liberates 2850 mol of substrate.Enzyme Activity per minutes = 2850 = 285 mol/min/2gm of wheat 10Enzyme activity per minute = 285 = 142.5 mol/gm wheat. 2

Result:

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

Reagent:

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

Assay of lipase activity

SlNo.

Substrateml

Bufferml

Enzymeml

NaOHml

O.D. at405min

Mol ofsubstrate

VMol/ml/minute

1

2

3

0.1

0.1

0.1

0.9

0.7

0.7

0.0

0.2

0.2

Incu

bate

at

37

C

for

10

m

inut

es

5

5

557 142.5

23

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 35c for 5minutes. Add 2ml of enzyme solution. Shake well and incubate for 1Hcur at 37C. 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.

Lab-Report:

24

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.No.

Bufferml

Sub-strateml

Enzymeml

Ethanolml

Etherml

NaOHml

Differ-enceml

Activity(Unites)

Vmol/ml/min

1

2

27

26

5

5

Incu

bate

5m

in. 3

7C

-

1

1lit

re in

ocul

atio

n

5

5

5

5

4.2

4.20.6 300

25

26