Effect Of Substrate Concentration On α -amylase

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. Sl.No.

H2O O.D. ml 540nm Heat in Boiling water bath for 5min & cool

1 2 3 4 5 6 7

Substrate Enzyme Time DNS ml ml (minutes) ml

Mg of Maltose

Activity mol/ml/min

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. Sl.No.

Result:

H2O O.D. ml 540nm Heat in Boiling water bath for 5min & cool

1 2 3 4 5 6 7

Substrate Enzyme Time DNS ml ml (minutes) ml

Mg of Maltose

Activity mol/ml/min

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.

Substrate ml

Buffer ml

Enzyme ml

No. 1 2 3 4 5 6 7 8 9 10

0 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5

1 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

DNS ml

Incubate for 3 minutes minutuuuru

Determinat ion of Km by DNS Method

1 1 1 1 1 1 1 1 1 1

Heat in BWB for 5 min. & cool

LAB-REPORT:

H2O ml 8 8 8 8 8 8 8 8 8 8

O.D. 540 nm

µ g of Maltose

V M/L

1/V M/L

1000 1400 1400 1560 1680 1760 1760 1840 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.

S 1/S

Km from graph

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.

0 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5

Buffer ml

Enzyme ml

SSR ml

0.1N H2So4 ml

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

5 5 5 5 5 5 5 5 5 5

1 1 1 1 1 1 1 1 1 1

1 0.4 0.35 0.2 0.25 0.2 0.15 0.1 0.05 0

Heat in BWB for 15 min. & cool

1 2 3 4 5 6 7 8 9 10

Substrate ml

Incubate for 3 minutes

Sl. No

Burette Reading I ml

F ml

Difference ml

µ g of maltose

V M/L

1/V M/L

S M/L

0 800 1250 1500 2000 2225 2500 2600 2650 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.

1/s M/L

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. 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 2x10

∴ 1/s =

1

-5

= 0.5x10 mole/litre

5

= 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.

Buffer ml

1 2 3 4 5 6 7 8 9 10

1 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

0 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5

Lab-Report.

Enzyme ml 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

DNS Water ml ml Incubate for 3 minutes

Sl. Substrate No. ml

1 1 1 1 1 1 1 1 1 1

8 8 8 8 8 8 8 8 8 8

O.D. 540nm

Mg of Maltose 520 1060 1300 1580 2080 2480 2800 3160 3580

V M/L

1/v M/L

S 1/S

Km From graph

9

0 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5

Buffer ml

1 0.4 0.35 0.2 0.25 0.2 0.15 0.1 0.05 0

Enzyme ml

0.1N H2So4 ml

SSR ml

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

coolHeat in BWB for 15 min. &

1 2 3 4 5 6 7 8 9 10

Substrate ml

Incubate for 3 minutes

Sl. No.

5 5 5 5 5 5 5 5 5 5

µ g of maltose

Burette Reading I ml

F ml

Difference ml

1 1 1 1 1 1 1 1 1 1

V M/ L

1/V M/L

S M/L

1/s M/L

275 1150 1150 1100 1400 1450 1825 1925 2000

Km = 90.9 from graph. Vmax = 2

Lab-Report Inhibition by 0.1mM Inhibitor

1 2 3 4 5 6 7 8 9 10

0 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5

0.5 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

Enzy -me ml

DNS ml

Water ml

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

1 1 1 1 1 1 1 1 1 1

7.9 7.9 7.9 7.9 7.9 7.9 7.9 7.9 7.9 7.9

Heat in BWB for 5 min. & cool

Buffer ml

0.1 mM I ml 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

Incubate for 3 minutes

Sl. No.

Subs trate ml

O.D. 540nm

µ g of maltose 0 740 940 1180 1220 1500 1530 1980 2010 2280

V M/L

1/V M/L

S M/L

1/s M/L

10 Lab-Report: Inhibition by 0.4 mM salicylic acid Buffer ml

1 2 3 4 5 6 7 8 9 10

0 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5

0.5 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

0.4 mM I ml 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4

Enzyme ml

DNS ml

Water ml

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

1 1 1 1 1 1 1 1 1 1

7.6 7.6 7.6 7.6 7.6 7.6 7.6 7.6 7.6 7.6

Heat in BWB for 5 min. & cool

Subs trate ml

Incubate exactly for 3 minutes

Sl. No.

O.D. 540nm

µ g of maltose

V M/L

1/V M/L

S M/L

1/s M/L

V M/L

1/V M/L

S M/L

1/s M/L

710 800 1100 1260 1420 1610 1610 1910 1910

Lab-Report: Inhibition by 0.6mM salicylic acid Buffer ml

1 2 3 4 5 6 7 8 9 10

0 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5

0.5 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

Results: -

0.6 mM I ml 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6

Enzyme ml

DNS ml

Water ml

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

1 1 1 1 1 1 1 1 1 1

7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4

Heat in BWB for 5 min. & cool

Subs trate ml

Incubate exactly for 3 minutes

Sl. No.

O.D. 540nm

µ g of maltose 400 480 630 830 750 1100 900 1020 1060

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. 2.

The enzyme shows maximum activity at 40c. 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. Different Substrate Enzyme No. Temperature ml ml

DNS ml

Water ml

O.D. 540nm

Activity µ g of µ Moles/m Maltose l

1 2 3 4 5 6 7 8

Control 0 10 20 30 (RT) 40 50 60

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 0.5

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:

Incubate exactly for 3 minutes

12 1 1 1 1 1 1 1 1

8 8 8 8 8 8 8 8

0 480 680 960 1120 1240 1200 1100

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. 2.

The Enzyme shows Maximum Activity at pH 6.5. 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. PH No. Range

Substrate ml

Buffer Enzyme ml ml

DNS ml

Activity Water O.D. µ g of µ moles/ml/mi ml 540nm Maltose n

4.5

0.5 0.5

5

0.5 0.5

6

1 1

0.5

1 1

1 1

0.5

1 1

0.5 0.5

1 1

0.5

6.5

0.5 0.5

1 1

0.5

7

0.5 0.5

1 1

0.5

7.5

0.5 0.5

1 1

0.5

Incubate exactly for 3 minutes

1 C T 2 C T 3 C T 4 C T 5 C T 6 C T

1 1 1 1 1 1 1 1

Heat in BWB for 5 minutes & cool

14 360

7 7

0.0 0.09

7 7

0.0 0.0

-

-

7 7

0.0 0.0

-

-

7 7

0.0 0.12

460

0.89

7 7

0.0 0.12

460

0.89

7 7

0.0 0.08

320

0.70

0.62

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

NO.

ml

ml

ml

ml

ml

ml

540nm Maltose

16 µ moles/ ml/min

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

Heat in BWB for 5 min & cool

1

Incubate exactly for 3 minutes

10.5

-

10.26

500

10.62

490

9.54

400

8.58

300

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

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

Urea ml 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)

DNS ml

Water ml

1

10

0

8.5

0.073

7

0.01

5.5

0.001

4

0

2.5

0

1

0

0

0

1 1 1 1 1 1 1

Heat in BWB for 5 minutes & cool

1

Substrate Enzyme ml ml

Incubate for 3minutes

Sl. NO.

O>D. G of V 540nm Maltose Moles/ml/min 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 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 Phosphate Buffer ml

1

0

1

2

0.2

0.8

3

0.3

0.7

4

0.5

0.5

1% Casein

5% TCA Solution Shake and Incubate for 20minutes

Standard Enzyme ml

5minutesIncubate at 37C for

Sl. No.

1 1 1 1

3 3 3

After 1 hour Centrifuge And test for supernatant for Tyrosine

3

PART – B Tyrosine Calibration Curve Sl. No.

Tyrosine ml

Water ml

Alkaline Cu-reagent

FCR ml

µ g of Tyrosine

O.D. at 660nm

19 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Incubate for 20 minutes

ml 1 2 3 4 5 6 7 8

5 5 5 5 5 5 5 5

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

20 40 60 80 100 130 140

0.2 0.39 0.51 0.7 0.84 1.0 1.2

Lab-Report: PART - C Assay of Enzyme Activity

II III IV

Water ml

-

1

Alkaline Cu Reagent ml 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

5 5 5 5 5 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

O.D. at 660nm

FCR ml 0.5 0.5 0.5 0.5 0.5 0.5 0.5

minutesIncubate for 20

I

Enzyme Extract ml

minutesIncubate Fro 20

Sl. No.

Mean OD. 0.225

0.275 0.405

Concentration Of ((g) Unknown 1year

V µ g/ml /hour

-

-

25

375

28

277.2

46

276.0

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 pnitrophenol 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: PNP ml 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Sl No.

Substrate Buffer ml ml

1

0.1

0.9

2

0.1

0.7

3

0.1

0.7

Concentration Of PNP µ moles 0 10 20 30 40 50 60 70 80 90 100

0.2M NaOH ml 6 5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5

Enzyme ml 0.0 0.2 0.2

NaOH ml Incubate at 37°C for 10 minutes

Sl. No. 1 2 3 4 5 6 7 8 9 10 11

O.D. at 405 nm 0.0 0.04 0.09 0.15 0.2 0.23 0.27 0.3 0.35 0.38 0.41

O.D. at 405min

Mol of substrate

V Mol/ml/minute

57

142.5

5 5 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.

2 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.

1

5

2

27 26

5

Differ- Activity Ethanol Ether NaOH ence (Unites) ml ml ml ml

Enzyme ml

1

1litre inoculation

Substrate ml Incubate 5min. 37°C

T.T. Buffer No. ml

5

5

4.2 0.6

5

5

4.2

300

V µ mol/ ml/min

26