ENZYMES AND METABOLISM
EXPERIMENT-1
N SANJANA SHYAM
SE22UBIT018
BIOTECHNOLOGY
27/8/23
AIM:- To ascertain extracellular lipase activity.
PRINCIPLE:- If the bacterium can manufacture the lipase enzyme, it can degrade
tributyrin when grown on tributyrin agar. This shows that extracellular lipase is
produced by the bacteria.
Substrate + Enzyme —----------> Product
By measuring the decrease in the pH, we can determine the enzyme activity.
EA = vol of NaOH consumed(ml) x Molarity of NaOH
vol of enzyme(ml) x Reaction time(min)
APPARATUS:- Glass beakers, falcon tubes, pH meter, magnetic stirrer, centrifuge,
micropipette, weighing balance, electronic pipette controller, dropper.
Tributyrin, gum acacia crystals, bacterial colonies, 0.1M NaCl, 2% CaCl2, 0.01M NaOH,
dH2O.
PROCEDURE:-
1) Prepare 150 ml of 1% tributyrin and 1% gumacacia solution. This is a substrate.
2) To balance the pH of the reaction, prepare 0.01M NaOH solution.
3) In a beaker, add 14 m of substrate, 0.5ml 0.1 M NaCl, 0.5ml 2% CaCl2..
4) Measure the pH of the above solution. Place it on a magnetic stirrer and add NaOH
accordingly to set the pH to 7.0.
5) Add 0.1 ml of 1% gum acacia solution to balance the volume of the reaction.
6) Check for any decrease in the pH for the next 3 minutes. If any change is observed,
add the required amount of 0.1M NaOH to set the pH to 7.0 again. Record the volume
of NaOH added.
7) Calculate the enzyme activity using the formula.
CALCULATIONS:-
1g of tributyrin in 100ml of dH2O
1g of Gum Acacia in 100ml of dH2O
2% NaCl 100 ml solution
2g in 100ml
X g in 10 ml
x = 2 x 10
100
1M NaCl in 10ml
1 = y x 1000 = 0.585g
58.5 x 10
0.001M NaOH in 50ml
0.01= z x 1000 = 0.02g
40 x 50
EA = vol. of NaOH consumed(ml) x molarity of NaOH
vol. Of enzyme x reaction time
= 0 ml x 0.01 M = 0 M min-1
0 ml x 3 min
OBSERVATIONS:- No change is observed in the pH of the solution.
CONCLUSIONS:- Therefore, it may be said that there hasn't been any enzyme
activity detected in the solution.
ENZYMES AND METABOLISM
LAB REPORT 2
AIM:- To ascertain intracellular lipase activity.
PRINCIPLE:- Enzymes called lipases are in charge of the breakdown of
lipids. They are very important for the breakdown and metabolism of dietary
lipids. Here, we'll break the cell open with a sonicator and watch the enzyme
activity.
Substrate + Enzyme —----------> Product
By measuring the decrease in the pH, we can determine the enzyme activity.
EA = vol of NaOH consumed(ml) x Molarity of NaOH
vol of enzyme(ml) x Reaction time(min)
APPARATUS:- Glass beakers, falcon tubes, pH meter, magnetic stirrer, centrifuge,
micropipette, weighing balance, electronic pipette controller, dropper.
Tributyrin, gum acacia crystals, bacterial colonies, 0.1M NaCl, 2% CaCl2, 0.01M NaOH,
dH2O.
PROCEDURE:-
1) Prepare 150 ml of 1% tributyrin and 1% gumacacia solution. This is a substrate.
2) To balance the pH of the reaction, prepare 0.01M NaOH solution.
3) In a beaker, add 14 m of substrate, 0.5ml 0.1 M NaCl, 0.5ml 2% CaCl2..
4) Measure the pH of the above solution. Place it on a magnetic stirrer and add NaOH
accordingly to set the pH to 7.0.
5) Add 0.1 ml of 1% gum acacia solution to balance the volume of the reaction.
6) Check for any decrease in the pH for the next 3 minutes. If any change is observed,
add the required amount of 0.1M NaOH to set the pH to 7.0 again. Record the volume
of NaOH added.
7) Calculate the enzyme activity using the formula.
CALCULATIONS:-
1g of tributyrin in 100ml of dH2O
1g of Gum Acacia in 100ml of dH2O
2% NaCl 100 ml solution
2g in 100ml
X g in 10 ml
x = 2 x 10
100
1M NaCl in 10ml
1 = y x 1000 = 0.585g
58.5 x 10
0.001M NaOH in 50 ml
0.01= z x 1000 = 0.02g
40 x 50
EA = vol. of NaOH consumed(ml) x molarity of NaOH
vol. Of enzyme x reaction time
= 0 ml x 0.01 M = 0 M min-1
0 ml x 3 min
OBSERVATIONS:- No change is observed in the pH of the solution.
CONCLUSIONS:- Therefore, it may be said that there hasn't been any enzyme
activity detected in the solution.
