This experiment is entitled analysis of enzyme activity with the purpose to

knowing the effect of trypsin enzyme concentration to their activities, knowing the effect
of substrate concentration on trypsin enzyme activity, knowing the principle of
determining Km and Vmax on enzyme, knowing the difference in lipase enzyme activity
because extract buffer differences. (Mulyani, 2022). Experimental analysis is:
A. Extraction and Assay of Peanut Lipase Activity
This experiment is purpose to determine differences in lipase enzyme activity due
to differences in extracting materials. The working principle of this experiment was
carried out with 5 grams of ground peanuts, then added 50 mL of 0.1M NaCl and allowed
to stand for 30 minutes. Take 8 mL of milk, cool it in a water bath for 15 minutes then
add 2 mL of peanut filtrate and shake it. The, incubate for 10 minutes and then add 40
mL of ethanol. Add 5 drops of PP to the blank as well as the sample. titrate with 0.1M
NaOH then repeat the experiment again by replacing 0.1M NaCl with phosphate buffer.
Based on the experimental results in peanut sample reacted with NaCl, it
produced a cloudy white solution and after allowed to stand, it produced 2 layers. The top
layer is cloudy white and the bottom layer is white ppt. Then filtered the result is a cloudy
white filtrate. After adding milk to a milky white solution, incubated, adding ethanol, and
5 drops of PP indicator to make a white solution. After titration, the solution can become
pink in color and requires 15.3 mL of NaOH for the titration process. Then in the blank
solution, cow's milk mixed with NaCl produces a white solution, after adding 5 drops of
ethanol and PP indicator it becomes a faded white solution. Then after being titrated it
becomes a pink solution with the required volume of NaOH 14.5mL.
Based on the experimental results, in Peanut sample reacted with phosphate buffer
can produced a cloudy white solution and after allowed to stand, it produced 2 layers, the
top layer is cloudy white and the bottom layer is white ppt. Then filtered it produces a
cloudy white filtrate. After adding milk is to milky white solution, incubated, adding
ethanol, and 5 drops of PP indicator to make a white solution. After titration has the
solution becomes pink and requires 20.5 mL of NaOH for the titration process. In the
blank solution, cow's milk mixed with NaCl can produces a white solution and after
adding 5 drops of ethanol and pp indicator it becomes a faded white solution. Then after
titrated it becomes a pink solution with the required volume of NaOH 17mL.
Based on the calculations, the lipase enzyme activity in NaCl was 4μmol/minute/1
mL of the enzyme and in phosphate buffer of 17.5 mol/minute/1 mL of the enzyme.
According to the theory, the lipase enzyme works optimal at a pH slightly below 7 which
indicated by the value of the enzyme activity. So, the experiment is accordance with the
theory because the phosphate buffer environment is more optimum than NaCl for lipase
enzyme activity. Phosphate buffer solution > 0.1M NaCl solution (17.5 mol/min/1 mL
enzyme > 4μmol/min/1 mL enzyme).
Addition function:
a. NaCl to extract lipase enzyme
 b. Phosphate buffer to extract lipase enzyme
c. Ethanol for enzyme activation before titration
d. NaOH as titrant to neutralize acid (lipase in a fatty acid)

B. Trypsin Enzyme Activity Test

a. Effect of Enzyme Concentration on its Activity
This experiment is purpose to determine the effect of trypsin enzyme
concentration on its activity. The experiment was carried out by filling 5 mL of casein,
then heated for 5 minutes (350C) in 5 test tubes and then adding the volume according to
the table. Incubate (35 0C) for 20 minutes. Then add 3 mL of 20% TCA and let stand for
5 minutes in an ice bath, filter, then measure the absorbance.
In this experiment, in tubes 1A-5A, make a same observation results in each
treatment. Casein is clear when heated it became clearer and when added with phosphate
buffer and trypsin, 1A solution remains clear, 2A-4A is clear yellow solution, and 5A is
clear yellow (more concentrated). When incubated in a water bath for 20 minutes it was
no change and 2 mL of 20% TCA was added and stirred there was also no color change,
only the sediment was still scattered. Then left for 30 minutes in ice water, solution 1A
the result in top layer is clear and a bottom layer is white ppt , 2A-4A in top layer is clear
yellow and bottom layer is pale yellow ppt, and 5A solution has result a more
concentrated in the top layer of yellow more concentrated and the bottom layer is yellow
ppt. After centrifugation all tubes are clear. then 2 mL of the supernatant was taken plus 4
mL of 0.5M NaOH all tubes are clear. When 1 mL of aqueous solution of phenol folin
ciocalteu reagent is added, the tubes 1A becomes clear blue, 2A is blue, 3A is dark blue,
4A is blackish blue, and 5A blackish blue is more concentrated. After that left for 10
minutes it was no change and the absorbance was calculated with a wavelength of 650
nm.
In tube 1B-5B, the results of 1B is clear, 2B is clear yellow, 3B is clear yellow
slightly to an orange, 4B is cloudy orange, 5B is cloudy orange is more concentrated
when the phosphate buffer is added to trypsin. Then it incubated at a temperature of 350C
for 5 minutes didnt cause any changes. then added 3 mL of 20% TCA and stirred the
entire tube formed 2 layers, it is 1B white solution + ppt, 2B is cloudy white + ppt; 3B is
clear yellow + ppt, 4B is clear yellow+ppt and 5B is clear orange + ppt. When it was
added 5 mL of 1% casein, stirring, and incubation for 20 minutes the result show no
change. Then it is left for 30 minutes at low temperature to form ppt, namely 1B white
ppt, 2B is cloudy white ppt, 3B is yellowish white ppt, 4B is pale yellow ppt, and 5B is
white yellow ppt. After being centrifuged for 3 minutes, all tubes are clear. 2 mL of the
supernatant was taken and added 4 mL of 0.5M NaOH all tubes are clear. Then when 1
mL of aqueous solution of phenol folin ciocalteu reagent is added, the tubes was become
1B is clear blue, 2B is blue, 3B-5B are dark blue. Then let it stand for 10 minutes without
showing any change. The absorbance was calculated with a wavelength of 650 nm.
Table of experimental results and calculation results :

Solution Volume (mL Absorbance

Casein Buffer Trypsin Total

1A 5 2 0 7 0,572

2A 5 1,5 0,5 7 0,1328

3A 5 1 1 7 1,553

4A 5 0,5 1,5 7 1,620

5A 5 0 2 7 1,602

1B 5 2 0 7 0,371

2B 5 1,5 0,5 7 0,556

3B 5 1 1 7 1,004

4B 5 0,5 1,5 7 1,284

5B 5 0 2 7 1,276

After calculating the enzyme activity, the following data were obtained :

Reaction tube V 1/ V ( y ) S 1/ S ( x )

I 0,0100 100 0 Tak terhingga

II 0,0386 25,907 0,0714 14,006

III 0,0274 36,496 0,1428 7,003

IV 0,0168 54,524 0,2142 4,668

V 0,0163 61,350 0,2857 3,500

Based on the table, it can be seen that the rate of enzyme activity fluctuates as the
volume of trypsin increases. According to the theory, the higher the concentration of the
trypsin enzyme, the higher the rate of activity, it can be concluded that the experiment is
not in accordance with the theory. This may be due to the lack of thoroughness of the
practitioner in each step of his work, the lack of clean tools used so that the material is
contaminated, and other factors. From the graph, we get the equation y= -4.3683x +
69.76 and from the calculation we get Vmax= 0.0143 and Km= 0.0625.

Addition function:
- Casein as substrate
- Trypsin as an enzyme whose activity was tested
- TCA to stop enzymatic reactions
- Phosphate buffer to maintain pH

b. Effect of substrate concentration on its activity

This experiment is purpose to determine the effect of substrate concentration on
the activity of the trypsin enzyme.The experiment was carried out by filling 5mL of
casein, heated for 5 minutes (35 0C) in 5 test tubes and then adding the volume according
to the table. incubated (35 0C) for 20 min. then add 20% TCA in order of volume (1, 2, 3,
4, 5) ml in tubes 1-5 then let stand for 5 minutes in an ice bath, filter, then measure the
absorbance.
In this experiment, in 1C-5C tubes, the same observation results were obtained in
each treatment, namely casein (clear) when heated it became clearer and then when added
with phosphate buffer, 1C tube was clear solution, 2C tube was clear a little cloudy, 3C
tube was clear with a little ppt is cloudy, tube 4C clear solution is cloudy and the ppt is
cloudy, tube 5C is clear solution is cloudy and produces more cloudy deposits. Then,
when trypsin is added, 1C is clear yellow, 2C is a clear yellow solution with a little
precipitate, 3C is clear yellow with more white deposits, 4C is 3 layers formed, the top
layer is cloudy white, the middle layer is clear, the bottom layer is there are more deposits
When incubated for 20 minutes in a water bath at 35 degrees Celsius, the results
obtained in tube 1C is clear yellow with ppt, 2C is clear yellow with white ppt, 3C is
clear yellow with white ppt, 4C is clear yellow with a little white ppt, 5C is clear yellow
with a little white ppt. When added TCA 2% 3 ml, the results obtained in tube 1C is pale
yellow a little bit cloudy, 2C is pale yellow a little bit cloudy, 3C is pale yellow a little bit
cloudy, 4C is pale yellow a little bit cloudy, 5C is pale yellow a little bit cloudy.
When left for 30 minutes in ice water in a solution, all tubes produce a
precipitate. After centrifugation for 3 minutes, all tubes are clear. then 2 mL of the
supernatant was taken plus 4 mL of 0.5M NaOH all tubes are clear yellow. When added 1
mL of aqueous solution of phenol folin ciocalteu reagent in tube 1C is dark blue, 2C is
greenish blue, 3C is dark blue, 4C is Dark green, 5C is dark blue. After being left for 10
minutes there was no change and the absorbance was calculated with a wavelength of 650
nm.
 In tubes 1D-5D, when phosphate buffer was added trypsin, all tubes were light
milk brown in color and after being incubated in a water bath for 5 minutes, all tubes
were milk brown in color. When adding 3 ml of 20% TCA to each tube, the 1D and 2D
tubes were turmeric yellow solution, 3D turmeric yellow with very little ppt, 4D tubes
were turmeric yellow with lots of ppt and 5D was turmeric yellow with more ppt. The
addition of 1% casein makes 1D and 2D tubes cloudy white on top and clear yellow on
bellows with floating precipitate; on the 3D tube clear on top while in bottom is clear
yellow with ppt; tube 4D-5D more white on top and clear yellow in bottom with ppt float.
Incubate in a water bath for 20 minutes to form 3 layers in each tube with a brown milk
precipitate in the middle. Leaving for 30 minutes at low temperature (ice water) didn't
make any change in tube. After centrifuged also have no changes on tube. 2 ml of the
centrifuged supernatant after adding 4 ml of 0.5 M NaOH and 1 ml of phenol folin
ciocalteu reagent in the 1D-5D tube produced 2 layers with dark blue on top and green on
bottom except on tube 4D which produced dark blue on bottom color. After stirring all
solutions are dark blue. Leave it for 10 minutes the color is still the same and the
absorbance is calculated with a wavelength of 650 nm.
Table of observations and calculations:

Solution Volume (mL Absorbance

Casein Buffer Trypsin Total

1C 1 4 2 7 1.523

2C 2 13 2 7 1. 456

3C 3 2 2 7 1.569

4C 4 1 2 7 1.495

5C 5 0 2 7 1.620

1D 1 4 2 7 1.482

2D 2 3 2 7 1.585

3D 3 4 2 7 1.523

4D 4 1 2 7 1.509

5D 5 0 2 7 1.432

After calculating the following data were obtained :

 Reaction tube V 1/ V ( y ) S 1/ S ( x )

I 9,1 x 10-3 476,190 0,0014 714,285

II 6,45 x 10-3 155,038 0,0028 357,142

III 2,3 x 10-3 439,782 0,0042 238,095

IV 7 x 10-4 1428,571 0,0057 175,4385

V 9,4 x 10-3 106,382 0,0071 140,845

Based on the table, it can be seen that the rate of substrate activity increased and
decreased by increasing the volume of casein so that it can be said to be fluctuating.
According to the theory, increasing the volume of the substrate will increase the rate of
the reaction, so this experiment is not theoretical. In this experiment, the maximum rate
was found in the addition of 5 ml of casein. From the graph, the equation y= -0.3747x +
642.04 is obtained and from the calculation it is obtained that Vmax= 0.00155 and Km=
-0.005807.
Addition functions:
- Casein as substrate
- Trypsin as an enzyme was tested for its activity and aims to catalyze the
hydrolysis reaction of the peptide bonds present in albumin
- TCA to stop enzymatic reactions
- Phosphate buffer to maintain pH so that the enzyme can work optimally, in this
experiment the enzyme in question is the trypsin enzyme
- Phenol folin-ciocalteu as a reagent to form a colored complex so that the
absorbance can be measured using a spectrophotometer