VIETNAM NATIONAL UNIVERSITY – HO CHI MINH CITY
INTERNATIONAL UNIVERSITY
REPORT
Experience 2: EXPLORING THE ENZYMATIC OF BROMELAIN
Instructor: Msc. Nguyen Hong Lan
Group: 1
Nguyễn Minh Hân BTFTIU20082
Lưu Khương Yến Nhi BTBTWE20012
Nguyễn Thị Diễm Quyên BTFTIU20097
Nguyễn Thế Minh BTFTIU21048
1
Table of contents:
I. INTRODUCTION...................................................................................................2
II. EQUIPMENT AND CHEMICALS.....................................................................2
III. PROCEDURES....................................................................................................3
IV. RESULTS ........................................................................................................… 8
V. DISCUSSION AND CONCLUSION .................................................................12
VI. REFERENCES .......................................................................................................
I. INTRODUCTION.
In the second practical lab, our group studied the role of enzymes, particularly Bromelain, in
cellular function. In this experiment, the enzymatic activity of bromelain, a protease that is
commonly found in pineapple, will be measured. We all know that bromelain is frequently
used to catalyze substrates that are naturally proteins, including hemoglobin and casein. The
process also yields peptides and mixtures of amino acids, including tyrosine. In this practical
study, our team then analyzes bromelain using the Folin-Ciocalteu reagent. Additionally, this
reagent is utilized to increase the rate of color development. Tyrosine levels can also be
estimated using Folin-Ciocalteu. In addition, our team need the assistance of a few devices
that have been used in the previous lab, including a centrifuge and a spectrophotometer. Our
team was capable of understanding the enzymatic activity of bromelain once this experiment
was complete. While performing practical work, we can also identify and fix errors. As a
result, our team will be able to solve issues and collect knowledge for the other particular
experiments' foundation.
II. EQUIPMENT AND CHEMICALS:
1. Equipments:
Name Quantity
Test tubes
Pipettes and pumps 1mL; 2mL; 5mL
Beakers 500mL; 250mL; 100mL
Falcons 50mL
Filter paper 11mm
Spectrophotometer
Centrifuges
2. Chemicals:
2
Chemicals Required volume
0.5M Sodium hydroxide (NaOH) solution 50mL
0.2M Hydrochloric acid (HCl) solution 50mL
5% Trichloroacetic acid (TCA) solution 50mL
2% Casein solution 50mL
1mM Tyrosine solution 20mL
Folin-Ciocalteu reagent
III. PROCEDURES:
1. Extraction:
- Step 1: Take grinded pineapple with 100ml distilled water.
- Step 2: Put both the pieces of pineapple and distilled water into a blender to grind down.
- Step 3: Filter the extract into a beaker.
- Step 4: Put the extract in the centrifuge tube (falcons).
- Step 5: Put the falcons into the centrifuge.
2. Survey the enzymatic activity:
3
Test tubes Ctr Ctr Ctr St1 St2 St3 Un Un Un
1 2 3 1 2 3
Step 1: Add 0.65% Casein in phosphate 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
buffer (mL)
Step 2: Keep tubes at room temperature for 5 min.
Step 3: Add 5% Trichloroacetic acid (TCA) 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0
(mL)
Step 4: Add Distilled water (mL) 0.5 0.5 0.5 0.0 0.0 0.0 0.5 0.5 0.5
Step 5: Add Tyrosine standard solution 0.0 0.0 0.0 0.5 0.5 0.5 0.0 0.0 0.0
(mL)
Step 6: Add pineapple extract (mL) 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.2 0.2
Step 7: Shake the tubes well and keep at room temperature for 10 min.
Step 8: Add 5% Trichloroacetic acid (TCA) 0.0 0.0 0.0 0.0 0.0 0.0 5.0 5.0 5.0
(mL)
Step 9: Add Pineapple extracts (mL) 0.2 0.2 0.2 0.2 0.2 0.2 0.0 0.0 0.0
Step 10: Shake the tubes well and keep at room temperature for 10 min and then filter.
Step 11: Transfer 2.0 mL of the filtered solution form each tube into new tubes.
Step 12: Add 5.0ml of 0.5M NaOH solution and 1mL of Folin-ciocalteu reagent.
Step 13: Shake the tubes well and keep at room temperature for 15 min.
Step 14: Measure the optical density (OD) at the 578nm & 620nm.
- Step 1+2: 0.65% Casein
4
- Step 3:
- Step 4:
Distilled water
- Step 5:
5
- Step 6:
- Step 7: Shake the tubes well and keep at room temperature for 10 min.
- Step 8:
6
- Step 9:
- Step 10: Shake the tubes well and keep at room temperature for 10 min and then
FILTER.
- Step 11:
- Step 12:
7
- Step 13: Shake the tubes well and keep at room temperature for 15 min.
- Step 14: Measure the OPTICAL DENSITY (OD) at the 578nm & 620nm.
IV. RESULTS:
- Step 3:
- Step 7:
8
- Step 10:
- Step 11:
9
- Step 13:
- Step 14:
10
Tube Tubes of water Tube of Tyrosine (mL) Tube of sample
(mL) (mL)
Ctr1 Ctr2 Ctr3 St1 St2 St3 Un1 Un2 Un3
OD 0.3053 0.3117 0.3085 0.5019 0.4821 0.4782 0.6576 0.6524 0.6363
Average 0.3085 0.4874 0.6488
ΔOD 0 0.1789 0.3403
Calculate
We are using the data that was recorded while doing the experiment to calculate the
average OD and ΔOD of each type of test tubes.
To calculate the average OD:
0.3053+0.3117+0.3085
Average ODCtr = = 0.3085
3
0.5019+0.4821+0.4782
Average ODSt = = 0.4874
3
0.6576+0.6524+0.6363
Average ODUn = = 0.6488
3
Then, ΔOD can be calculated by subtracting the average OD value of water tube from
the average OD value of Tyrosine tube and sample tube.
ΔODCtr: 0.000
ΔODSt: 0.4874 – 0.3085 = 0.1789
11
ΔODUn: 0.6488 – 0.3085 = 0.3403
Thus, the enzymatic activity is calculated by the following formula:
450 × ∆𝑂𝐷𝑀 1 𝑥1 450 × 0.3403 1 0.5
× × = × × = 213.9743 (IU/mL)
∆𝑂𝐷𝑇 10 𝑥 2 0.1789 10 0.2
where
x1: Tyrosine solution (mL)
x2: Enzyme solution (mL)
ΔODM: delta optical density of sample
ΔODT: delta optical density of Tyrosine
V. DISCUSSION AND CONCLUSION:
1. Discussion
Possible factors leading to errors:
+ Leaving the solutions with folin-ciocalteu exposed to light for too long
+ Prepared chemicals are not standard
+ Improper chemical transmission
+ Real volumes of chemicals in samples differ from required volumes
+ Incorrect measuring process
2. Conclusion
+ Measuring, transferring and of chemicals and calculation must be done precisely in
order to gain the most exact result throughout the experiment
+ Prior to the experiment, from data and according to the enzymatic activity formula,
we have: Enzyme activity= 26.330 (UI/mL)
+Gained resulted after calculations differs from public data mainly because of errors
while processing the experiment, but still within range, therefore not a failure.
VI. REFERENCES:
Biochemistry Lab Manual – MSc. Nguyen Hong Lan – Department of Biotechnology -
International University – VNU HCMC.
BioRender
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