Lab report

Module 2: BROMELAIN EXTRACTION AND CHARACTERIZATION

VIETNAM NATIONAL UNIVERSITY

UNIVERSITY OF SCIENCE
FACULTY OF BIOLOGY & BIOTECHNOLOGY
DEPARTMENT OF BIOCHEMISTRY

Report

Module 4: BROMELAIN EXTRACTION AND

CHARACTERIZATION BY SDS-PAGE

Course name Labwork on fundamental biochemistry

Lecturer Phạm Thị Mỹ Bình
Date experiment completed 23/05/2023
Group: 1 Subgroup: B
Student’s information Thái Hoàng Duy - 20157061
Nguyễn Nhật Bình - 20157060
Pham Nguyễn Phụng Nhi - 21157076
Representative’s student email 20157061@student.hcmus.edu.vn
20157060@student.hcmus.edu.vn
21157076@student.hcmus.edu.vn

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Module 2: BROMELAIN EXTRACTION AND CHARACTERIZATION

I. Introduction
What is bromelain?
o Bromelain is defined as a natural complex mixture of various proteolytic enzymes
which has a cysteine amino acid side chain. It is also stated that bromelain is a
combination of various thiol endopeptidases and other constituents like
peroxidase, phosphatase, cellulase, glucosidase, and several protease inhibitors.
Bromelain is mainly derived from pineapple (Ananas comosus), which is a well-
known plant of the Bromeliaceae family that is widely cultivated in tropical and
subtropical areas. It is used for edible purposes and the fruit has been popularly
used in different cuisine. Moreover, pineapple is considered a nutrient-rich plant
and has been traditionally known for having a wide array of potential biological
compounds among which bromelain is of great importance. Bromelain has been
used in folk medicine for so many years to address various health problems which
cause persistent interest in this protease. It possesses a wide range of therapeutic
applications like fibrinolytic, anti-thrombotic, anti-cancer, anti-inflammatory, and
antibacterial activity. In addition to this, bromelain has various industrial
applications also that include the pharmaceutical, food, textile, and cosmetic
industry.
o Bromelain belongs to the group of cysteine proteases. The enzyme maintains its
activity on casein at 50°C for 24 hours in the pH range of 4 to 10 and has an
optimal pH of 7 (Inagami and Murachi, 1963). The enzyme is stable in solutions
of 25% of methanol (v/v) at 25°C for 20 min, and it loses 50% of its activity when
the enzymatic solution is heated to 55°C for 20 min at pH 6 (Whitaker and El-
Gharbawi, 1963). Lyophilization causes a 27% loss in activity. (Murachi et al.,
1964)

What sources can you use to obtain bromelain?

o Bromelain is abundant in the stem and fruit of the pineapple plant and it can also
be isolated in small amounts from pineapple waste such as core, leaves, peel, etc.
(Hossain et al. 2015). Bromelain present in the fruit of pineapple has been
assigned the EC number EC 3.4.22.33 and is regarded as fruit bromelain.
Likewise, bromelain present in the stem of a pineapple is called stem bromelain
and its EC number is EC 3.4.22.32. Stem bromelain possess different biochemical
properties and composition as compared to fruit bromelain (Pavan et al. 2012) and
contains a variegated blend of different thiol-endopeptidases.

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Module 2: BROMELAIN EXTRACTION AND CHARACTERIZATION

What source did you use to extract bromelain in this module?

o In this module, the source used to extract bromelain is crown pineapple.

What are the objectives of the experiments?

o From the experiment, the objectives we accomplished include: measuring protein
concentration in pineapple, measuring bromelain activity, and SDS-PAGE
electrophoresis to determine the molecular weight of bromelain or to determine the
size of the protein.

I. Materials and Methods:

1. Materials:
Equipment Needed:
 Glass test tube
 100mL, 50mL volumetric flask
 1mL, 2mL, 5mL,10mL,20mL pipette
 Water bath, blender
 Beakers
 Cuvettes for spectrophotometer and spectrophotometer
Solutions needed:
 Pineapple
 HCl 0.2N, NaOH 1N, TCA 5%, Casein 1%, Tyrosine
 Deionized water
 Ethanol
 Bradford Reagent, Bovine serum albumin
 Loading buffer, SDS Running buffer
2. Methods:

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II. Results:
Calculation
o How can you calculate the protein concentration?
The graph with equation: y = 0.0021x + 0.0023 (1)
And ∆ OD 595 nmof Solution (I) = 0.059 which is y => y=0.059 and substitute y into equation
(1):
 0.059=0.0021x + 0.0023
 x = 29.19 and X is protein concentration of solution (I)

The graph with equation: y = 0.0021x + 0.0023 (1)

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Module 2: BROMELAIN EXTRACTION AND CHARACTERIZATION
And ∆ OD 595 nmof Solution (II) = 0.082 which is y => y=0.082 and substitute y into
equation (1):  0.082=0.0021x + 0.0023
 x = 40.143 and X is protein concentration of solution (II)

o How can you calculate the bromelain activity?

450 ∗ 0.5∗ ∆ OD M
Bromelain activity = ∗dilution factor (UI/ml)
∆ OD T ∗ 0.2 ∗10
Note: Volume of sample: 0.2
Time: 10 min
450 ∗ 0.5∗ 0. 211
Solution (I) = ∗ 1 = 98.496 (UI/ml)
0. 241∗ 0.2∗ 10
∆ OD M =∆ OD 620nm Sample of Solution ( I )=0. 211
∆ OD T = ∆ OD 620 nm Tyrosine of Solution ( I )=0.241

450 ∗ 0.5∗ 0. 076

Solution (II) = ∗ 1 = 48.305 (UI/ml)
0. 177 ∗0.2 ∗10
∆ OD M =∆ OD 620nm Sample of Solution ( II ) =0.076
∆ OD T = ∆ OD 620 nm Tyrosine of Solution ( II )=0.1 77

Data report and analysis

o Calculate the concentration of protein (µg/ml) in each of the five samples that
make up the standard curve and draw a calibration curve of Bradford assay.
Table 1. Bradford protein assay standard curve
Vol of BSA Vol of Protein Corrected
Test
0.1 mg/mL deionized concentration OD595nm OD595nm
tubes
(mL) water (mL) (µg/mL) (∆OD595nm)
Blank 0 10 0 0.118 0
1 1 9 10 0.140 0.022
2 2 8 20 0.146 0.028
3 3 7 30 0.176 0.058
4 4 6 40 0.206 0.088
5 5 5 50 0.216 0.098
∆OD Blank = 0
∆OD1 = OD1 – OD Blank = 0.140 – 0.118 = 0.022

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Module 2: BROMELAIN EXTRACTION AND CHARACTERIZATION
∆OD2 = OD2 – OD Blank = 0.146 – 0.118 = 0.028
∆OD3 = OD3 – OD Blank = 0.176 – 0.118 = 0.058
∆OD4 = OD4 – OD Blank = 0.206 – 0.118 = 0.088
∆OD5 = OD5 – OD Blank = 0.216 – 0.118 = 0.098

Bradford protein assay standard curve

0.12

0.1
f(x) = 0.00205142857142857 x − 0.00228571428571427
R² = 0.972356558150062

0.08

0.06
OD

0.04

0.02

0
0 10 20 Protein concentration
30 (µg/mL)
40 50 60

 The equation obtained was: Y=0.0021x + 0.0023 with R2=0.9724

 Calculate the protein concentration in samples (I-II) using the standard curve.
Table 2. Determination of protein concentration in unknown samples

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Module 2: BROMELAIN EXTRACTION AND CHARACTERIZATION
Protein
concentration
OD595nm OD595nm OD595nm Dilution Actual protein
OD595nm (Average) calculated based on
Samples (1) (2) (3) ∆OD595nm factor concentration
the standard curve
(b) (µg/ml)
(µg/ml)
(a)
Solution 0.148 0.149 0.154 = =0.150-0.091 Y=0.0021x+0.0023 50 =(a)*(b)
(I) 0.1 48+0.1 49+ 0.154
=0.059 0.059=0.0021x =29.19*50
3 +0.0023 = 1459.5
= 0.150 X =29.19
Solution 0.163 0.177 0.178 = =0.173-0.091 Y=0.0021x+0.0023 50 =(a)*(b)
(II) 0.163+ 0.177 +0.1=0.082
78 0.082=0.0021x =40.143*50
3 +0.0023 =2007.15
= 0.173  x=40.143
Blank 0.082 0.095 0.096 = 0
0. 082+ 0. 095+0. 096
3
= 0.091

o Calculate the enzyme activity in samples (I-II)

Table 3. Determination of bromelain activity in unknown samples

Solution (I) Solution (II)

Samples Blan
Tyrosine Sample Blank Tyrosine Sample
k
0.165 0.494 0.403 0.036 0.161 0.072
OD620nm 0.196 0.377 0.394 0.022 0.192 0.077
0.190 0.403 0.388 0.023 0.258 0.159
OD620nm (Average) 0.184 0.425 0.395 0.027 0.204 0.103
∆OD620nm 0 0.241 0.211 0 0.177 0.076
Dilution factor 1 1
3 3
45 ∗ 0.5∗ 10 45 ∗ 0.5∗ 10
Amount of 100 100
tyrosine (µg) in 0.5 =225 =225
mL solution used
in “tyrosine” batch

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Module 2: BROMELAIN EXTRACTION AND CHARACTERIZATION
45 ∗ 0.5∗ 10 ∗ ∆OD M 45 ∗ 0.5∗ 10 ∗ ∆OD M
Amount of ∆ OD T ∆ OD T
tyrosine (µg) = 45 ∗ 0.5∗ 10 ∗0. 076
=
produced in 45 ∗ 0.5∗ 10 ∗0. 211 0.177
“sample” batch 0. 241 =96.61
= 196.992
450 ∗ 0.5∗ ∆ OD M 450 ∗ 0.5∗ ∆ OD M
* Dilution factor * Dilution factor
∆ OD T ∗ 0.2 ∗10 ∆ OD T ∗ 0.2 ∗10
Bromelain activity 450 ∗ 0.5∗ 0 .211 450 ∗ 0.5∗ 0. 076
= ∗1 = ∗1
(UI/ml) 0. 241∗ 0.2∗ 10 0. 177 ∗0.2 ∗10
= 98.496 = 48.305

 Show the results of protein and enzyme activity assays from the protein
purification in Table 4.
Table 4. Bromelain purification table

Bromelain
Volu Total Total Specific Protein
Purificati activity
Step me protein activity activity recove
on fold recovery
(ml) (mg) (UI) (UI/mg) ry (%)
(%)
Homogenat 50 1459.5* 50* =98.496*5 4924.8 1 100% 100%
¿
e /(crude 10
−3 0 729.75
= 729.75 =4924.8 ¿ 6.74 9
extract) (I)
10 2007.15*10* 48.305*10 483.05 2.407 =
=
Solvent 10
−3 =483.05 200.715 6.749 200.715 =
∗ 100 %
= 200.715 ¿ 2.40 7 = 0.357 729.75 483.05
precipitatio ∗ 100 %
=27.51 4924.8
n (II) % =9.81%

Total protein (mg) = Actual protein concentration * Volume

Total activity (UI) = Bromelain activity * Volume
Total activity
Specific activity (UI/mg) =
Total protein
Specific activity (II )
Purification fold (II) =
Specific activity (I )
Total protein(II)
Protein Recovery (%) = ( ) * 100%
Total protein(I )

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Total activity (II )
Bromelain activity recovery (II) (%) = ( ¿ ∗100%
Total activity ( I )

 Show the result of SDS-PAGE: Label the protein ladder and the target protein, and
estimate the size of target protein based on the protein ladder

The result of SDS-PAGE, the molecular weight of the protein obtained ranged from 15 to
20 kDa (approximately 20 kDa).
III. Discussion
- Based on the protein concentration obtained from the Bradford protein test
standard curve and the dilution factor, we can calculate the total protein content of
solutions I and II gradually decreasing as 729.75 mg and 200.715 mg. As a rule,
the total protein will be reduced by removing more protein during precipitation.
- The total activity of Solution I (4924.8 UI) is higher than the Solution II (483.05
UI). According to hypothesis, after collecting Bromelain and removing unneeded
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proteins, total activity would increase since many unnecessary proteins were
filtered away during the experiments. Consequently, the total activity of Solution
II is lower than Solution I. This happens because in the process of diluting a
solution there is an error or maybe the manipulation has an error, which affects our
data well.
- Because of the error in total activity, the purification fold was 0.357 and less than
1 (the theory result predicted is ≥1). Theoretically, protein recovery is less than
bromelain activity recovery. However, our results show that protein recovery is
more than bromelain activity recovery (the protein recovery is 27.51% and
bromelain activity recovery is 9.81%).
- The result of SDS-PAGE, the molecular weight of the protein obtained ranged
from 15 to 20 kDa (approximately 20 kDa).

IV. Conclusion
 Solution I:
- The total protein concentration: 729.75 (mg)
- The total activity: 4924.8 (UI)
- Specific activity: 6.749 (UI/mg)
 Solution II:
- The total protein concentration: 200.715 (mg)
- The total activity: 483.05 (UI)
- Specific activity: 2.407 (UI/mg)
- Purification fold: 0.357
- Protein recovery: 27.51%
- Bromelain activity recovery: 9.81%
- The result of SDS-PAGE, the molecular weight of the protein obtained ranged
from 15 to 20 kDa (approximately 20 kDa).

V. Reference
[1] Anum Gul, Sadaf Khan, Hanzala Khan, Maryam Siddiqui, Habiba Arain, Urooj
Ishrat, 23/2/2021, Extraction, Partial Purification and Characterization of
Bromelain from Pineapple (Ananas Comosus) Crown, Core and Peel Waste,
BRAZILLIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY.
[2] Zoya Manzoor, Ali Nawaz, Hamid Mukhtar, Ikram Haq (2016) Brazilian Archives
of Biology and Technology, Bromelain: Methods of Extraction, Purification and
Therapeutic Applications, Vol.59.

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