Kinetic Studies of Purified Bromelain from Pineapple (Ananas

comosus [L.])Merr) Core with Hidroxyapatite and CM Sephadex C-50

Ion Exchange Chromatography

Nofa Rahayu Desi Putri

, Sumi Hudiyono

and Siswati Setiasih

Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok, Indonesia

Keywords: Pineapple core, bromelain, specific activity,Michaelis-Menten constant, maximum reaction velocity.

Abstract: Bromelain is a protease enzyme that functions to break peptide bonds. in the health field bromelain can

function as anti-inflammatory, anti-thrombotic and fibrinolytic. Here we purposed a new purification step to

collect enzyme from pineaplle. The purification of the enzyme from Ananas comosus was carried out by

precipitation with varying concentration of acetone and followed by column chromatography using

hidroxyapatite ion exchage and CM sephadex C-50 resin. The enzyme activities were evaluated using casein

as substrate. The highest specific activity of bromelain was gained from acetone fractination as 51,51 U/mg

in the range of 50-80% saturation with purity level of enzyme as 11 times from its crude extract. The

highest specific activity from last fraction which run on hidroxyapatite ion-exchange column

chromatography was 87,49 units/mg with purity level of enzyme resulted 19 times higher compared to the

crude extract. Later on, the purification with CM sephadex C-50 resulted in increasing the specific activity

to 200 U/mg with purity level of enzyme as 45 times higher from its crude extract. Hydrolysis of various

casein concentration with purified bromelain was carried out at optimum reaction condition of pH 7,0 and

C. The results obtained revealed the Km and Vmax value were 0,94% (w/v) and 0,023 U/min

respectively. From the various purification steps that have been done, it can be observed the increasing of

bromelain specific activity from each stage.

1. INTRODUCTION

Bromelain is a complex mixture of compounds that

differ from one another namely thiol endopeptidase

and other components that are not yet fully

characterized such as phosphatase, glucosidase,

peroxidase, cellulase, glycoproteins, carbohydrates,

some protease inhibitors, and calcium organically

bound, among others. Bromealin is non-toxic

compounds and a proteolytic enzyme that can

catalyze the hydrolysis reaction of protein.

Bromelain is a collective name for proteolytic

enzymes or proteases found in tissues including the

stem, fruit, and leaves of pineapple plants, the family

Bromeliaceae. (A.D Rowan 1990)

Bromelain is usually distinguished as bromelain

stem (EC. 3.4.22.32) or bromelain fruit (EC.

3.4.22.33) depending on the source. This proteolytic

enzyme has unique functions useful to the food,

pharmaceutical and cosmetic industries. As a drug,

bromelain has been used for the treatment of various

diseases, including thrombosis, rheumatoid arthritis,

inflammatory diseases such as atherosclerosis,

cancer treatments and others (Ketnawa et al., 2012) (

Jeung, A 1980)

In the process of isolation and purification of

enzymes, to obtain fractions with proteolytic activity

and high purity, it is necessary to know the

properties of the enzymes to be isolated. This is

needed so that in the process there is no

denaturation. Some techniques are often used such

as sedimentation, filtering solvent extraction, affinity

chromatography, ionic exchange and gel filtration.

Although this methods has a high concentration of

power but still has a low purity level. (M.A Desai,

2000)

Therefore, in this study protein isolation and

purification were carried out using various methods

to obtain increased purity and high activity of

bromelain from pineapple core.

Putri, N., Hudiyono, S. and Setiasih, S.

Kinetic Studies of Puriﬁed Bromelain from Pineapple (Ananas comosus [L.])Merr) Core with Hidroxyapatite and CM Sephadex C-50 Ion Exchange Chromatography.

DOI: 10.5220/0009843300002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

2. MATERIALS AND METHODS

2.1 Materials

The materials used for the process of isolation and

fractionation by precipitation include, Phosphate

buffers pH 7, prepared from NaH

PO4 (Merck) and

HPO

(Merck), Acetone for precipitation. For

ion column exchange chromatography,

hydroxyapatite resin and CM sephadex C-50 are

required, Tris–HCl buffers, NaCl. Furthermore, for

the test of bromelain activity with Kunitz method

(Kunitz, 1947 ) and for the determination of protein

content by Bradford method (Bradford, 1976)

2.2 Methods

2.2.1 Preparation of Crude Extract

Core was cut into pieces and weighed as much as

500 gram, then blended and filtered using a filter

cloth. Furthermore, core solution that has been

obtained centrifuged at 6000 rpm for 15 minutes at

4°C. Supernatant was filtered and the filtrate taken

as a crude extract of bromelain

2.2.2 Bromelain Purification from Crude

Extract by Acetone Precipitation

Bromelain crude extract obtained proceed to the

purification process by precipitation method using

acetone. The concentration level was divided into

three fractions, fraction 1 (0-20)%, fraction 2 (20-

50)% and fraction 3 (50-80)%. Acetone was added

to the crude extract at 4°C. The extracts was kept for

overnight. Extract was centrifuged with a speed of

6000 rpm for 15 minutes at 4°C. The precipitate

obtained re-suspended using 0.2 M phosphate buffer

pH 7. The filtrate was resumed to higher

concentration levels.

2.2.3 Measurements of Protein Content and

Enzymatic Activity

Protein content was measured using Bradford

method and the standard used was bovine serum

albumin (Bradford, 1976). All measurements were

performed in duplicate. All measurements are done

in duplicate. While enzyme activity was measured as

follows: 1% casein (w / v) at 0.9 M Tris - Hcl buffer

(pH 8.0) was used as a substrate. Aliquots of 100 µL

of sample were added to a centrifuge tube containing

1.9 mL of casein buffer solution. The mixture was

held for 30 minutes in a water bath at 37 ° C.

Furthermore, 3 ml of trichloroacetic acid (TCA)

solution was added and after 30 minutes at 4 ° C.,

the mixture was centrifuged at 4000 rpm. for 10

minutes (Kunitz, 1947). The supernatant absorbance

was determined at 280 nm using a UV / visible

spectrophotometer (Shimadzu UV-2450)

2.2.4 Hydroxyapatite Chromatography

Following acetone precipitation, the sample was

applied to a hydroxyapatite column (50 cm × 3,3 cm

size), pre-equilibrated with using 0.05 M phosphate

pH 7buffer containing at a flow rate of 5 ml/4 min.

The enzyme was eluted by a linear gradient of 50–

400 mM phosphate buffer. The bound enzyme was

eluted by a linear gradient of 50 – 400 mM NaCl

in phosphate buffer . Each fraction was assayed for

protein and enzyme activity.

2.2.5 CM Sephadex C-50 Chromatography

Fractions with the highest specific activity will be

purified by CM sephadex C-50 ion chromatography.

The sample was applied to a cm sephadex c-50

column (50 cm × 3 cm size), pre-equilibrated with

using 0.05 M Tris - Hcl pH 8 buffer containing at a

flow rate of 5 ml/4 min. The enzyme was eluted by a

linear gradient of 50– 400 mM Tris-Hcl buffer. The

bound enzyme was eluted by a linear gradient of 50

– 400 mM NaCl in phosphate Tris - Hcl buffer .

Each fraction was assayed for protein and enzyme

activity.

2.2.6 Kinetic Studies

Determination of kinetic parameters such as

Michaelis-Menten constant (Km), maximum

reaction speed (Vmax) is determined by measuring

and distributing enzyme activity data at optimum pH

and temperature as a function of substrate

concentration, based on the Lineweaver-Burk

method (Alves et al., 2014) (Lehninger, 1982).

3. RESULT AND DISCUSSION

3.1 Acetone Precipitation

In this study, acetone (p.a) was used as the enzyme

precipitator. Acetone has been widely used for the

process of protein deposition and bromelain enzyme

(Sharma et al 2014); (Rowan et al., 1990), (Heinicke

& Gortner 1957) with high values of specific

enzyme activity. The addition of acetone to the

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

pineapple solution is then stirred until homogeneous

to precipitate the protein, including the bromelain

enzyme contained in the pineapple. The principle of

this protein precipitation process is to reduce water

activity in core pineapple solution (Scopes, 1994).

The dielectric constant of acetone (20.7) is smaller

when compared to the air dielectric constant (80.1).

It will be more of the protein contained in the air.

Acetone is less polar than air, the more confusing it

is from proteins as well because the polar air surface

of polarity, mixed with less polar acetone, will be

more interested in interacting with the same protein

molecules wiped with acetone. Acetone capable of

interacting with the hydrophobic part of the protein

also further increases the reduced solubility of the

mixture and increases the precipitation protein

The largest proteolytic activity, protein content, and

specific activity of enzyme were obtained from the

third fraction of bromelain enzyme (50-80%

saturation level of acetone) with a value of

proteolytic activity 7,717 Units and protein content

0.149 mg. Specific activity of 51.513 U / mg. The

purity level obtained is 11 times purer than the crude

enzyme

3.2 Hidroxyapatite and CM Sephadex

C-50 Ion Exchange

Chromatography

Ion exchange chromatography is an advanced

purification step for separating bromelain from other

proteins to obtain enzyme with higher specific

activity. Purification by ion exchange

chromatography uses the principle of charge

difference between proteins and charged groups

present in the column matrix. Proteins with the same

charge as the matrix will elute first from the column.

If the protein charge is different from the matrix,

then the protein will interact electrostatically on the

matrix and out of the column at different times

depending on the strength of the bond

After precipitation with acetone, the sample is

loaded onto a ion exchange chromatography

columns hidroxyapatite and collected fractions were

analyzed for proteins content and proteolytic

activity. This chromatography step resulted in a 15

fold purification. The pooled fractions was further

purified using CM sephadex C-50 chromatography.

In the second chromatographic step, the pooled

active fraction was loaded onto a CM Sephadex C-

50 ion-exchange column and the bound enzyme was

eluted with a linear gradient of 50-400mM NaCl.

The last step chromatography resulted in a 45 fold

purification (tabel 1)

The effluent (protein and eluent molecules

coming out of the column) is accommodated on

tubes that hold 5 mL of effluent on each tube. And

all fractions of protein uptake at 280 nm wavelength.

This absorbance measure aims to detect and separate

spreading proteins in each part of the column

chromatographic results (Burgess, 2008). All

fractions also measure their proteolytic activity by

Kunitz method.

The following Figure shows that Elution profiles

of chromatography steps related to the purification

of bromelain from pineapple core.

Figure 1: Hidroxyapatite chromatography

Figure 2: CM Sephadex C-50 chromatography

At the elution stage, the bond between the

protein and the resin will be replaced by the ions

from the salt eluent, the salt ion ions binding to the

resin and the protein molecules will be released out

of the column. The protein molecule that binds to

the matrix will release more mixture through the

lower salt, and the higher the salt concentration, the

stronger the binding protein can also get out of the

column. Separation occurs in protein molecules

based on different times, depending on the strength

of the molecular charge attached to the resin.

Kinetic Studies of Puriﬁed Bromelain from Pineapple (Ananas comosus [L.])Merr) Core with Hidroxyapatite and CM Sephadex C-50 Ion

Exchange Chromatography

Table 1 Summary of purification and yields of bromelain

Step

Volume

Proteolitc

Activity

(U/mL)

Protein

Content

(mg/mL)

Total

Specific

Activity

(U/mg )

Fold

Proteolitc

Activity

(U)

Protein

Content

(mg)

Core solution

350

0,507

0,171

177,45

59,85

2,968

Crude Enzyme

200

0,733

0,163

146,60

32,60

4,490

Acetone

850

7,717

0,149

6559,45

126,65

51,513

Hidroxyapatite

2,116

0,031

179,86

2,635

68,258

CM Sephadex

C-50

0,400

0,002

18,00

0,09

200,00

Enzyme solution of each fraction were

proteinolytic activity and protein levels. Table 1

shows the value of proteolytic activity of each

fraction obtained in the purification stage. The

highest proteolytic activity was obtained in fraction

CM Sephadex C-50 with a value of 200 Units and

protein levels of 0,09 mg (figure 3). From the data of

proteolytic activity and protein levels, the highest

specific activity was obtained by a purity level

reached 45 times.

Figure 3 : an increase in purity levels of bromelain after

going through various stages of purification

3.3 Kinetic Studies

The Michaelis-Menten (Km) constant, maximum

reaction velocity (Vmax) is determined by passing

data of activity enzyme at pH and temperature

optimum as a function of substrate concentration,

based on the Lineweaver-Burk method plot as

shown in Figure 4

The Michaelis-Menten (Km) constant is the

amount of substrate required to achieve half of the

maximum rate of reaction. This constant can also be

used to show the enzyme specificity to a substrate

(Corzo et al., 2012). While Vmax is the maximum

rate of enzymatic reactions in the enzyme state that

has been saturated by the substrate. In this study, the

Michaelis-Menten (Km) constant for fraction AP1

purification with CM sephadex c-50 was 0.943% (w

/v) casein with a maximum velocity value (Vmax)

0.0234 U / min.

Figure 4 : Lineweaver-Burk Plot for Casein Hydrolysis by

Bromelain Fraction AP1 CM sephadex

4 CONCLUSIONS

Bromelain was successfully purified through a series

of purification steps, starting from extraction of

crude extract and precipitation of bromelain with

acetone and purification with chromatography

column ion exchange. The result of bromelain after

precipitation by acetone and purification with

hidroxyapatite and CM Sephadex C-50 gave higher

specific activity than before crude enzyme

ACKNOWLEDGEMENTS

This work was funded by Hibah Kompetensi

Publikasi Internasional Terindeks Untuk Tugas

Akhir Mahasiswa (PITTA), Universitas Indonesia

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

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Kinetic Studies of Puriﬁed Bromelain from Pineapple (Ananas comosus [L.])Merr) Core with Hidroxyapatite and CM Sephadex C-50 Ion

Exchange Chromatography