ISOLATION OF PROTEASE ENZYME FROM CHAYOTE FRUIT (Sechium edule

(Jacq.) Sw.) WITH AMMONIUM SULFATE FRACTINATION METHOD

Ketut Ratnayani* and Lia Kusumaningrum

Chemistry Department, Faculty of Mathematics and Natural Science, Udayana University
*Corresponding author : ratnayaninew@gmail.com

ABSTRACT

Protease is an enzyme that is capable to hydrolyze (breakdown) protein molecules into

simpler compounds such as small peptides and amino acids. The aim of the research was to
isolate protease enzyme from chayote (Sechium edule (Jacq.) Sw.) using fractination
ammonium sulfate method and to find out the optimum saturation level of the ammonium
sulfate. P rotease activity examination of each fraction of ammonium sulfate was performed
using Anson method. Protein content assay was determined using Biuret method. The results
showed that crude extract protease of chayote had specific activity of 3,7338 x 10-3 U/mg. The
optimal saturation levels of ammonium sulfate for protease chayote precipitation was 40-
50%. At this saturation level, the highest enzyme spesific activity were 16,00 x 10-3 U/mg,
with four times purifying of protease enzyme from the crude extract protease.

Keyword : protease, protease activity, ammonium sulfate, specific activity

INTRODUCTION papaya’s (Joseph, 2010). Juwarni et al (2014)

found that the chayote latex contained
Protease is an enzyme that is capable
protease with protease activity as much as
to hydrolyze (breakdown) protein molecules
0.0264 U/mL. However, there are some
into simpler compounds such as small
problems in latex collection from chayote
peptides and amino acids. The source of
fruit. The latex is rapidly agglomerated, few
protease enzymes comes from
in number and easily sticking to the blade
microorganism, animal and plant. Plant is the
when tapped from the fruit. So it is
biggest source (43.85%) followed by bacteria
necessary to find another alternative method
(18.09%), fungi (15.08%), animal (11.15%),
for obtaining proteases from chayote fruit, in
algae (7.42%) and viruses (4.41%) (Mahajan
this case by directly squeezing the flesh
and Shamkant, 2010). Plant protease has
without collecting the sap.
higher activity and stability on various
For characterization and next
temperature, pH, inhibitor and metal ion
application purposes, protease need to be
(Mehrnoush et al.,, 2011).
isolated from chayote fruit with fractionation
One of the subtropical plant in
using ammonium sulfate salt. Ammonium
Indonesia is labu siam or chayote (Sechium
sulfate salt often used to fractionate enzyme
edule (Jacq.) Sw) which is commonly served
and protein due to its high solubility, giving
as cooked vegetable. Plant latex that causes
stabilizing effect on the enzyme and do not
itching may due to its protease component
disturb protein structure (GE Healthcare Life
(Lavinka and Dong, 2013). It is predicted
Sciences, 2011). Protease enzyme isolation
that chayote fruit has protease activity
using ammonium sulfate have been reported
because its latex causes itching as well as
by some researchers. Noda, Koyanagi and

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 ISOLATION OF PROTEASE ENZYME FROM CHAYOTE FRUIT (Sechium edule (Jacq.) Sw.) WITH AMMONIUM SULFATE
FRACTINATION METHOD | Ketut Ratnayani and Lia Kusumaningrum

Kamiya (1994) isolated protease from melon A hundred mL of the supernatant I

fruit using ammonium sulfate with 50% (crude extract protease) was fractionated with
saturation level. Asakura et al., (1997) ammonium sulfate. Determination of the
extracted and purified oryzasin from rice amount of ammonium sulfate that is used to
seed using ammonium sulfate 30-60% achieve the expected saturation level refers to
saturation level. a table compiled by the Scopes (1982). The
The aim of the research was to isolate percentage of saturation of ammonium
protease enzyme from chayote fruit (Sechium sulfate used was 0-20%, 20-50% and 50-
edule (Jacq.) Sw.) using fractination 70%. The addition of ammonium sulfate salts
ammonium sulfate method and to find out the was gently done using a magnetic stirrer until
optimum saturation level of the ammonium a certain saturation level was achieved. The
sulfate. precipitation obtained by centrifugation at
5000 rpm and a temperature of 4 ° C for 15
minutes. Then it dissolved in 12 mL of 0.05
MATERIAL AND METHODS M of cold phosphate buffer (pH 7) containing
1% sodium metabisulfite. Ammonium sulfate
Material
fraction with the highest specific activity
Chayote fruit, aquades, ammonium occurred prior to fractionation protease.
sulfate ((NH4)2SO4), bovine serum albumin
(BSA), tyrosine, natrium metabisulfite 1%, Assay of Protease Activity
casein 0,65% (b/v), K2HPO4 0,05 M, Determination of enzyme activity
KH2PO4 0,05 M, Na2CO3 0,5 M, TCA using the method of Anson (Anson, 1938)
(Trichloroacetic Acid) 0,11 M, Biuret with a colorimetric technique that utilizes
reagent and Folin-Ciocalteau reagent. absorption of the blue complex formed by
reaction of tyrosine with Folin Ciocalteu
Crude Extract Preparation
reagent (Folin and Ciocalteu, 1927). Casein
The crude protease extract was solution 0.65% (w/v) of 2.5 mL in the pre-
obtained by separating the enzyme from incubation at 37°C for 4 minutes added 1.0
chayote fruit. Two hundreds grams of mL sample of precipitant. This mixture
chayote fruit was added with 90 mL of cold incubated at 37°C for 30 minutes. Hydrolysis
buffer fosfat 0.05 M ( pH of 7) which reaction was stopped by addition of 2.5 mL
contained 1 % of natrium metabisulfite. Then of 0.11 M TCA and allowed to stand for 5
it homogenized and filtered using a filter minutes. This mixture was centrifuged for 15
paper. The filtrate then was centrifuged at minutes at a speed of 5000 rpm. Protease
5000 rpm for 15 minutes, at a temperature of activity was determined by measuring the
4oC. The supernatant was collected and levels of tyrosine generated using
separated from the sediment. Supernatant colorimetric reagent Folin-Ciocalteau. As
obtained from this step referred as many as 2.0 mL of the supernatant was
supernatant I, which will be used for the reacted with 5.0 mL of 0.5 M Na2CO3 and
fractionation process. 1.0 mL reagent Folin-Ciocalteau then the
mixture was left for 30 minutes. Read of the
Protease Isolation Method Using absorbance of this solution was done using a
Ammonium Sulfate Fractionation UV-Vis spectrophotometer at 755.4 nm. As a
blank, the addition of 0.11 M TCA conducted

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INTERNATIONAL JOURNAL OF BIOSCIENCE AND BIOTECHOLOGY  VOL. II NO. 2  APRIL 2015 ISSN: 9 772302 257 000
ISSN ONLINE: 9 772303 337 008

before the addition of a protease extract

sample. Protease activity (U / mL) is
RESULT AND DISCUSSION
expressed in units of activity. one unit (U) is
expressed as the amount of protease that can In the preparation phase of the crude
hydrolyze a substrate (casein) and produce a extract of the fruit flesh of squash obtained
color equivalent to 1 µmol tyrosine product filtrate (filtering results juice) is solid green.
(181 µg) every minute incubation period in The filtrate was centrifuged resulting in
experimental conditions. Protease enzyme crude protease extract (supernatant I) light
activity can be determined by the formula green, 200 grams of chayote fruit obtained
(Sigma, 1999): crude extract as much as 200 mL.
The determination of the specific
activities carried out to determine the level of
purity of the enzyme, the higher the value of
= total volume used on sample protease specific activity, the higher the purity of
activity assay; = volume of crude protease enzymes that have been isolated (Lehninger,
extract sample ; = volume of 1990). The specific activity of the protease
supernatan used on tyrosin concentration enzyme is an enzyme activity for each
analysis; t= incubation time. milligram of total protein extracts of chayote.
The specific activity of chayote protease in
Determination of Total Protein Content the crude extract, fractions of 0-20%, 20-
(AOAC, 1995) 50% and 50-70 can be seen in Table 1.
The highest specific activity found in
Precipitate of 1.0 mL sample was fractions of 20-50%, so it was conducted the
reacted with 5.0 mL of Biuret reagent and next fractionation using ammonium sulfate
then allowed to stand for 20 minutes at room saturation level with a more narrow range
temperature. The absorbance of mixture was that is 20-30%, 30-40% and 40-50%. The
measured using a UV-Vis spectrophotometer results of the specific activity value can be
at 547 nm. Total protein content is calculated seen in Table 2.
by converting the absorbance to the standard
linear regression equation of BSA.

Table 1. Specific activity of chayote protease on crude extract, 0-20% fraction, 20-50%
and 50-70% fraction

Description Protease Activity Total Protein Level Specific Activity

(U/mL) (mg/mL) (U/mg)
Crude extract 5,13 x 10-3 ± 0,0775 1,3782 ± 0,0822 3,7338 x ± 0,0018
0-20% fraction 1,09 x 10-3± 0,0000 0,8431 ± 0,0125 1,2931 x ± 0,00002
20-50%fraction 25,30 x 10-3 ± ,0002 1,4938 ± 0,0125 16,9000 x 10-3 ± 0,0003
50-70%fraction 4,05 x 10-3 ± 0,0000 0,6551 ± 0,0000 6,1823 x ± 0,0000

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 ISOLATION OF PROTEASE ENZYME FROM CHAYOTE FRUIT (Sechium edule (Jacq.) Sw.) WITH AMMONIUM SULFATE
FRACTINATION METHOD | Ketut Ratnayani and Lia Kusumaningrum

Table 1 shows that there was an increasing of molecules bind to the salt ions, so that the
protease activity in fractions of 20-50% i.e. amount of water that bind to protein is
25.30 x 10-3 U/mL, whereas in Table 2 it reduced and resulting precipitation of
appears that protease activity as a whole has proteins (Seidman and Mowery, 2006).
increased from crude extract (5.13 x 10-3 Although the value of the highest
U/mL). From the crude extract to the protease activity was found in fractions 20-
addition of salt (NH4)2SO4 30-40% rise and 30% and the highest protein content was
drop upon addition of salt (NH4)2SO4 40- found in fractions 30-40%, but the fractions
50%. The decrease and increase in the has a lower specific activity. The specific
activity of this protease can be caused by activity showed a purity level of protease
variations in the amount of protease enzyme enzyme. The more pure protease enzyme
protein and non-protein enzyme that settles obtained, would have a value of specific
in each fraction as protease results activity increasing (Lehninger, 1990). During
fractionation was still consists of protein the process of purification, enzyme
enzymes and non-enzyme proteins (Wang, concentration increased relative to the total
2004), as well as due to the respective each protein content to a certain extent
type of protein has a different solubility (Lehninger, 1990). The purity of protease
(Scopes, 1982). obtained in fractions of 40-50% (16.00x10-3
The total protein content shows a U/mg) reach 4 times than the specific activity
decrease in the fraction of 0-20% and 50- of the crude extract (3.7338 x 10-3 U/mg)
70% and an increase at the fraction of 20-
50% compared to the protein content of the CONCLUSION
crude extract (Table 1). The total protein It can be concluded that the value of
content in decreased in each additional level the specific activity of the crude extract
of salt saturation (NH4)2SO4 (Table 2). This protease (before the stage of salting out) was
is caused by each protein has a different 3.7338 x 10-3 U/mg. The most optimal of
solubility (Scopes, 1982) and in this case a ammonium sulfate saturation level to
protein thought to have much precipitate in precipitate chayote protease was 40-50%.
the fractions of 20-30%, so that the protein is
not too much precipitate in the fraction of 30-
40% and 40-50% , The addition of
ammonium sulfate salt resulted in water

Tabel 2. Specific activity of chayote protease

Protease Activity Total Protein Specific
Description
(U/mL) Content (mg/mL) activity(U/mg)
20-30% fraction 9,6933 x 10-3 ± 0,0074 0,7563 ± 0,0821 12,30 x 10-3 ± 0,0119
30-40% fraction 13,7700 x 10-3 ± 0,0124 0,4671 ± 0,0102 11,05 x 10-3± 0,0303
40-50% fraction 5,4933 x 10-3 ± 0,0039 0,3588 ± 0,2622 16,00 x 10-3 ± 0,0049

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ISSN ONLINE: 9 772303 337 008

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