Agricultural Science Research Journals Vol. 2(8), pp.

434-440, August 2012

Available online at http://www.resjournals.com/ARJ
ISSN-L:2026-6073 2012 International Research Journals

Full Length Research Paper

Isolation and characterization protease enzyme from

leguminous seeds
M. Akhtaruzzaman1, *N.H.M. Rubel Mozumder2, Ripa Jamal3, Atikur Rahman4
and Tanjina Rahman3
1

Institute of Nutrition and Food Science, University of Dhaka, Dhaka-1000, Bangladesh

Department of Food Science and Nutrition, Hajee Mohammad Danesh Science and Technology University, Dinajpur5200, Bangladesh
3
Institute of Nutrition and Food Science, University of Dhaka, Dhaka-1000, Bangladesh
4
Department of Food Processing and Preservation, Hajee Mohammad Danesh Science and Technology University,
Dinajpur-5200, Bangladesh.

*Corresponding Authors Email: rubel.infs@gmail.com

ABSTRACT

The present study was conducted to isolate and characterize the proteases from seven leguminous
seeds: soybean, lentil, black gram, green gram, bengal gram, groundnut and pea bean. We elaborated
the easy procedure for isolation of protease from leguminous seeds by using (NH4)2SO4 precipitation.
The effect of pH and temperature of protease activity were determined. This study revealed that the
protein concentration of crude extract ranged between 2.215 to 2.40 mg/ml in which groundnut was
highest protein concentration (2.40 mg/ml) and lentil accounted was lowest concentration (2.21 mg/ml).
The pH profile of proteases showed maximum specific activity at pH 7.5 to 9.0 with one main peak at pH
9.0. Among the seeds, bengal gram showed highest specific activity (0.007659 U/mg of protein) at pH 7.5
but lowest activity was observed in pea bean (0.001681 U/mg of protein) at pH 9.0. The temperature vs.
specific and catalytic activity of all proteases relationship demonstrated a symmetrical distribution with
one main peak and optimum at 37oC except black gram which showed two main peaks at 37oC and 70oC
respectively. The paper concludes that leguminous seeds can be source of proteases for industrial
purposes.
Key Words. Protease activity, Characterization, Industrial purposes, leguminous seeds.

INTRODUCTION
Proteases are one of the largest groups of industrial
enzymes that catalyze the hydrolytic reactions by
cleaving peptide bonds in protein. Proteases may be
classified as two major groups; exopeptidase and
endopeptidase based on their ability to degrade N- or Cterminal peptide bond. Endopeptidases, which have more
potent industrial applications than exo-peptidases, can be
divided into four types (aspartic, cysteine, metallo and
serine protease) on the basis of their active site and
sensitivity to various inhibitors (Al- sherhi and Mostafa,
2004; Sandhya et al., 2004). Proteases have been used
in processing of various foods such as calf rennet or
chymosin in cheese making in which chymosin hydrolyze

the specific peptide bond (the Phe-15-Met 106 bond) to

generate park-k-Casein and macro peptides (Smit et al.,
2005). They are also used in preparation of soy sauce
and other hydrolysates (Wang & Wang, 2004) and used
of papain for meat tenderization (Soper, 1998). The
bitterness of protein hydrolysate in soybean is a major
barrier of uses as a food and health care product. The
intensity of bitterness is increased due to the number of
hydrophobic bonds present in the protein. The proteases,
which can cleave hydrophobic bond into amino acids, are
valuable in debittering of protein hydrolysates in soybased products (Rao et al., 1998). In leather processing
steps such as soaking, dehairing and baiting, the use of

Akhtaruzzaman et al

proteases as alternatives to chemical detergent powder

has proved successful modification to remove protein and
bloodstain from the skin and improving environmental
pollution
(Nadafi
and
Deobagkar,
2005).
In
pharmaceutical industry, they offer a gentle and selective
debridement, supporting the natural healing process in
the successful local management of skin ulcerations by
the efficient removal of necrotic material (Sjodahl et al.,
2002).
Proteases are mainly obtained from microbial sources for
industrial purposes Some examples of microbial
proteases include: microbial rennin like enzyme from
Mucor miechi, pepsin like acid protease from Aspergillus
spp. and Rhizopus spp., acid protease from thermophilic
Penicillium sp. and a neutral metalloprotease, Pseu-216A
from Aspergillus oryzae (Kumar et al., 2005; Tremacoldi
et al., 2004). Though microbial protease are the
predominant source of industrial enzyme due to their
broad
biochemical
diversity,
rapid
growth
of
microorganisms and limited space required for cell
cultivation, it involves advanced technology in the field of
biotechnology and microbiology ( Rao et al., 1998). In our
country, there are few opportunities to do a research on
microbial enzyme for industrial purposes.
In view of unrestricted availability, the plant sources
would be a possible alternative of microbial and animal
proteases. Beside this, to study the mechanism of protein
mobilization process and in-vitro degradation of protein,
several studies have been conducted on purification and
characterization of proteases and peptidases, some of
which occur only transiently in germinating leguminous
seeds (Ashton, 1976; Davy et al., 1998; Shutov and
Vaintraub, 1987). The understanding of such facts
influenced us to conduct this study. In the present study,
we tried to establish an easy procedure for the isolation
and characterization of protease enzyme obtained from
leguminous seeds.
METHODS AND MATERIALS
Materials
The present study was conducted in the laboratory of the
Institute of Nutrition and Food Science, University of
Dhaka (DU) on seven kinds of leguminous seeds
collected from six market places of Dhaka city. These
were soybean (Glycine max), lentil (Lens esculenta),
black gram (Vigna mungo), green gram (Vigna radiate),
bengal gram (Cicer arietinum,) groundnut (Arachis
hypogaea) and pea bean (Phaseolus vulgaris). Bovine
serum albumin (BSA), casein, hemoglobin and Folin
ciocalteau reagent (FCR) were collected from E. Merk
(Germany) and Tricholoacetic acid (TCA) from BDH
(England). The other chemicals and reagents used in this
experiment were analytical grade and obtained from
Institute of Nutrition and Food Science (INFS), University
of Dhaka without further purification.

435

Methods
Isolation and preparation of protease enzyme (crude
extract)
The seeds (soybean, lentil, black gram, green gram,
bengal gram, groundnut and pea bean), approximately
fifty grams (50g) in amounts, were washed separately
and then soaked in distilled water at room temperature
for overnight germination. After that, all the seeds except
soybean and groundnut were ground by electric
homogenizer without using acetone, because of their low
fat content. Soybean and groundnut were homogenized
with cold acetone to remove the fat. Then the
homogenates were finely powdered in a pre-chilled
mortar and mixed with chilled 10mMTris-HCl buffer at pH
8.0 containing 2M NaCl for 3 hours. The extracted
mixtures were filtered through gauge and filtrates were
centrifuged at 10000 rpm for 10 minutes below 4oC. The
collected supernatant was used for the estimation of
extracellular concentration of protein and further
purifications.
(NH4)2SO4 precipitation: The collected supernatants
were saturated with 50% solid (NH4)2SO4 for overnight
precipitation. After precipitation, they were centrifuged at
10000 rpm for 30 min below 4oC. The collected
precipitated were dissolved in 10 nm Tris-HCl buffer (pH
8) and dialyzed against the same buffer and finally
centrifuged at 5000 rpm for 10 min. The supernatant was
used as crude enzyme for the assay of specific activity of
enzyme and characterization.
Protein measurement
Protein concentration was determined by the method of
Lowry et al., 1951 using bovine serum albumin (BSA) as
standard protein. The amount of the soluble protein was
calculated from the standard curve as mg of protein per
ml of test samples.
Assay of protease enzyme: Determination of catalytic
activity of protease
Assay of protease was determined by the method of
Anson (1938). The reaction mixture consisted of 250 l of
crude enzyme solution (supernatant) obtained by
centrifugation and 1 ml of substrate (2% hemoglobin for
acidic pH and 2% casein for alkaline pH in various
o
buffers) was incubated at 37 C for 30 minutes. The
reaction was stopped by the addition of 500 l of 10%
trichloroacetic acid (TCA). For the control, the substrate
was precipitated with 500 l 10% TCA before adding the
enzyme solution and then treated as described above.
The resulting precipitate was removed by centrifugation
and collected 300 l supernatant was added to the 2.5 ml

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Agric. Sci. Res. J.

Table 1. Concentration of protease (crude extract) from leguminous seeds

Common Name
Soybean
Lentil
Black gram
Green gram
Bengal gram
Groundnut
Pea bean

Botanical Name

volume of (NH4)2SO4
precipitation (ml)

Glycine max
Lens esculenta
Vigna mungo
Vigna radiata
Cicer arietinum
Arachis hypogaea
Phaseolus vulgaris

97.0
98.0
97.0
97.0
97.0
97.5
97.0

cu-alkaline solution using vortex and allowed to stand for

15 minutes. The mixture was then similarly mixed well
with 250 l of double times diluted folin ciocalteau
reagent (FCR). The absorbance was measured at 660
nm by spectrophotometer. The activity of proteases is
designated as endopeptidases activity since casein
(Kunitz, 1946) or haemoglobin (Bergmeyer, 1984) used
as a substrate. The protease activity was expressed as
the difference of absorbance at 660 nm between the
control sample and the test sample. A unit of protease
activity is defined as the amount of enzyme required to
release TCA- soluble fragment giving a blue colour
equivalent to one microgram of product under the same
condition of assay.
Calculation of specific activity
Specific activity of the protease was calculated by the
activity of protease per milligram of protein per minute
specifically dividing the determined protease activity
values on the protein content results (Equation 1)
Specific activity= Absorbance at 660 nm X (Protein
concentration)-1 X (min)-1 (1)
Effect of pH and temperature specific activity of
proteases: Partial characterization
Optimum temperature and pH of protease activity were
determined using various temperatures (10oC, 20oC,
37oC, 50oC and 70oC) and pH (2.0, 4.0, 7.5, 9.0 and
11.0).
RESULTS AND DISCUSSIONS
The present study investigated the protease activity
obtained from seven leguminous seeds by establishing
an easy assay system using two substrates: casein for
alkaline protease and haemoglobin for acidic protease. In
this study, the most widely used ammonium sulfate
fractionation was carried out directly from the crude
extract of germinating seeds and maximum amount of

Protein Concentration
(mg/ml)
2.35
2.21
2.30
2.33
2.39
2.40
2.38

protease enzyme was recovered in the precipitation

obtained by fractionating with 50% (NH4)2SO4 saturation.
The protein concentrations of extracellular proteases
isolated from seven leguminous seeds were shown in
Table 1. The concentration of extracellular soluble protein
ranged between 2.215-2.40 mg/ml in which groundnut
showed highest protein concentration (2.40 mg/ml) and
lentil accounted lowest concentration (2.21 mg/ml). Dahot
M U. (1992) investigated on proteases present in some
plant seeds and found the ranged of protein
concentration between 1.10-2.76 mg/ml with highest
value in soybean seed (Glycine max) (2.76 mg/ml) which
is in a good agreement with our value reported for
soybean seeds (2.35 mg/ml). Ericson and Chrispeels
(1973) observed that the storage proteins of leguminous
seeds have recently been shown to be glycoproteins
containing both mannose and glucosamine.
The effect of different pH (2.0, 4.0, 5.0, 7.5, 9.0 and
11.0) on the specific proteases activity (Units/mg) of
germinated leguminous seeds using casein (alkaline
protease) and haemoglobin (acidic protease) as a
substrate was depicted in Table 2. All the enzymes
showed maximum specific activity at pH 7.5 and 9.0
when they were treated by 2% casein. From the table,
Bengal gram showed highest specific activity (0.007659
U/mg of protein) at pH 7.5 but lowest activity was
observed in pea bean (0.001681 U/mg of protein) at pH
9.0. The second highest specific activity was observed in
green gram, (0.005722 U/ mg protein) that was lower
than the previous studies (Dahot MU, 1992; Lin and Yao,
1996; Rahman et al., 2007; Vidyavati et al. 1983). With
this assay system, the pH vs. protease (catalytic) activity
relationship was depicted by line graph in Figure 1. The
pH profiles showed one main peak at 9.0. A very similar
situation (pH maxima at 8.4 and 9.2) was also reported
by Evans et al., 2011 for the extract of palm weevil
(Rhynchophorous palmarum).The results from effect of
pH indicate that the alkaline proteases involved in all
seeds were more potent than the acidic proteases. This
alkaline protease may be playing an important role in
industrial food applications such as production of soy
sauce, digestion of soy bean protein and in leather and
detergent industries (Kamini et al., 2004; Nadafi and

Akhtaruzzaman et al

Table 2. Effect of pH on specific activity (U/mg) of proteases at 37 C

Sample

Specific protease activity (U/mg) at different pH

4.0
7.5
9.0
0.002553
0.002723
0.005106
0.002287
0.003612
0.003070
0.003362
0.003536
0.005217
0.004063
0.004578
0.005722
0.007476
0.007659
0.005523
0.002222
0.002666
0.002167
0.001232
0.001569
0.001681

2.0
0.001986
0.001204
0.001101
0.000572
0.001116
0.00027
0.000448

Soybean
Lentil
Black gram
Green gram
Bengal gram
Ground nut
Pea bean

11.0
0.002156
0.001505
0.001159
0.001030
0.001283
0.000722
0.001457

Table 3. Effect of temperatures on specific activity (U/mg) of proteases at pH 4.0 and pH 9.0 isolated from leguminous seeds
0

Sample

Temperature ( C)

Soybean

10
20
37
50
70
10
20
37
50
70
10
20
37
50
70
10
20
37
50
70
10
20
37
50
70
10
20
37
50
70
10
20
37
50
70

Lentil

Black gram

Green gram

Bengal gram

Ground nut

Pea bean

pH 4.0
Specific activity
(U/mg)
0.001191
0.002156
0.002723
0.003574
0.001021
0.000361
0.000722
0.002287
0.001746
0.000903
0.000638
0.001739
0.003536
0.002203
0.000522
0.001946
0.003891
0.004578
0.005036
0.002289
0.000614
0.001116
0.007755
0.003347
0.000725
0.000500
0.001000
0.002222
0.001722
0.000556
0.000168
0.001008
0.001232
0.000728
0.00448

pH 9.0
Specific activity
(U/mg)
0.000511
0.000965
0.005106
0.001702
0.001135
0.000783
0.001204
0.003070
0.001023
0.000542
0.001043
0.001449
0.005217
0.001739
0.001217
0.001602
0.002518
0.005722
0.004464
0.002232
0.000725
0.004351
0.005523
0.002957
0.000391
0.001000
0.000889
0.002167
0.001111
0.001056
0.000560
0.000672
0.001681
0.001176
0.000392

437

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Agric. Sci. Res. J.

Catalytic (protease) activity (Units/ml)

0.16

Soybean

0.14

Lentil

0.12
0.1

Black gram

0.08
0.06

Green gram

0.04

Bengal gram

0.02
0

7.5

11

Ground nut

PH
0

Figure 1: Effect ofFigure

pH on catalytic
of catalytic
proteaseactivity
at 37 C.
1. Effect activity
of pH on
of protease at 370C.

0.16

Catalytic activity (Units/ml)

0.14
0.12
Soy bean

0.1

Lentil

0.08

Black gram
Green gram

0.06

Bengal gram

0.04

Ground nut
Pea bean

0.02
0
10

20

37

50

70

Temperature (oC)

Temperature ( 0C)

Figure 2a: Effect of temperature on catalytic activity of protease at pH 4.0

Figure 2a. Effect of temperature on catalytic activity of protease at pH 4.0

Deobagkar, 2005).
The effect of temperature on specific protease activity
at pH 4.0 and 9.0 from leguminous seeds was studied
and presented in Table 3. At pH 4.0, all the enzymes in
the present study demonstrated maximum specific

activity at 37 C except soybean and green gram; both

o
showed maximum activity at 50 C. But the specific
activity of protease from all leguminous seeds expressed
their maximum activity at 37oC and pH 9.0. Dahot MU,
1992 investigated on plant protease and found that the

Akhtaruzzaman et al

439

0.12

Catalytic activity (Units/ml)

0.1

0.08

Soy bean
Lentil

0.06

Black gram
Green gram

0.04

Bengal gram

Ground nut
0.02

Pea bean

0
10

20

37

50

70

Temperature (oC)
Figure2b.
2b:Effect
Effect of
of temperature
catalytic
activity
of protease
at pH at
9.0pH 9.0
Figure
temperatureonon
catalytic
activity
of protease

optimum temperature for enzyme reaction was 35oC,

which was a good consistent with our results but Evans
et al., 2011 reported optimum temperature for the palm
weevil was 23oC on 0.03% casein. Kamini et al., 2004
and Aoki et al., 2004 have shown that the protease
activity with optimum temperature less than 20oC is
considered as a cold protease. So, the proteases from
present study can be excellent source for industrial
purposes where requires low or mild (optimum)
temperature as a vital important factors in food
processing steps.
The effect of temperature on catalytic activity of
protease (temperature vs. catalytic activity relationship) at
pH 4.0 and 9.0 was mapped out at Figure 2a and Figure
2b. From Figure 2a, the protease enzyme from soybean
seed showed a symmetrical distribution with one main
peak at 37oC but enzyme from black gram showed two
main peaks at 37oC and 70oC. This characteristic
indicates that enzymes are ampholytes (having carboxyl
and amino group) and undergo change in respect to
solubility, osmotic pressure, viscosity etc. The change in
enzymatic activity with varying pH levels is due to
changes in ionization of the enzyme, the substrate or the
enzyme substrate complex (Gerald Reed, 1975). All the
enzymes showed their symmetrical distribution curve at
o
37 C under pH 9.0 (Figure 2b) whereas catalytic activity
from pea bean was less significant. 0
CONCLUSION
In present study, we extracted the proteases from
overnight imbibed leguminous seeds and tried to make

an easy assay of protease activity procedure for the

leguminous seeds using hemoglobin and casein as
substrate. Using this easy procedure during the
germination, it seems to be important to purify and
characterize these acidic and alkaline proteases from the
leguminous seeds. The study clearly expressed that all
the protease enzymes isolated from seeds were edible
and showed higher activities in both acidic and alkaline
conditions. They have the potentiality in our food
industries.
ACKNOWLEDGEMENTS
This work was supported through a grant provided by
University Grant Commission of Bangladesh (UGC).
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