Religiosin C, a cucumisin-like serine protease from Ficus religiosa

Article abstract of DOI:10.1016/j.procbio.2012.02.015

A serine protease was purified to homogeneity from the latex of Ficus religiosa. The enzyme, named religiosin C is a monomer with molecular mass of 80 kDa. The enzymatic activity of the protein was inhibited by serine protease inhibitors. Isoelectric poin

Full text of DOI:10.1016/j.procbio.2012.02.015

Process Biochemistry 47 (2012) 914–921
Contents lists available at SciVerse ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Religiosin C, a cucumisin-like serine protease from Ficus religiosa
∗
Anurag Sharma, Moni Kumari, M.V. Jagannadham
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A serine protease was purified to homogeneity from the latex of Ficus religiosa. The enzyme, named
religiosin C is a monomer with molecular mass of 80 kDa. The enzymatic activity of the protein was
inhibited by serine protease inhibitors. Isoelectric point of the enzyme is pH 4.6 with optimum pH and
Received 2 December 2011
Received in revised form 9 February 2012
Accepted 20 February 2012
Available online 27 February 2012
◦
1%
temperature of pH 6–8 and 45–50 C, respectively. The specific extinction coefficient (ε 80) of the enzyme
2
is 14.68 with 16 tryptophan, 20 tyrosine and 7 cysteine residues in its molecular structure. The enzyme
shows broad substrate specificity and hydrolyzes both natural and synthetic substrates. The enzyme is
highly stable over a broad range of pH and temperature as well as in the presence of high concentration of
chemical denaturants, organic solvents and metal ions. The N-terminal residues of religiosin C exhibited
considerable homology with cucumisin and other cucumisin/subtilisin-like serine proteases. The high
milk-clotting ability of religiosin C supports its probable use in the food and other biotechnological
industries.
Keywords:
Cucumisin-like serine protease
Ficus religiosa
Latex
N-terminal sequence
Milk-clotting activity
©
2012 Elsevier Ltd. All rights reserved.
1
. Introduction
is available about the features of the plant subtilisins, also referred
as cucumisin-like proteases. Cucumisin from melon fruit (Cucumis
melo) was the first plant serine protease characterized to date [3,4].
Subsequently, more cucumisin-like proteases were isolated from
other plants, such as Taraxacum officinale Webb, Euphorbia supine
and Benincasa hispida var. Ryukyu, and characterized for their broad
substrate specificity and optimum temperature and pH [5].
Proteolytic enzymes are ubiquitous in biological systems and
play numerous cellular and extracellular processes. Serine pro-
teases are one of the largest groups of proteolytic enzymes involved
in several biological processes. In plants, they are widely distributed
among different taxonomic groups and involved in physiological
processes such as protein degradation and processing, microsporo-
genesis, symbiosis, hypersensitive response, signal transduction
and differentiation, and senescence. Despite being their prevalence,
the functions and regulatory roles of plant serine proteases are
poorly understood, probably due to a lack of identification of their
physiological substrates [1].
Plant lattices are valuable reservoir of biomolecules including
organic and inorganic compounds, waxy materials, and enzymes
such as proteases. These extracellular proteases have been shown
to play important defensive roles against herbivores, insects, and
pests. Besides, plant latex could be a potential cost-effective nat-
ural source of enzymes in terms of easy purification methods,
low levels of interfering substances during purification, and good
yield. Moreover, the substantial uses of plant-derived proteases in
food and biotechnological industries have been well documented.
These proteases are found to be suitable under working condi-
tions of different industries due to their broad substrate specificity,
high stability in extreme conditions, good solubility, and activity
over a wide range of pH and temperature. Some cysteine pro-
teases such as papain, bromelain, ficin, and calotropins are utilized
widely in several processes in the food and dairy industries. In
addition, several plant serine proteases have been studied for their
medicinal and industrial applications. Their enzymatic activity over
a wide range of natural protein substrates is one of the factors
which have made them suitable for commercial applications in
industries. Cucumisin, a plant serine protease, could be an exam-
ple of milk-clotting enzyme similar to papain, but also produced
less bitter cheese than those formed by the cysteine proteases
[6]. One of the most important applications of proteases in the
Serine proteases are grouped into six classes, and the second
largest class being the subtilisins [2]. Nearly, all the members of the
subtilisins are tripeptidylpeptidases or endopeptidases and best
characterized in microorganisms. However, very little information
Abbreviations: BSA, bovine serum albumin; CBB, coomassie brilliant blue;
DEAE, diethylaminoethyl; DMSO, dimethyl sulfoxide; DTNB, 5,5-dithiobis (2-
nitrobenzoicacid); DTT, di-thiothreitol; EDTA, ethylenediaminetetraacetic acid;
EGTA, ethylene glycol-bis (raminoethyl ether) tetraacetic acid; GuHCl, guanidine
hydrochloride; IAA, iodoacetic acid; IEF, iso electric focusing; PAGE, polyacrylamide
gel electrophoresis; PMSF, phenylmethanesulfonyl fluoride; SBTI, soybean trypsin
inhibitor; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; TEMED, N,N,N,N-
tetramethylethylenediamine; ␤-ME, ␤-mercaptoethanol.
∗ Corresponding author. Tel.: +91 542 2367936; fax: +91 542 2367568.
E-mail addresses: anuragmbu@rediffmail.com (A. Sharma),
monimbu@rediffmail.com (M. Kumari), jvm@bhu.ac.in, jaganmv@sifymail.com
(
M.V. Jagannadham).
1
359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2012.02.015

A. Sharma et al. / Process Biochemistry 47 (2012) 914–921
2.3. Protein concentration
915
food industry is the use of rennet in cheese formation. Numer-
ous attempts have been made to find out a suitable alternative
of calf rennet because of its limited supply, ethical issues and
increasingly high prices. In this direction, attention has been drawn
to enzymes from plant sources for the production of cheese and
other food products. Recently, many proteases have been char-
acterized from the different parts of the plants and their uses
in food industries have been publicized. Milk-clotting enzymes
have been found in almost all kinds of plant tissues. Therefore,
the search for new potential plant proteases still continues in
order to make them industrially applicable and cost effective
The protein concentration was measured by absorbance at 280 nm as well as by
the method of Bradford using BSA as standard [11].
2.4. Protease assay
The proteolytic activity of the enzyme during purification was monitored using
natural substrates casein and hemoglobin. For the assay, 10 ␮g of enzyme in 0.5 ml
of 0.05 M Tris–HCl buffer, pH 8.0 was added to 0.05 ml of 1% substrate in the same
◦
buffer and the reaction was allowed to proceed for 30 min at 37 C. The reaction was
terminated by the addition of 0.5 ml of 10% TCA and kept for 10 min. The resultant
precipitate was removed by centrifugation and TCA soluble peptides in the super-
natant were measured by absorbance at 280 nm. A control assay, without enzyme
in the reaction mixture was used as blank. One unit of enzyme activity is defined
as the amount of enzyme that gave rise to an increase of one unit of absorbency at
280 nm per min of substrate digestion. The specific activity is the number of units
of activity per milligram of protein.
[
7].
Multiple proteases of the same family are quite often reported
in latex bearing plants. However, the cause of such multiplicity of
proteases has not been extensively highlighted. These multiple pro-
teases from the same source show different behaviors in terms of
stability, activity and specificity [8]. Such reports about the multi-
plicity of proteases have prompted further screening of the latex of
Ficus religiosa and during the process one more milk-clotting serine
protease has been identified which is more active than other pro-
teases of the same source. The other proteases namely religiosin
and religiosin B have already been characterized for their milk-
clotting activity [9,10]. Also, all the three enzymes show distinct
features from one other as well as from other well-known serine
proteases in various terms. In this respect it is essential to pursue
studies on these enzymes to get a better understanding of their fea-
tures. This manuscript describes the identification, purification and
biochemical properties of a new serine protease from the latex of
F. religiosa.
2
.5. Electrophoresis and zymography
Homogeneity, intactness and molecular mass (Mr) of the purified enzyme were
determined by 15% SDS-PAGE under reducing and non-reducing conditions. After
electrophoresis, proteins in the gel were stained by coomassie R-250. Gelatin zymog-
raphy was performed to confirm the proteolytic activity of religiosin C in the gel
using the protocol of Tomar et al. [7]. After electrophoresis, protein in the gel was
stained by coomassie G-250.
2.6. Isoelectric focusing
The isoelectric point (pI) of the purified enzyme was determined by isoelectric
focusing on polyacrylamide disc gel as described by Tomar et al. [7]. Electrophoretic
run was carried out with ampholine carrier ampholytes in the pH range 4–6 at 5 mA
current for 2 h using 0.1 M NaOH as catholyte and 0.1 M orthophosphoric acid as
anolyte. The protein band in the gel was visualized by coomassie G-250 staining.
2
.7. pH and temperature optima
2. Materials and methods
pH and temperature affects the activity of an enzyme and are of the prime impor-
2
.1. Materials
tance when choosing an enzyme for industrial processes. The optimum protease
activity of religiosin C was measured at different pH and temperature. The assays
Acetonitrile, acrylamide, bovine serum albumin, casein, chymostatin, coomassie
◦
were carried at 37 C as described above. Below pH 4.0, casein could not be use as
brilliant blue, DEAE-sepharose fast flow, DTNB, DTT, EDTA, EGTA, glycerol, GuHCl,
hemoglobin, hen egg white lysozyme, HgCl2, IAA, N,N-methylene bis-acrylamide,
o-phenanthroline, papain, PMSF, rennin, ribonuclease A, SBTI, TCA, trypsin, urea,
substrate due to its insolubility. Therefore, hemoglobin was used as substrate below
pH 4.0. A control assay at same pH without enzyme in reaction mixture was used as
blank. Effect of temperature on the activity of purified enzyme was also investigated
and the activity assay was performed at different temperatures.
␤
-mercaptoethanol and all synthetic amides were purchased from Sigma Chemical
Co., USA. Ampholine carrier ampholites were from LKB. All other chemicals were of
the highest purity and commercially available.
2
.8. Stability
2
.2. Purification of enzyme
The stability of an enzyme dictates its applicability therefore; the effect of pH
1.0–12.0) and temperature (20–90 C) as well in the presence of different concen-
◦
(
◦
All the experiments of purification were carried out at 4 C unless stated other-
tration of denaturants, and organic solvents detergents and metal on the proteolytic
activity of religiosin C was examined. The enzyme was incubated under specified
condition of pH, denaturants, organic solvents and metal ions for 24 h, whereas in
the case of temperature and detergents the enzyme was incubated for 15 min and
6 h, respectively. The residual proteolytic activity was assayed as described above.
wise.
Step 1. Gum removal
Fresh latex was collected from the stem by making longitudinal incisions in to
.01 M acetate buffer, pH 4.5 and frozen at −20 C for more than 48 h. Subsequently,
◦
0
the latex was thawed to room temperature and centrifuged at 24,000 × g for 30 min
to remove gum and other debris. The resulting clear supernatant was termed as
crude latex and used in the next step.
2.9. Effect of various inhibitors on the activity
Effect of different inhibitors on the activity of purified enzyme was studied to
classify the protein. Effect of various protease inhibitors (PMSF, chymostatin, IAA,
Step 2. Ammonium sulfate precipitation
The crude latex (devoid of gum and any insoluble material) was subjected to 70%
ammonium sulfate precipitation. The clear supernatant after ammonium sulfate
precipitation showed good amount of proteolytic activity and was used in the next
step of purification.
HgCl , EDTA, EGTA, o-phenanthroline) on hydrolyzing activity of religiosin C was
2
monitored. Ten micrograms of enzyme was incubated with increasing concentration
of specific inhibitor (0–50 mM) in 0.05 M Tris–HCl buffer pH 8.0 for 30 min at 37 ◦C
and assayed. A control assay was performed without inhibitor, and the activity was
considered as 100%.
Step 3. Anion exchange chromatography
The supernatant from previous step was subjected to anion exchange chromatog-
raphy on DEAE-sepharose fast flow in a column pre-equilibrated with 0.01 M Tris
buffer, pH 8.5. The column was washed thoroughly with the same buffer until no
protein or activity was seen in the eluate. The bound proteins were eluted with
a linear salt gradient from 0 to 0.6 M NaCl. All the fractions were monitored by
absorbance at 280 nm for protein content and assayed for enzymatic activity with
casein as substrate. Intactness and homogeneity of the enzyme in all the fractions
were also assessed by SDS-PAGE.
2
.10. Assay for amidolytic activity towards synthetic substrates
Protease activity was determined by measuring p-nitroaniline liberation
from the chromogenic synthetic peptide substrates such as N␣-benzoyl-
dl-arginine-p-nitroanilide (BAPNA), l-alanine-p-nitroanilide, l-alanine-alanine-
p-nitroanilide, l-leucine-p-nitroanilide, N-succinyl-l-phenylalanine-p-nitroanilide
and l-␥-glutamyl-p-nitroanilide. In every case, a stock of 20 mM solution of syn-
thetic substrate was prepared by dissolving the required amount of substrate in a
minimum volume of DMSO and made up to the final volume with 0.05 M Tris–HCl
buffer, pH 7.5. The reaction mixture contained approximately 15 ␮g of enzyme in
Step 4. Gel-filtration chromatography
Active fractions from previous column were subjected to gel filtration chro-
matography on superdex-200 pre-equilibrated with 0.01 M Tris buffer, pH 8.5
containing 0.2 M NaCl and the column was eluted isocratically. All the fractions
were analyzed as in the above step. The active and homogenous fractions were
0.5 ml of Tris–HCl buffer, pH 7.5, and 0.5 ml of peptidyl pNA. After 30 min of incu-
bation at 37 ◦C, the reaction was terminated by addition of 0.2 ml of 30% acetic acid
◦
−1 −1
and the liberated p-nitroaniline (ε = 8800 M cm at 410 nm) was monitored by
pooled, dialysed and stored at 4 C for further experiments.

9
16
A. Sharma et al. / Process Biochemistry 47 (2012) 914–921
absorbance at 410 nm, against a reaction blank without enzyme. One unit of enzyme
activity is defined as the amount of enzyme that gives rise to an increase in one unit of
absorbency at 410 nm per min substrate digestion, under standard assay conditions.
2.17. Milk-clotting activity
Milk-clotting activity was determined according to the methods described by
Arima et al. [14] with a slight modification. The substrate (10% skim milk in 0.01 M
CaCl2) was prepared and the pH was adjusted to 6.0. The substrate (2.0 ml) was pre-
2.11. Kinetic parameters
◦
incubated for 5 min at 37 C, and 0.2 ml of enzyme was added, and the curd formation
◦
The effect of increasing substrate concentration on the velocity of the enzyme-
was observed at 37 C while manually rotating the test tube from time to time. The
◦
end point was recorded when discrete particles were discernible. One milk-clotting
unit is defined as the amount of enzyme that clots 10 ml of the substrate within
40 min.
catalyzed reaction was studied synthetic substrate at pH 8.0 and 37 C. The
concentration of synthetic substrates (l-Leu-pNA, l-␥-glutamyl-pNA, l-Ala-pNA, l-
Ala-Ala-pNa) was studied in the range of 0.001–40 mM. Kinetic constants of the
purified enzymes were calculated from the product accumulation curves with molar
absorption coefficient for p-nitroaniline determined in the reaction buffer for differ-
ent synthetic peptide substrates. The value of Michaelis constant, Km was calculated
by fitting the values in excel plot. The value of the catalytic constant (Kcat) was
obtained by dividing Vmax by molar concentration of enzyme. The specificity con-
stant was calculated by dividing kcat/Km.
2
400
MCA (U/ml) =
× dilution factor
clotting time in sec
3. Results and discussion
2.12. Estimation of tryptophan and tyrosine content
3.1. Protein purification
Total numbers of the tryptophan and tyrosine residues in the enzyme molecule
A new serine protease was purified to homogeneity from the
latex of F. religiosa by combination of procedure using ammonium
sulfate precipitation, anion exchange and gel filtration chromatog-
raphy. The crude latex (devoid of gum and any insoluble material)
was subjected to 70% ammonium sulfate precipitation. The clear
supernatant after ammonium sulfate precipitation showed good
amount of proteolytic activity and was used in the next step of
purification. The supernatant was applied to DEAE-sepharose fast
flow column pre-equilibrated with 10 mM Tris buffer, pH 8.5. The
bound proteins were eluted with a linear salt gradient of 0–0.6 M
NaCl. The column elution profile resolved in to two peaks and
denoted as peaks I and II as shown in Fig. 1A. Fractions of both
the peaks were assayed for proteolytic activity and subjected to
SDS-PAGE to check the purity.
were measured as described by Sharma et al. [12]. An absorbance spectrum of
the purified enzyme in 0.1 M NaOH was recorded from 300 to 220 nm and the
absorbance values at 280 and 294.4 nm were deduced from the spectra. For cal-
culations, the following formula was used:
A280 − x · εy
w =
εw − εy
where A280 is the absorbance at 280 nm from the protein spectra; w is the molar
concentration of tryptophan; εw and εy are the molar extinction coefficients of tryp-
tophan and tyrosine in 0.1 M NaOH at 280 nm (εw = 5225 and εy = 1576), respectively.
x, the total molar concentration of total tyrosine and tryptophan content, was calcu-
lated using ε294.4 = 2375. The number of a particular amino acid residue per molecule
of the protein was calculated from the ratio of the molar concentrations of the amino
acid residues to that of the total protein. To validate the current estimation, papain,
ribonuclease and lysozyme were used as standards.
2
.13. Estimation of total and free cysteine content
The magnitude of activity as well as purity of the fractions
of peak I was higher than the fractions of peak II. Therefore, the
fractions of ascending limb of pool I (125–160) were pooled and
subjected to further purification to gel filtration chromatography
on Superdex-200. The elution profile constitutes of a major sym-
metrical peak followed by a small peak as shown in Fig. 1B. The
active and homogenous fractions of the former peak were pooled,
concentrated and dialyzed for further use. The purification fold of
the purified protein is 2.49 with 11% yield and specific activity of
55 U/mg. The purified protein is named as religiosin C according to
protease nomenclature.
The free and total cysteine residues of the enzyme were estimated by Sharma
et al. [12]. For the free cysteine content estimation, the enzyme was reduced with
.01 M ␤-ME, whereas, for the total cysteine content estimation the enzyme was
0
first denatured in 6 M GuHCl and then reduced with 0.05 M DTT. The excess reducing
agents were removed by dialysis against 0.1 M acetic acid. An aliquot of the dialyzed
enzyme was added to DTNB solution and the liberated TNB anions were monitored
by absorbance at 412 nm. The number of disulfide bonds per molecule of the protein
was calculated using the number of total and free cysteine residues in the molecule.
To validate the current estimations, papain, ribonuclease, and lysozyme were used
as standards.
2
.14. Estimation of specific extinction coefficient
The purification result of religiosin C is summarized in Table 1.
The purification protocol is simple, highly reproducible with the
consistent yield and specific activity of the enzyme.
The extinction coefficient of the enzyme was determined by spectrophotomet-
ric method as described by Tomar et al. [7]. The specific extinction coefficient was
determined by using the formula,
3.2. Homogeneity and physical properties of purified protease
1
0(5690nw + 1280ny + 120nc)
1
%
ε
=
2
80
M
Religiosin C showed a single band when assayed by SDS-PAGE
where nw, ny, and nc are the number of tryptophan, tyrosine, and cysteine residues
in the protein, respectively; M is the molecular mass of the protein; 5690, 1280, and
under both reducing and non-reducing conditions with estimated
molecular mass of 80 kDa (Fig. 2A). The mobility pattern of the
enzyme by SDS-PAGE indicates that the protein is a monomer
and consists of single subunit of 80 kDa. The molecular mass of
the purified enzyme is similar to those of cucumisin-like serine
proteases from Cucumis metuliferus, and Pleioblastus hindsii, while
different from the other proteases of the same source [9,10,15,16].
The molecular masses of plant serine proteases vary from 19 to
10 kDa where majority of proteases fall in the range of 60–80 kDa
1].
Gelatin zymography confirmed the proteolytic nature of the
protein, where digested gelatine appeared as well-resolved white
band against a dark background, corresponding to the position of
the enzyme in gel (Fig. 2B). Although the zymogram gel contained
1
20 are the extinction coefficients of tryptophan, tyrosine and cysteine, respectively.
The total numbers of tryptophan, tyrosine and cysteine residues in the protein were
determined as described above.
2.15. Autolysis
Proteases are prone to autolysis. Autolysis depends upon concentration of
enzyme, pH, temperature, and any type of activator, if any. Extent of autolysis of
◦
the religiosin C was monitored at 37 C. The enzyme at different concentrations in
1
[
◦
the range of 0.01–1.0 mg/ml was incubated in 50 mM Tris–HCl, pH 8.0 at 37 C. An
aliquot of enzyme was used for the determination of remaining proteolytic activity
with casein as substrate. Reaction mixture without enzyme was used as a blank.
The activity of the enzyme after the first 2 h was taken to be 100% for calculating the
residual activity.
2.16. N-terminal sequencing
0
.1% SDS and the sample was treated with 1% SDS, the enzyme still
displayed activity, indicating that it was resistance to SDS dena-
turing. As recently reported, SDS resistance is a property often
associated with heat-stable proteases of thermo-stable archaea and
The protein sample for sequencing was electrophoresed according to the proce-
dure given by Matsudaria [13] and transferred to PVDF membrane. The N-terminal
sequence was determined an Applied Biosystems ABI 470 protein sequencer.

A. Sharma et al. / Process Biochemistry 47 (2012) 914–921
917
Table 1
Purification of religiosin C.
Steps
Total protein (mg)
150
30.22
15.1
12
Total activitya (units)
Specific activity (units/mg)
Purification fold
Yield (%)
Crude latex
3599.97
900.43
605.40
400.01
23.99
29.79
40.09
55.01
1.00
1.24
1.67
2.29
100
25
16.8
11.1
(
NH4)2SO4 supernatant (70%)
DEAE-sepharose
Superdex-200
a
One unit of enzyme activity is defined as the amount of enzyme that gives rise to an increase in one unit of absorbance at 280 nm per min of casein digestion, under
standard assay conditions.
bacteria [17]. SDS resistance is the most striking property of puri-
fied enzyme among all reported plant serine proteases except for
cucumisin [18].
The isoelectric point (pI) of the purified enzyme was pH 4.6
which confirms the acidic nature of the protein (Fig. 2C). Most of
the recently isolated serine proteases from plants have isoelectric
points in the range of pH 4.0–7.0 [12]. However, serine protease
from melon fruit was reported to have a highly basic isoelectric
point [19].
moiety while the other has no detectable glycosylation in their pro-
tein architecture [9,10]. The different biochemical properties of the
enzyme are compared with the other serine proteases as shown
in Table 2. A single band obtained by SDS-PAGE and isoelectric
focusing demonstrates the high purity of the enzyme.
3.3. Effect of pH on the activity and stability of religiosin C
The proteolytic activity of religiosin C was monitored at differ-
The result of Schiff’s staining confirms that there is no detectable
carbohydrate moiety in the molecular architecture of the protein,
as majority of the reported plant serine proteases are glycoproteins
ent pH from 1.0 to 12.0. Religiosin C acted optimally and shows
00% activity from pH 6 to 8 (Fig. 3A). Moreover, religiosin C shows
more than 80% and 50% of proteolytic activity in the pH range of pH
.5–8.5 and pH 4–10, respectively. The above observations suggest
1
[
1]. However, one of the previously reported serine proteases from
5
the same source is highly glycosylated with 12% of carbohydrate
that religiosin C is active from acidic to basic pH range. The other
cucumisin-like serine proteases show their optimal pH from acidic
to basic ranges are MCA-protease and serine protease from P. hind-
sii [15,20]. However, most of the other plant serine proteases show
their optimal activity in the alkaline range, pH 7–11 [1].
The stability of the enzyme dictates its usefulness in various
applications. Religiosin C is stable under a wide range of pH 4–11,
retains full activity within the same pH range when incubated for
0
0
0
0
.6
.4
.2
.0
A
0.6
0.4
0.2
I
1
and 24 h (Fig. 3A). Besides, the enzyme retains more than 80%
of activity from pH 3.5 to 11.5. The pH stability of the enzyme is
more comparable to other cucumisin-like serine proteases from
Cucumis trigonus Roxburghi, C. melo L. var. Prince, and a milk-
clotting enzyme from Solanum dubium [4,5,21]. However, only 30%
II
0.0
0
50
100
150
200
250
Fraction number
B
0
0
0
0
.6
.4
.2
.0
a
0.6
0.4
0.2
b
0
.0
0
20
40
Fraction number
60
Fig. 1. Elution of the crude latex on cation exchange chromatography. (A) DEAE-
sepharose fast flow, pre-equilibrated with 10 mM Tris buffer, pH 8.5. The bound
proteins were eluted with a linear salt gradient of 0–0.6 M NaCl. Fractions of 3 ml
volume at a flow rate of 3 ml/min were collected. The fractions of ascending limb
of pool I (125–160) were pooled. (B) Superdex-200 column, pre-equilibrated with
Fig. 2. Biochemical properties of religiosin C. (A) Assessment of homogeneity and
molecular weight of the enzyme by 15% SDS-PAGE. Gel electrophoresed lanes 1–3
represent: marker, religiosin C (20 ␮g) under nonreducing and reducing conditions,
respectively. (B) Zymogram (in-gel activity) of religiosin C. The unstained region in
the gel (white colour band) showed the hydrolysis of gelatin by the enzyme. (C) Iso-
electric focusing was performed by 5% polyacrylamide disc gel electrophoresis with
ampholine carrier ampholyte, pH 4–6, at constant current of 5 mA. The isoelectric
point of the purified protein is indicated by an arrow.
0
.01 M Tris buffer, pH 8.5 containing 0.2 M NaCl and the column was eluted iso-
cratically. The fractions were assayed for the protein concentration (ꢀ) and the
proteolytic activity (᭹).

9
18
A. Sharma et al. / Process Biochemistry 47 (2012) 914–921
Table 2
Comparison of biochemical properties of religiosin C with other known serine proteases.
1
2
%
80
Enzyme (source)
Mol. mass
kDa)
Optimum
pH
Stability
pH
pI
Trp
Tyr
Cys
ε
Glycosylation
(%)
(
◦
◦
Temp. ( C)
Temp. ( C)
Religiosin C (F. religiosa)
80
43.3
63
66
50.5
54
6–8 45–50
4–11.5
5.5–10
5.5–11
3–12
6–12
4–11
20–60
20–65
20–70
60–70
10–75
50
4.6
3.8
7.6
9.3
6
16
16
23
NR
15
NR
17
20
26
15
NR
41
NR
31
7
11
7
NR
8
NR
9
14.68
29.47
23.8
NR
26.4
NR
No
12
No
Yes
6–7
Yes
10–12
Religiosin (F. religiosa) [9]
Religiosin B (F. religiosa) [10]
Dubiumin (S. dubium) [24]
Cryptolepain (C. buchanani) [27]
Cucumisin (C. melo) [18]
8–8.5
8–8.5
11
60
55
70
8–10.5
7.1
70–75
70
NR
4.4
Benghalensin (F. benghalensis) [12]
47
8
55
5.5–10
20–80
29.25
NR in the table represents data not reported.
of the activity was retained when the enzyme was incubated at
pH 2.5 and no activity was observed at or below pH 2.0, probably
due to denaturation of the protein at highly acidic pH. The stabil-
ity of religiosin C in regard to its pH is more comparable to other
cucumisin-like serine proteases (Table 2). In this respect, the iso-
lated enzyme is unique, and might therefore be suitable for uses in
industry under alkaline conditions. These characteristics are impor-
tant, because most enzymes are catalytically unstable at alkaline pH
values, thus limiting their usefulness in the food industry especially
as cheese-making coagulants [22]. An exception to this general rule
is represented by the aqueous extract and aspartic proteases from
the flower of Cynara cardunculus, which have been employed suc-
cessfully for the manufacture of traditional cheeses from ovine and
caprine milk [23].
3.4. Effect of temperature on the activity and stability of religiosin
C
The proteolytic activity of religiosin C was monitored in the tem-
◦
perature range of 20–90 C and the optimal activity was observed
◦
at 45–50 C (Fig. 3B). More than 80% of the activity was observed
◦
from 40 to 55 C. However, the activity decreases steadily as the
◦
◦
temperature rose over 60 C and no activity was observed at 80 C.
The optimum temperature for other plant serine proteases may
vary from 30 to 80 C but most of the plant serine proteases act
◦
◦
optimally in the range of 20–50 C [1].
The temperature stability of religiosin C was monitored in range
◦
◦
of 20–90 C and the enzyme showed 100% activity up to 60 C
◦
(Fig. 3B). Moreover, 87% of activity was observed at 70 C when
incubated for 30 min which is significantly higher as compared
to other subtilisin-like serine proteases [5]. The activity sharply
◦
reduced as the temperature increased higher than 70 C proba-
A
bly due to thermal denaturation of the protein. The temperature
profile of the purified enzyme was similar to those of other serine
proteases from C. trigonus Roxburghi, C. melo L. var. Prince, and a
milk-clotting enzyme from S. dubium [4,5,24].
100
7
5
2
5
0
5
0
3
.5. Effect of denaturants, organic solvents and metal ions on the
activity of religiosin C
The purified enzyme exhibited remarkable stability under
various conditions. Religiosin C retains full activity at higher con-
centrations of denaturants, up to 4 M GuHCl and 8 M urea at pH 7.0.
The retention of activity at such a high concentration of denatu-
rants has also been observed in the case of religiosin and religiosin B.
Moreover, the enzyme retains its complete activity in 50% methanol
whereas in 50% of other organic solvents such as ethanol, butanol,
acetonitrile, and dioxane, the residual activity observed was more
than 60% (Table 3). Thus, the enzyme is fairly stable at high temper-
atures, broad range of pH, at high concentrations of denaturants as
well as organic solvents. Such observation of stability may be a dis-
tinct feature of this enzyme. The high stability of religiosin C against
0
2
4
6
pH
8
10
12
B
1
00
7
5
2
5
0
5
0
Table 3
Stability of religiosin C under different conditions.
Condition
Concentration
Residual
activity (%)
pH
pH 4.0–11.5
>90
>90
◦
Temperature
GuHCl
Urea
20–60
4.0 M
8 M
C
99.89 ± 0.16
100.10 ± 0.21
100.04 ± 0.45
75.09 ± 0.18
60.03 ± 0.58
68.24 ± 0.45
65.35 ± 0.22
100.01 ± 0.3
Methanol
Ethanol
Acetonitrile
Butanol
Dioxane
65%
50%
50%
50%
50%
10 mM
2
0
40
60
80
Temperature (ºC)
Fig. 3. Effect of pH (A) and temperature (B) on the proteolytic activity (᭹) and sta-
bility (ꢀ) of religiosin C. The assay protocols are described in Section 2. Each value
in all figures represented as mean ± SD (n = 3).
Metal ions (Na , K , Mg and Ca2+
+
+
2+
)
Residual activities shown in the table as mean ± SD (n = 3).

A. Sharma et al. / Process Biochemistry 47 (2012) 914–921
919
Table 4
Table 5
Effect of inhibitors on the activity of religiosin C.
Kinetic parameters of religiosin C with different synthetic substrates.
−1
−1
s )
Inhibitor
Concentration
Residual activity
(%)
Substrate
Km (mM)
Kcat (s−1)
Kcat/Km (mM
(mM)
Ala-pNA
0.002 ± 0.001
0.004 ± 0.001
0.273 ± 0.03
0.093 ± 0.01
32.13 ± 2.42
25.48 ± 3.89
207.80 ± 5.60
39.35 ± 1.30
13,906.42 ± 414.66
6888.29 ± 120.97
749.73 ± 43.13
PMSF
0.5
1
5
1
2
5
0.5
1
20.02 ± 0.21
12.55 ± 0.11
5.12 ± 0.34
Ala-Ala-pNA
␥-Glu-pNA
Leu-pNA
422.19 ± 33.24
Chymostatin
22.11 ± 0.15
6.54 ± 0.31
All values in the table are represented as mean ± SD (n = 3).
2.45 ± 0.11
DIFP
14.34 ± 0.39
2.34 ± 0.17
amidolytic activity against synthetic substrates such as
l-alanine-p-nitroanilide, l-alanine-alanine-p-nitroanilide, l-␥-
glutamyl-p-nitroanilide and l-leucine-p-nitroanilide, while fails
to hydrolyze N␣-benzoyl-dl-arginine-p-nitroanilide, N-succinyl-
l-phenylalanine-p-nitroanilide. The results indicated that the
purified protease preferred both hydrophilic and hydrophobic
amino acid residues at the P1 position, whereas, the activity of
enzyme over bulky aromatic groups at position P is not detectable.
Thus, the specificity of religiosin C differs from that of cucumisin,
a well known and characterized serine protease from the latex
SBTI
5
96.02 ± 0.35
94.12 ± 0.41
99.21 ± 0.06
98.97 ± 0.12
100.00 ± 0.35
98.34 ± 0.31
99.05 ± 0.24
97.54 ± 0.34
100.10 ± 0.14
98.35 ± 0.34
100.10 ± 0.12
99.76 ± 0.34
96.09 ± 0.28
97.56 ± 0.38
10
5
10
5
10
5
10
5
10
5
10
5
10
HgCl2
IAA
DTT
1
EDTA
EGTA
of C. melo [28]. The preference of hydrophobic residue at the P1
position of this enzyme was comparable to that of chymotrypsin,
and subtilisin. Moreover, aliphatic neutral residues (e.g., Ala) are
preferred at the P2 site as seen also for the other proteases [4].
o-Phenanthroline
Residual activities shown in the table as mean ± SD (n = 3).
3
.8. Kinetic parameters
pH and temperature along with the high stability under denaturing
conditions facilitates the possibility of utilization of the enzyme to
elucidate the structure–function relationship as well as in indus-
trial and biotechnological applications. Metal ions, the monovalent
cations (K , Rb , and Li ), and the divalent cations (Ca , Mg ) do
not show any considerable inhibitory effect on the activity of reli-
giosin C up to 10 mM, as shown in Table 3. The high stability of
religiosin C without perturbing the enzymatic activity by various
agents could make it beneficial to work under various conditions.
The Km values for the enzyme were estimated to be
.002, 0.004, 0.273 and 0.093 mM against Ala-pNA, Ala-Ala-
0
+
+
+
2+
2+
pNA, ␥-glutamyl-pNA and Leu-pNA, respectively. As the speci-
ficity constant (Kcat/Km) values of religiosin C with various
synthetic substrates are 13,906.42 ± 414.66, 6888.29 ± 120.97,
−
1
−1
s against Ala-pNA, Ala-Ala-
7
49.73 ± 43.13, 422.19 ± 33.24 mM
pNA, ␥-glutamyl-pNA and Leu-pNA, respectively. The specific
constant with Ala at P1 position was higher than with Leu and Glu
at the same position. These results indicated that the enzyme pre-
3.6. Effect of inhibitors and metal ions on activity of religiosin C
ferred a small and non-polar residue at the P position to a charged
1
residue at the same position. One of the notable aspects of serine
proteases is their wide diversity of substrate specificities coupled
to a single catalytic mechanism [24]. The kinetic parameters and
specificity constant (Kcat/Km) values of religiosin C with various
synthetic substrates are shown in Table 5.
Inhibition studies can provide a first insight into the nature of
the enzyme, its cofactor requirement, and the nature of the active
centre [25]. Effect of inhibitors on the activity of the enzyme is rep-
resented in Table 4. The inhibitors of cysteine protease (HgCl , IAA,
2
DTT) and metalloprotease (EDTA, EGTA) did not affect the enzy-
matic activity significantly up to the concentration of 10 mM. The
strongest inhibition was observed with the inhibitors of serine pro-
tease such as PMSF, chymostatin, and DIFP. The above-mentioned
inhibition profile classified the isolated protease as a member of the
serine protease class. The inhibition profile of the purified enzyme
is consistent with those reported for bamboo serine protease [15]
and cucumisin [26]. Strong inhibition by PMSF was also reported for
some plant serine proteases, such as cucumisin-like protease from
latex of Cryptolepis buchanani [27] and subtilisin-like protease from
C. trigonus Roxburghi [5].
It is noticeable that a proteinaceous inhibitor such as soybean
trypsin inhibitor (SBTI), which is present in a typical protein-rich
food such as soybean, did not inhibit activity of the purified enzyme.
This property could, therefore, pave the way for the application of
the purified enzyme in food industries. Generally, proteinaceous
inhibitors known so far inhibit activity of either bacterial or animal
serine proteases, but fail to do so in the case of plant serine pro-
teases such as cucumisin, bamboo sprout proteases, benghalensin
3.9. Estimation of amino acid contents and specific extinction
coefficient
The tryptophan and tyrosine contents of the protein are
16 (measured value 15.76 ± 0.03) and 20 (measured value
20.07 ± 0.04), respectively. The total cysteine content is found to
be 7 (measured value 6.89 ± 0.11) with one free cysteine (mea-
sured value 1.22 ± 0.05) and six cysteine forming three disulfide
bridges. The specific extinction coefficient of religiosin C measured
by spectrophotometric method is 14.68.
3.10. Autolysis
Generally, proteases are prone to autolysis which becomes
hindrance in the utilization of the same. The loss of activity of
religiosin C in the concentration range 0.01–1.0 mg/ml, after 72 h
of incubation at room temperature was studied. The magnitude of
loss of activity decreases with increase in the enzyme concentra-
tion from 0.01 to 0.5 mg/ml and further increase in loss of activity
was not observed. Religiosin C retains more than 90% activity even
at very low concentration up to 0.03 mg/ml. This, in turn, indicates
its high stability, and thus its possible application in food, textile,
and biotechnological industries. In our experience, the enzyme is
[
12,15,22].
3
.7. Substrate specificity
The enzyme hydrolyzes denatured natural substrates such as
casein and hemoglobin. Religiosin C also exhibits significant

9
20
A. Sharma et al. / Process Biochemistry 47 (2012) 914–921
Table 6
Comparison of N-terminal sequences of religiosin C with other serine proteases.
Enzyme
N-terminal sequence (first 10 residues)
% identity
Religiosin Ca
Cucumisin
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
R
R
R
R
R
R
H
H
H
S
S
S
S
S
T
T
T
T
P
D
D
D
D
T
L
D
S
F
F
F
F
F
F
Y
F
F
L
L
L
L
L
L
L
L
L
G
G
G
N
G
G
G
G
S
F
F
F
F
L
L
I
100
90
90
80
80
70
60
50
50
W
W
W
P
P
P
Kiwano proteaseb
White gourd protease
Tomato P69B
Arabidopsis ARA12
Lily LIM9
Tomato P69A
Alnus ag12
S
P
L
L
R
a
This report.
Ref. [16].
b
All other sequences are from Ref. [5].
◦
stable for four months at 4 C, under neutral conditions without
loss in activity. Besides, religiosin C can also be stored for a longer
time at neutral conditions and low temperatures with retention of
full activity.
Religiosin C shows striking physico-chemical differences with
religiosin and religiosin B in terms of molecular mass, isoelectric
point, extinction-coefficient, carbohydrate contents and mobility
pattern in SDS-PAGE gel. Temperature optimum of religiosin C is
slightly lower than the other two proteases from the same source.
In addition, pH optimum of religiosin C is slightly towards the acidic
region as compared to religiosin and religiosin B. The Michaelis
constant of religiosin C is significantly lower than religiosin and
religiosin B with Leu-pNA as a common substrate used in the study
of all the three enzymes may dictate wide diversity in substrate
specificity coupled to a single catalytic mechanism. Therefore, reli-
giosins could be ideal model systems to study structure–function
relationship. The stability of the enzyme in the presence of denatu-
rants, organic solvents, and metal ions as well as over a wide range
of temperature and pH is comparable to religiosin and religiosin
B; therefore this protease may turn out to be an efficient choice in
food, pharmaceutical, and biotechnological industries. Religiosin
C shows highest milk-clotting activity out of the three proteases
form the same source. Moreover, the detergent activity of reli-
giosin is significantly higher than the other two proteases (data not
shown). Therefore, the use of religiosin could be possible in deter-
gent and food industry whereas religiosin B and C could be more
ideal choices for food industry.
3.11. N-terminal sequencing
The sequence of 10 amino acids at the N-terminus of religiosin
C was determined, and compared with other plant serine proteases
Table 6). The N-terminal sequence of religiosin C showed high
(
similarity with other plant serine proteases. The highest similar-
ity (90%) was with cucumisin and kiwano protease. The sequence
similarity was 80% with white guard protease and tomato P69B.
Besides, the N-terminal residues of religiosin C are also identical
with other subtilisin/cucumisin like serine proteases. Therefore,
religiosin C may be a member of subtilisin/cucumisin like serine
proteases. Additional sequence analysis, catalytic site studies and
structural determination may refine the classification of religiosin
C.
3.12. Milk coagulation
The enzyme coagulates skimmed milk and forms a white and
firm curd. Moreover, the ratio of milk-clotting activity to proteolytic
activity of religiosin C is determined to be 950.42 ± 43.21 U/OD
Acknowledgments
6
60 nm comparable to those of (387 ± 12.67, 4989 ± 109.771,
67 ± 5.90, 3.6 ± 0.025, and 393 ± 6.12 U/OD 660 nm) religiosin,
3
Financial assistance to AS and MK in the form of a research
fellowship from CSIR (Council of Scientific & Industrial Research)
Government of India is gratefully acknowledged. Funding assis-
tance to AS from BIF (Boehringer Ingelheim Fonds, Germany) for
three months is also gratefully acknowledged.
rennin, papain, trypsin and ficin, respectively [9]. The ratio of milk-
clotting activity to proteolytic activity is a useful indicator of the
protease efficiency to be used as a coagulant for cheese making [14].
The capacity of religiosin C to produce milk curds together with its
high ratio of milk clotting to proteolytic activity, could make it use-
ful as a new milk coagulants, although, more studies about quality
of both milk curds and the cheese formed should be carried out in
the future to confirm its usefulness in the dairy industry. However,
the calf rennet used for cheese production is a relatively expensive
enzyme due to its limited availability and ethical considerations
associated with its use; therefore, the search for new enzymes from
other sources still continues [29].
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