Religiosin B, a milk-clotting serine protease from Ficus religiosa

Article abstract of DOI:10.1016/j.foodchem.2011.09.122

A novel milk-clotting serine protease, named religiosin B, is purified from Ficus religiosa. The molecular mass of the protein is 63,000 with pI value of pH 7.6. The proteolytic activity of the enzyme is strongly inhibited by phenylmethanesulfonyl fluorid

Full text of DOI:10.1016/j.foodchem.2011.09.122

Food Chemistry 131 (2012) 1295–1303
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Religiosin B, a milk-clotting serine protease from Ficus religiosa
⇑
Moni Kumari, Anurag Sharma, 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:
Received 28 April 2011
Received in revised form 25 July 2011
Accepted 22 September 2011
Available online 8 October 2011
A novel milk-clotting serine protease, named religiosin B, is purified from Ficus religiosa. The molecular
mass of the protein is 63,000 with pI value of pH 7.6. The proteolytic activity of the enzyme is strongly
inhibited by phenylmethanesulfonyl fluoride (PMSF) and chymostatin. Religiosin B acts optimally at pH
ꢀ1
ꢀ1
8
2
.0–8.5 and temperature 55 °C. The molar absorption coefficient of the enzyme is 149,725 M cm with
3 tryptophan, 15 tyrosine and 7cysteine residues per molecule of the enzyme. The enzyme shows broad
substrate specificity with natural as well as synthetic substrates. Religiosin B is highly stable against
denaturants and metal ions as well as over a wide range of pH and temperature. The de novo sequencing
confirms the novelty of the enzyme. In addition to its high milk-clotting ability, it could be used in the
cheese industry, as well as other food and biotechnological industries.
Keywords:
Ficus religiosa
Latex
Milk-clotting enzyme
Religiosin B
Ó 2011 Elsevier Ltd. All rights reserved.
Serine protease
1
. Introduction
Proteases constitute one of the most important groups of indus-
ability of crude, easy purification processes and in isolation of
natural coagulant. Also, the use of plant coagulant could be advan-
tageous in improving the nutritional intake of vegetarian people
worldwide (Tavaria, Sousa, & Malacata, 2001). In recent years,
much research interest has been directed towards finding several
new proteases from the plants and their milk coagulating activity
has been explored.
Proteolytic enzymes from the plant sources have received spe-
cial attention because of their broad substrate specificity, activity
in a wide range of pH values, temperature, and presence of organic
compounds as well as other additives. Many operations in food
industry are carried out at high temperature such as, hydrolysis
of proteins at high temperature, enzymatic production of aspar-
tame and other peptides, baking and brewing (Sharma, Kumari, &
Jagannadham, 2009). Therefore, the search for valuable proteases
with distinct activity and specificity is always a continuous chal-
lenge for varied industrial applications.
Multiple proteases of the same family are quite often reported
in latex bearing plants. However, the reason for such multiplicity
of proteases has not been extensively highlighted. These multiple
proteases, from the same source, show different behaviours in
terms of stability, activity and specificity (Nallamsetty, Kundu, &
Jagannadham, 2003). Such reports about multiplicity of proteases
have prompted further screening of the latex of Ficus religiosa. Dur-
ing the process one more milk-clotting serine protease has been
identified which is more active relative to other protease of the
same source. The other protease namely religiosin, has been al-
ready characterised for its milk-clotting activity (Kumari, Sharma,
trial enzymes, accounting for about 60% of the total enzyme mar-
ket. Several useful proteases have been obtained from various
sources such as plants, animals and microorganisms. Proteases
have a large variety of applications in food industry. They have
been routinely used for various purposes such as, cheese making,
baking, meat tenderization and preparation of soya hydrolysates
(Tomar, Kumar, & Jagannadham, 2008). The major application of
proteases in dairy industry is in the manufacture of cheese. The
milk coagulating enzymes fall into three main categories; animal
rennet, microbial milk coagulants and genetically engineered chy-
mosin. However, the increasing cheese production and consump-
tion, the high price and reduced supply of rennets, as well as the
associated ethical issues have led to the search for alternative or
additional rennet substitutes produced either from plants or from
genetically modified microorganisms (Ahmed, Morishima, Babiker,
&
Mori, 2009a, 2009b). Moreover, the use of plant as a source of
proteases could be preferred over other sources in terms of avail-
Abbreviations: BSA, bovine serum albumin; CBB, coomassie brilliant blue; DEAE,
diethylaminoethyl; DMSO, dimethyl sulfoxide; DTNB, 5,5-dithiobis (2-nitrobenzo-
icacid); 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 electro-
phoresis; PMSF, phenylmethanesulfonyl fluoride; SBTI, soybean trypsin inhibitor;
SDS, sodium dodecyl sulphate; TCA, trichloroacetic acid; TEMED, N,N,N,N-tetra-
methylethylenediamine; b-ME, b-mercaptoethanol.
&
Jagannadham, 2010). Also, both the enzymes show distinct fea-
⇑
Corresponding author. Tel.: +91 542 2367936; fax: +91 542 237568.
E-mail addresses: jvm@bhu.ac.in, monimbu@rediffmail.com (M.V. Jagannadham).
tures from each other and from other well-known serine proteases
in various terms. In this respect, it is essential to pursue studies on
0
308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.09.122

1
296
M. Kumari et al. / Food Chemistry 131 (2012) 1295–1303
these enzymes to get a better understanding of their features. This
manuscript describes the identification, purification and biochem-
ical properties of a new serine protease from the latex of F.
religiosa.
2.5. Electrophoresis and zymography
r
Homogeneity, intactness and molecular mass (M ) of the puri-
fied enzyme were determined by SDS–PAGE under reducing and
non-reducing conditions as described by Laemmli (1970). The gel
was cast in 1 mm slab consisting of 15% separating gel
2
. Materials and methods
(
8 cm ꢁ 10 cm) and 5% stacking gel (2 cm ꢁ 10 cm). Electrophore-
sis was carried out at constant current of 20 mA, until the tracking
dye (bromophenol blue) reached till the bottom of the gel (ca. 1 h
running time). After electrophoresis, proteins in the gel were
stained by coomassie R-250. Gelatin zymography was performed
to confirm the proteolytic activity of religiosin B in the gel using
the protocol of Tomar et al. (2008). After electrophoresis, protein
in the gel was stained by coomassie G-250.
2
.1. Materials
Acetonitrile, acrylamide, bovine serum albumin, casein, chymo-
statin, coomassie brilliant blue, DEAE–Sepharose fast flow, DTNB,
DTT, EDTA, EGTA, glycerol, GuHCl, haemoglobin, hen egg white
lysozyme, HgCl , IAA, N,N-methylene bis-acrylamide, o-phenan-
2
throline, papain, PMSF, rennin, ribonuclease A, SBTI, TCA, trypsin,
urea, b-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 com-
mercially available.
2.6. Isoelectric focusing
The isoelectric point (pI) of the purified enzyme was deter-
mined by isoelectric focusing on polyacrylamide disc gel, as de-
scribed by Pande, Dubey, Yadav, Jagannadham (2006).
&
2
.2. Purification of enzyme
Electrophoretic runs were carried out with ampholine carrier
ampholytes in the pH range 7–9, at 5 mA current for 2 h. A 5% poly-
acrylamide gel containing 2% desired ampholine was cast in tube
gels. Anodic and cathodic chamber buffers were 0.1 M orthophos-
phoric acid and 0.1 M sodium hydroxide, respectively. The gels
were subjected to a pre-run at a constant current of 1 mA per
All the experiments of purification were carried out 4 °C unless
stated otherwise.
2
.2.1. Gum removal
Fresh latex was collected from the stem by making longitudinal
rod, for 2 h, to develop the pH gradient. Protein samples (100 lg)
incisions into 0.01 M acetate buffer, pH 4.5 and frozen at ꢀ20 °C for
more than 48 h. Subsequently, the latex was thawed to room tem-
perature and centrifuged at 24,000g for 30 min to remove gum and
other debris. The resulting clear supernatant was termed as crude
latex and used in the next step.
containing 10% (v/v) ampholine and 25% (v/v) glycerol were loaded
on each gel and electrophoresed at a constant current of 2 mA per
rod for 4 h. After the run, protein band was stained with 0.04% (w/
v) coomassie G-250 dissolved in 6% perchloric acid.
2.7. pH and temperature optima
2.2.2. Cation exchange chromatography
The crude latex was subjected to cation exchange chromatogra-
phy on SP-Sepharose fast flow, in a column pre-equilibrated with
.01 M acetate buffer, pH 4.5. 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 1.0 M NaCl. All the fractions were monitored by absor-
bance, 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.
pH and temperature affects the activity of an enzyme and are of
the prime importance when choosing an enzyme for industrial
processes. The optimum protease activity of religiosin B was mea-
sured at different pH and temperature. The assays were carried at
0
3
7 °C, as described above. Below pH 4.0, casein could not be used
as substrate due to insolubility. Therefore, haemoglobin was used
as substrate below pH 4.0. A control assay at the same pH, without
enzyme in reaction mixture, was used as blank. Effect of tempera-
ture on the activity of purified enzyme was also investigated and
the activity assay was performed at different temperatures.
2
.3. Protein concentration
2.8. Stability
The protein concentration was measured by absorbance, at
2
80 nm, as well as by the method of Bradford using BSA as stan-
The stability of an enzyme dictates its applicability therefore;
dard (Bradford, 1976).
the effect of pH (1.0–12.0) and temperature (20–90 °C), as well
as in the presence of different concentrations of denaturants, and
organic solvents detergents and metal ions on the proteolytic
activity of religiosin B 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, respec-
tively. The residual proteolytic activity was assayed as described
above.
2.4. Protease assay
The proteolytic activity of the enzyme during purification was
monitored using natural substrates, casein and haemoglobin. For
the assay, 10 g of enzyme in 0.5 ml of 0.05 M Tris buffer, pH
.0, was added to 0.05 ml of 1% (w/v) substrate in the same buffer
l
8
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% (w/v)
TCA and kept for 10 min. The resultant precipitate was removed
by centrifugation and TCA soluble peptides in the supernatant
were measured by absorbance at 280 nm. A control assay, without
enzyme in the reaction mixture, was used as blank. One unit of en-
zyme activity is defined as the amount of enzyme that gave rise to
an increase of one unit of absorbancy at 280 nm per min of sub-
strate digestion. The specific activity is the number of units of
activity per milligram of protein.
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 inhib-
itors (PMSF, chymostatin, IAA, HgCl , EDTA, EGTA, o-phenanthro-
2
line) on hydrolysing activity of religiosin B were monitored. Ten
micrograms of the enzyme was incubated, with increasing concen-
tration of specific inhibitor (0–50 mM), in 0.05 M Tris–HCl buffer

M. Kumari et al. / Food Chemistry 131 (2012) 1295–1303
1297
pH 8.0, for 30 min, at 37 °C and assayed. A control assay was per-
formed without inhibitor, and the activity was considered as 100%.
tured 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 dialysed enzyme was added to DTNB solu-
tion and the liberated TNB anions were monitored by absorbance
at 412 nm. The number of disulphide 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, pa-
pain, ribonuclease, and lysozyme were used as standards.
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), -alanine-p-nitro-
anilide, -alanine alanine-p-nitroanilide, -leucine-p-nitroanilide,
N-succinyl- -phenylalanine-p-nitroanilide and -glutamyl-p-
a
L
L
L
L
L-c
2.14. Estimation of molar absorption coefficient
nitroanilide. In every case, a stock of 20 mM solution of synthetic
substrate was prepared by dissolving the required amount of sub-
strate in a minimum volume of DMSO and made up to the final vol-
ume with 0.05 M Tris buffer, pH 7.5. The reaction mixture
The molar absorption coefficient of the enzyme was determined
by spectrophotometric method as described by Pace, Vajdos, Fee,
Grimsley, & Gray (1995). The molar absorption coefficient was
determined by using the formula,
contained approximately 15
pH 7.5, and 0.5 ml of peptidyl p-NA. After 30 min of incubation at
7 °C, the reaction was terminated by addition of 0.2 ml of 30%
lg of enzyme in 0.5 ml of Tris buffer,
3
e
ð280Þ ¼ 5500n þ 125n
w
þ 1490n
y
c
(
(
v/v)
e
acetic
acid
and
the
liberated
p-nitroaniline
= 8800 M 1cmꢀ1 at 410 nm) was monitored by absorbance at
ꢀ
where n , n and n are the number of tryptophan, tyrosine, and
w
y
c
4
10 nm, against a reaction blank without enzyme. One unit of en-
cysteine residues in the protein, respectively; the molar absorption
coefficients of tryptophan, tyrosine and cysteine are 5500, 1490,
zyme 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.
1
25, respectively. The total numbers of tryptophan, tyrosine and
cysteine residues in the protein were determined as described
above.
2.11. Kinetic parameters
The effect of increasing substrate concentration on the velocity
2
.15. Autolysis
of the enzyme-catalysed reaction was studied synthetic substrate
at pH 8.0 and 37 °C. The concentration of synthetic substrates (
Leu-pNA, -Glutamyl-pNA, -Ala-pNA, -Ala-Ala-pNa) were stud-
L
-
Proteases are prone to autolysis. Autolysis depends upon con-
L-
c
L
L
centration of enzyme, pH, temperature, and any type of activator,
if any (Tomar et al., 2008). Extent of autolysis of the religiosin B
was monitored at 37 °C. The enzyme at different concentrations,
in 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 deter-
mination 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 calculat-
ing the residual activity.
ied in the range of 0.02–40 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 different synthetic peptide substrates.
The value of Michaelis constant, K
values into an excel plot. The value of the catalytic constant (kcat
was obtained by dividing Vmax by molar concentration of enzyme.
The specificity constant was calculated by dividing kcat/K
m
was calculated by fitting the
)
m
.
2.12. Estimation of tryptophan and tyrosine content
2
.16. Milk-clotting activity
Total numbers of the tryptophan and tyrosine residues in the
enzyme molecule were measured as described by Sharma et al.
2009). An absorbance spectrum of the purified enzyme in 0.1 M
NaOH was recorded, from 300 to 220 nm, and the absorbance val-
ues at 280 and 294.4 nm were deduced from the spectra. For calcu-
lations, the following formula was used:
Milk-clotting activity was determined according to the methods
(
described by Arima, Ya, & Iwasaki (1970) with a slight modifica-
tion. The substrate (10% skim milk, w/v in 0.01 M CaCl ) was pre-
2
pared and the pH was adjusted to 6.0. The substrate (2.0 ml) was
pre-incubated for 5 min, at 37 °C, and 0.2 ml of enzyme was added,
and the curd formation was observed at 37 °C, while manually
rotating the test tube from time to time. The end point was re-
corded 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.
w ¼ ðA280 ꢀ x:
e
y
Þ=ðe
w
ꢀ
e
y
Þ
where A280 is the absorbance at 280 nm from the protein spectra; w
is the molar concentration of tryptophan; and are the molar
absorption coefficients of tryptophan and tyrosine in 0.1 M NaOH
at 280 nm ( = 5225 and = 1576), respectively. x, the total molar
concentration of total tyrosine and tryptophan content, was calcu-
lated using 294.4 = 2375. The number of a particular amino acid res-
e
w
e
y
e
w
e
y
MCAðU=mlÞ ¼ ð2400=clotting time in secÞ ꢁ dilution factor
e
idue 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, ribonu-
clease and lysozyme were used as standards.
2
2.17. Effect of CaCl concentration on MCA of the enzyme
The effect of CaCl
determined. The substrate (1% casein, w/v) was equilibrated at dif-
ferent concentrations of CaCl (0–0.8 M), for 5 min, at 30 °C. After
2
on the milk-clotting activity of enzyme was
2.13. Estimation of total and free cysteine content
2
The free and total cysteine residues of the enzyme were esti-
that the enzyme (0.15 ml) was added and further incubated for
30 min at the same temperature. Casein hydrolysing activity was
determined and expressed as a percentage of the activity measured
in the absence of the salt.
mated by Sharma et al. (2009). For the free cysteine content esti-
mation, the enzyme was reduced with 0.01 M b-ME, whereas, for
the of total cysteine content estimation the enzyme was first dena-

1
298
M. Kumari et al. / Food Chemistry 131 (2012) 1295–1303
2.18. de novo sequencing by MALDI TOF/TOF
by SDS–PAGE suggests highly purified and monomeric nature of
the enzyme. The relative molecular mass (M ) of the purified en-
r
Tryptic digestion of religiosin B was done using the protocol as
described by Kumari et al. (2010), with minor modifications. After
SDS–PAGE, gel pieces were excised, destained, washed, dehydrated
zyme is 63,000 by both MALDI–TOF and SDS–PAGE (Fig. 2A and
B). The molecular mass of the enzyme is similar to that of the
well-known plant serine protease cucumisin (Yamagata, Ueno, &
Iwasaki, 1989), and with other cucumisin-like serine proteases
(Asif-Ullah, Kim, & Yu, 2006; Uchikoba, Yonezawa, & Kaneda,
1998). The molecular mass of the purified enzyme was also similar
to those of subtilisin-like endoproteases from the tomato plant
(Messdaghi & Dietz, 2000). The proteolytic nature of religiosin B
was also confirmed by gelatin zymography, where digested gela-
tine appeared as well-resolved band against a dark background,
corresponding to the position of the protein in gel (Fig. 2C).
Although the zymogram gel contained 0.1% SDS (w/v) and the sam-
ple was treated with 1% SDS (w/v), the enzyme still displayed
activity, indicating that it was resistance to SDS denaturing. As re-
cently reported, SDS resistance is a property often associated with
heat-stable proteases of thermo-stable archaea and bacteria (Joo &
Change, 2005). SDS resistance is the most striking property of the
purified enzyme among all reported plant serine proteases, except
for cucumisin (Yamagata et al., 1989). As shown in fig. 2D, the puri-
fied enzyme was apparently resolved as a single band on isoelectric
focusing with an isoelectric point (pI) of pH 7.6 indicating that it is
a weakly basic protein. Noda, Koyanagi, & Kamyia (1994) found
similar results for plant serine protease from Cucumis melo L. with
a basic isoelectric point (pI) of 9.5. In contrast, most of the recently
isolated serine proteases from plants have isoelectric points in the
range of pH 4.0–7.0 (Tomar et al., 2008). Religiosin B was found to
contain no detectable carbohydrate moiety in the molecular archi-
tecture, as majority of the reported plant serine proteases are gly-
coproteins, as shown in table 1. Moreover, the previously reported
serine protease from the same source by Kumari et al. (2010), is
highly glycosylated with 12% of carbohydrate moiety in its protein
architecture. The different physicochemical properties of the en-
zyme are compared with the other serine proteases as shown in ta-
ble 2.
in CH
cooled on ice and soaked in digestion buffer containing 50 mM
NH HCO 5 mM CaCl and 12.5 ng/ of trypsin (Promega,
3
CN, and dried in a vacuum centrifuge. The gel pieces were
4
3
,
2
,
ll
sequencing grade), overnight at 37 °C. The digested peptides were
recovered from the gel by sonicating in a water bath for 10 min and
the process was repeated three times. The tryptic-digested sample
was mixed with the matrix (saturated solution of
a-cyano-4-
hydroxy-cinnamic acid in 0.1% (v/v) TFA and 50% (v/v) acetonitrile)
and spotted on MALDI plate. The mixture was allowed to dry at
room temperature and used for the MALDI analysis on an ultraflex
MALDI–TOF/TOF mass spectrometer (Bruker Daltonics).
3
. Results and discussion
3
.1. Purification of enzyme
A novel serine protease is purified to homogeneity from the la-
tex of F. religiosa using cation exchange chromatography. The pro-
tein elution profile resolves into four peaks (Fig. 1) and majority of
the total proteolytic activity is observed only in peak II. The
ascending limb of peak II (fractions 105–145) was homogeneous
and resulted in a single band by SDS–PAGE. The homogenous frac-
tions were pooled, dialysed and stored for further use. The enzyme
is purified up to 2.5-fold with 34% yield and a specific activity of
6
9 U/mg. The purification protocol is highly reproducible in terms
of purification fold, yield as well as specific activity. The purified
protein was named as religiosin B according to the nomenclature
of proteases. A simple purification procedure was developed in this
study to obtain a very active and stable enzyme from the latex of F.
religiosa.
3.2. Homogeneity and physico-chemical properties
3.3. Effect of pH and temperature on the activity of religiosin B
Homogeneity and intactness of the protein are judged by SDS–
PAGE under reducing and non-reducing conditions. A single band
The proteolytic activity of religiosin B was monitored at differ-
ent pH values from 1.0 to 12.0 and temperatures in the range of
2
1
1
0
0
.0
.5
.0
.5
.0
1.2
0.9
0.6
II
III
0.3
I
1
05 ─ 145
IV
0.0
0
50
100
150
200
250
Fraction number
Fig. 1. Elution of the crude latex on cation exchange chromatography. SP-Sepharose fast flow, pre-equilibrated with 10 mM acetate buffer, pH 4.5. The bound proteins were
eluted with a linear salt gradient of 0–1 M NaCl. Fractions of 4 ml, at a flow rate of 4 ml/min collected and assayed for the protein concentration (s) and the proteolytic
activity (d). The fractions of ascending limb of pool II (105–145) were pooled, as indicated by a horizontal line.

M. Kumari et al. / Food Chemistry 131 (2012) 1295–1303
1299
3.5. Effect of denaturants and organic solvents on the activity of
religiosin B
A
6
000
As shown in table 2, the purified enzyme exhibited remarkable
stability under various conditions. Religiosin B retains full activity
at higher concentrations of denaturants, up to 3.0 M GuHCl and
8
M urea. The enzyme also shows an increase in the proteolytic
activity at lower concentrations of GuHCl up to 2.0 M. The increase
in proteolytic activity of the enzyme is about twofold at 2.0–2.5 M
GuHCl, which is not found when the enzyme is treated with urea.
The enhanced activity might be correlated with high structural
integrity of the active site of the enzyme in the presence of the
denaturants, as reported for religiosin earlier by Kumari et al.
4
000
31637.037
6
3410.420
(
2010). The enzyme retains its complete activity in 50% (v/v) meth-
2
000
anol, where as in 50%, v/v (ethanol, butanol, acetonitrile, dioxane
and DMSO) the residual activity observed was more than 50%. Thus
the enzyme is fairly stable at high temperatures, at a broad range
of pH values, and at low concentrations of denaturant, where as or-
ganic solvent affects the activity of enzyme considerably. Such con-
trary observations of stability may be a distinct feature of this
enzyme. The high stability of the enzyme against pH and temper-
ature along with the high stability under mild denaturing condi-
tions facilitates the possibility of utilisation of the enzyme in
industrial and biotechnological applications.
0
2
0000 40000 60000 80000 100000 120000
m/z
kDa
1
2
3
pH 7
9
6
7
3.6. Effect of inhibitors and metal ions on activity of religiosin B
6.0
pH 7.6
43.0
Effect of inhibitors on the activity of the enzyme is shown in ta-
ble 2. The proteolytic activity was inhibited considerably by the
PMSF and chymostatin. Compared to other protease class inhibi-
tors this indicates that the enzyme belongs to the class of serine
proteases. However, the enzyme activity was inhibited by 20%
2
9.0
0.1
2
2
from HgCl (a cysteine protease inhibitor) and o-phenanthroline
(
a metalloprotease inhibitor), shows a distinct inhibition profile
1
4.3
for religiosin B, with a cysteine residue near its active site and con-
taining a metal in the protein architecture. Although, the complete
structure of protein should be necessary for better understanding
of the active site and catalysis of the enzyme. More inhibition stud-
ies can provide an insight into the nature of the enzyme, its cofac-
tor requirement, and the nature of the active centre (Sedmak &
Grossberg, 1977). It is noticeable that a proteinaceous inhibitor,
such as soybean trypsin inhibitor (SBTI), which is present in pro-
tein rich foods like soybeans, fails to inhibit the enzyme up to
6
.5
pH 9
B
C
D
Fig. 2. Biochemical and physico-chemical properties of religiosin B. (A) MALDI–TOF
analysis of religiosin B. (B) Assessment of homogeneity and relative molecular mass
(
M
r
) of the enzyme by 15% SDS–PAGE. Gel electrophorized lanes 1–3 represent:
marker, religiosin B (30 g) under nonreducing and reducing conditions, respec-
tively. (C) Zymogram (in-gel activity) of religiosin B. The unstained region in the gel
indicated by double head arrow) showed the hydrolysis of gelatin by the enzyme
D) Isoelectric focusing was performed by 5% polyacrylamide disc gel electropho-
l
(
(
5
mM, thus paving the way for its industrial usage. Generally, SBTI
inhibits the protease activity of animal and bacterial serine prote-
ases, but fails to do so in the case of plant proteases (Sharma et al.,
resis with ampholine carrier ampholyte, pH 7.0–9.0, at constant current of 5 mA.
The isoelectric point of the purified protein is indicated by an arrow.
2
009). Other inhibitors of cysteine protease (HgCl2, IAA, DTT) and
metalloprotease (EDTA, EGTA) did not affect the enzymatic activity
significantly up to the concentration of 5 mM.
2
0–90 °C. Religiosin B acted optimally at pH 8 and 55 °C. Moreover,
+
+
+
Metal ions, the monovalent cations (K , Rb , and Li ), and the
Religiosin B shows more than 80% of proteolytic activity in the pH
and temperature range of pH 7.0–9.5 and 50–60 °C, respectively.
2
+
2+
divalent cations (Ca , Mg ) do not show any considerable inhibi-
tory effect on the activity of religiosin B up to 10 mM, shown in ta-
ble 2. However, the activity was inhibited by some heavy divalent
2
+
2+
2+
2+
3.4. Effect of pH and temperature on the stability of religiosin B
cations such as Hg , Co , Zn , and Cu . The high stability of reli-
giosin B, without perturbing the enzymatic activity by various
agents, could make it beneficial to work under various conditions.
The enzyme retains more than 80% of the activity from pH 5.5–
1
1.0 and in the temperature range of 20–75 °C (Fig. 3A and B) and
its stability is more comparable to other cucumisin-like serine pro-
teases from Cucumis trigonus Roxburghi, Cucumis melo L. var. Prince
and Trichosantus kirrilowi A. (Asif-Ullah et al., 2006; Uchikoba,
Horita, & Kaneda, 1990; Yamagata et al., 1989). The thermostability
of the enzyme was found to be 75 °C. The temperature profile of
the purified enzyme was similar to those of subtilisin/cucumisin
like plant serine proteases from C. trigonus Roxburghi, C. melo L.
var. Prince, and T. kirrilowi A. (Asif-Ullah et al., 2006; Uchikoba
et al., 1990; Yamagata et al., 1989).
3.7. Substrate specificity
The enzyme hydrolyses denatured natural substrates such as
casein and haemoglobin. Religiosin B also exhibits significant
amidolytic activity against synthetic substrates such as,
p-nitroanilide, -alanine-alanine-p-nitroanilide, -glutamyl-
p-nitroanilide and -leucine-p-nitroanilide, while fails to hydrolyse
-benzoyl-DL-arginine-p-nitroanilide, N-succinyl- -phenylala-
nine-p-nitroanilide. The results indicated that the purified protease
L-alanine-
L
L-c
L
Na
L

1
300
M. Kumari et al. / Food Chemistry 131 (2012) 1295–1303
Table 1
Comparison of biochemical and physico-chemical properties of religiosin B with other selected serine proteases.
1%
Enzyme (Source)
Mol Mass (kDa)
Optimum
pH
Stability
pH
pI
Trp
Tyr
Cys
e
280
Glycosylation(%)
Temp (°C)
Temp (°C)
Religiosin B (F. religiosa)
Dubiumin (S. dubium)a
66
66
43.3
64
54
8–8.5
11
8–8.5
9
7.1
8
55
70
60
65
70
55
70
5.5–11
3–12
5.5–10
3–12.5
4–11
20–70
60–70
20–65
15–85
50
7.6
9.3
3.8
9.2
NR
4.4
6
23
NR
16
3
NR
17
20
15
NR
26
7
NR
31
75
7
23.80
NR
29.47
5.3
NR
29.25
36.4
no
yes
12
9.7
yes
10–12
8
NR
11
7
NR
9
b
Religiosin (F. religiosa)
Streblin (S. asper)c
Cucumisin (C. melo)
d
e
Benghalensin (F. benghalensis)
47
57.9
5.5–10
5.5–11.5
20–80
75–80
Wrightin (W. tinctoria)f
7.5–10
9
NR in the table represents data not reported.
a
Ahmed et al. (2009a, 2009b).
Kumari et al. (2010).
Tripathi et al. (2011).
Yagmata et al. (1989).
Sharma et al. (2009).
Tomar et al. (2008).
b
c
d
e
f
3.9. Estimation of amino acid contents and molar absorption
Table 2
Stability of religiosin B under different conditions.
coefficient
Condition
Concentration
Residual activity (%)
The tryptophan and tyrosine contents of the protein are 23
measured value 23.16 ± 0.03) and 15 (measured value
pH (6.0–10.5)
Temperature (20–70 °C)
GuHCl
Urea
Methanol
Ethanol
Acetonitrile
Butanol
Dioxane
DMSO
PMSF
>90
>90
(
1
5.07 ± 0.04), respectively. The total cysteine content is found to
3 M
8 M
100.12 ± 0.14
100.02 ± 0.11
100.04 ± 0.15
60.09 ± 0.18
64.03 ± 0.08
57.24 ± 0.15
55.35 ± 0.22
70.44 ± 0.12
8.55 ± 0.17
12.11 ± 0.15
95.02 ± 0.35
80.21 ± 0.06
100.02 ± 0.35
100.05 ± 0.24
100.20 ± 0.17
100.10 ± 0.12
80.09 ± 0.28
100.01 ± 0.01
be 7 (measured value 6.89 ± 0.11) with one free cysteine (mea-
sured value 1.22 ± 0.05) and six cysteine forming three disulphide
bridges. The molar absorption coefficient of religiosin B, measured
50% (v/v)
50% (v/v)
50% (v/v)
50% (v/v)
50% (v/v)
50% (v/v)
1 mM
2 mM
5 mM
5 mM
5 mM
ꢀ1
ꢀ1
by spectrophotometric method is 149,725 M cm
.
3.10. Autolysis
Chymostatin
SBTI
Generally protease undergoes autolysis, which is dependent on
protein concentration, temperature, time, and activators, if any.
Therefore, it is important to check the conditions for the storage
of the enzyme without any loss of proteolytic activity. The loss of
activity of religiosin B in the concentration range 0.01–1.0 mg/ml,
under neutral conditions at room temperature was studied. An ali-
HgCl
IAA
2
DTT
EDTA
EGTA
5 mM
5 mM
5 mM
5 mM
o-phenanthroline
Metal ions (Na , K , Mg and Ca2+
+
+
2+
)
10 mM
quot containing 10 lg of enzyme was used to measure the proteo-
lytic activity after 24 h, 48 h and 72 h of incubation. The magnitude
of loss of activity decreases with an increase in the enzyme concen-
tration from 0.01–0.30 mg/ml and no loss of activity was observed
at higher concentrations. Religiosin B retains more than 90% of
activity, even at very low concentration up to 0.05 mg/ml, after
Residual activities shown in the table as mean ± SD (n = 3).
preferred both hydrophilic and hydrophobic amino acid residues at
the P1 position, whereas, the activity of enzyme over bulky aromatic
groups at position P1 is not detectable. Thus, the specificity of
religiosin B differs from that of cucumisin, a well known and charac-
terised serine protease from the latex of C. melo by Arima, Yonezawa,
Uchikoba, Shimada, & Kaneda (2000). The preference for the
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 (Yamagata et al., 1989).
7
2 h of incubation. The result indicates that religiosin B shows high
resistant to autolysis. The similar results have also been reported
for other plant serine proteases like streblin and dubiumin
(Tripathi, Tomar, & Jagannadham, 2011; Ahmed et al., 2009b). In
contrast, many proteases studied undergo autolysis at low
concentrations, such as benghalensin, wrightin, procerain and
ervatamin A (Dubey & Jagannadham, 2003; Nallamsetty et al.,
2
003; Sharma et al., 2009; Tomar et al., 2008). The lower suscepti-
bility to autodigestion at very low concentrations, in turn, indicates
the possible use of religiosin B in food, textile, and other biotechno-
logical industries. In our experience, the enzyme is stable for four
months, at 4 °C, under neutral conditions, without loss in activity.
3
.8. Kinetic parameters
The K
.32 and 0.93 mM against Ala-pNA, Ala-Ala-pNA,
value of the enzyme, with Ala
m
values for the enzyme were estimated to be 0.04, 0.05,
1
c-glutamyl-pNA
and Leu-pNA, respectively. The K
m
2
3.11. Effect of CaCl on milk-clotting activity
at P1 position, was higher than with Leu and Glu at the same posi-
tion. These results indicated that the enzyme preferred a small and
non-polar residue at the P1 position to a charged 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 (Ahmed et al., 2009a, 2009b). The kinetic
Calcium has been described as an important substance in clot
formation during milk clotting, which happens when its concentra-
tion is high enough (Anema, Lee, & Klostermeyer, 2005). Calcium
helps coagulation by creating isoeletric conditions and by acting
as a bridge between casein micelles. Milk-clotting activity of religi-
parameters and specificity constant (kcat/K
m
) values of religiosin
2
osin B was highest at 0.2–0.3 M of CaCl . It is known that calcium
B with various synthetic substrates are shown in table 3.
has important function on casein aggregation during the second

M. Kumari et al. / Food Chemistry 131 (2012) 1295–1303
1301
100
1
00
7
5
7
5
2
5
0
5
0
50
25
A
B
0
0
2
4
6
8
10
12
20
40
60
80
pH
Temperature (
°
C)
100
C
D
100
7
5
2
5
7
5
0
5
0
50
25
0
4
6
8
10
12
25
50
Temperature (°C)
75
pH
Fig. 3. Effect of pH (A) and temperature (B) on the proteolytic activity (d) and stability (s) of religiosin B. Likewise, effect of pH (C) and temperature (D) on the milk-clotting
activity (d) and stability (s) of religiosin B. The assay protocols are described in the material and method section. Each value in all figures represented as mean ± SD (n = 3).
Table 3
OD 660 nm, comparable to those of (387 ± 12.67, 4989 ± 109.771,
367 ± 5.90, 3.6 ± 0.025, and 393 ± 6.12 U/OD 660 nm) religiosin,
rennin, papain, trypain and ficin, respectively (Kumari et al.
Kinetic parameters of religiosin B with different synthetic substrates.
cat (sꢀ1)
ꢀ1
ꢀ1
Substrate
K
m
(mM)
k
k
cat/K
m
(mM
s )
2
010). The ratio of milk-clotting activity to proteolytic activity is
Leu-pNA
Ala-pNA
Ala-Ala-pNA
0.928 ± 0.005
0.038 ± 0.001
0.052 ± 0.002
1.320 ± 0.020
64.81 ± 1.30
44.21 ± 2.42
43.64 ± 1.89
64.80 ± 2.10
69.85 ± 3.24
a useful indicator of the protease efficiency to be used as a coagu-
lant for cheese making (Arima et al., 1970). The capacity of religi-
osin B to produce milk curds together with its high ratio of milk
clotting to proteolytic activity, could make it useful 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.
1163.42 ± 34.66
837.76 ± 26.71
49.05 ± 4.13
c
-Glu-pNA
All values in the table are represented as mean ± SD (n = 3).
step (non-enzymatic) of milk clotting, caused by the neutralisation
of casein micelles’ negative residues (phosphoserine and carbox-
2
+
ylic groups) by Ca and calcium–phosphate complexes (Pires,
Orellana, & Gatti, 1999) and so that an increase in its concentration
leads to an increase in the coagulation rate (Arima et al., 1970;
Kumar, Sharma, Saharan, & Singh, 2005). Milk-clotting activity of
religiosin B decreased at concentrations higher than 0.3 M, probably
due to the increase of ionic force or to the saturation of negative
residues, of the micelles at increasing Ca2 concentration in the
medium. A similar behaviour was reported by Ahmed, Babikar, &
Mori (2010) and Preetha and Boopathy (1997), where the purified
proteases showed the maximum milk-clotting activity in the pres-
3
.13. Effect of pH and temperature on milk-clotting activity of
religiosin B
Highest milk-clotting activity of religiosin B was observed at pH
.0 and temperature 55–60 °C (Fig. 3C and D). Similar behaviour of
6
+
optimum pH was reported for the crude extract from Mucor pusil-
lus var. Lindt (Arima et al., 1970) and the precipitated extract from
Penicillium citrinum Abdel-Fattah, Mabrouk, & El-Hawwary, 1972),
at pH 5.5 and pH 6.0, respectively. Calf rennet exhibits the same
dependence towards pH, being weaker in alkaline conditions than
in acidic conditions (Richardson, Nelson, Lubnow, & Schwarberg,
1967). The similar temperature optimum (60 °C) was observed
in the case of the crude enzymatic extract from Penicillium
oxalicum (Hashem, 1999), the precipitated extract from P. citrinum
(Abdel-Fattah et al., 1972), and the purified protease from Rhizopus
oryzae (Kumar et al., 2005).
ence of 0.2 and 0.4 M of CaCl
.12. Milk coagulation
The enzyme coagulates skimmed milk and forms a white and
2
, respectively.
3
firm curd. Moreover, the ratio of milk-clotting activity to proteo-
lytic activity of religiosin B is determined to be 803.22 ± 23.67 U/

1
302
M. Kumari et al. / Food Chemistry 131 (2012) 1295–1303
The enzyme remains stable and shows more than 80% milk-
clotting activity in the range of pH 5–9 and temperature from
0–60 °C. On both sides of the pH range, the stability decreases
the form of a research fellowship from CSIR (Council of Scientific &
Industrial Research) Government of India is gratefully
acknowledged.
3
and at pH 10 the activity reduces to 40%. Similarly at 65 °C the sta-
bility of the enzyme reduces to 50% only. These observations con-
firm the high stability of the enzyme over a broad range of
temperature and pH. In this regard, the isolated enzyme is unique,
and therefore, might be suitable for uses in industry under alkaline
conditions. These characteristics are important, because most en-
zymes are catalytically unstable at alkaline pH values, thus limiting
their usefulness in the food industry especially as cheese-making
coagulants (Lamas, Barros, Balcao, & Malcata, 2001). 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 successfully for the manufacture of tradi-
tional cheeses from ovine and caprine milk (Sousa & Malcata,
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KLVMWSGMR, KGMGPWPGSAR, KFGGPVQLQW, DPFFEHPSADVR
and CHSVVPR. Due to the low intensity and weak MS/MS spectra
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and fragmentation of the peptides. These sequences when sub-
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4
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The present study describes the purification and characterisa-
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of a valuable medicinal plant, F. religiosa. We have optimised a sim-
ple and economic purification procedure, combined with the avail-
ability of the plant latex, could possibly be used for large-scale
production of the enzyme. The enzyme is stable in the presence
of denaturants, organic solvents, and metal ions as well as over a
wide range of temperature and pH; therefore this protease may
turn out to be an efficient choice in food, pharmaceutical, and bio-
technological industries. The enzyme is resistant to autolysis and
can be stored at low temperature for long time, without loss of
activity. Besides, high milk-clotting activity of the enzyme could
pave the way for its use as a potential cheese making enzyme from
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5
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