Decolorization of crude latex by activated charcoal, purification and physico-chemical characterization of religiosin, a milk-clotting serine protease from the latex of ficus religiosa

Article abstract of DOI:10.1021/jf101020u

The crude latex of Flcus religiosa is decolorized by activated charcoal. Decolorization follows the Freundlich and Langmuir equations. A serine protease, named religiosin, has been purified to homogeneity from the decolorized latex using anion exchange ch

Full text of DOI:10.1021/jf101020u

J. Agric. Food Chem. 2010, 58, 8027–8034 8027
DOI:10.1021/jf101020u
Decolorization of Crude Latex by Activated Charcoal,
Purification and Physico-Chemical Characterization
of Religiosin, a Milk-Clotting Serine Protease
from the Latex of Ficus religiosa
MONI
KUMARI, ANURAG
S
HARMA
, AND M. V. JAGANNADHAM*
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India
The crude latex of Ficus religiosa is decolorized by activated charcoal. Decolorization follows the
Freundlich and Langmuir equations. A serine protease, named religiosin, has been purified to
homogeneity from the decolorized latex using anion exchange chromatography. Religiosin is a
glycoprotein with a molecular mass of 43.4 kDa by MALDI-TOF. Religiosin is an acidic protein with a
pI value of 3.8 and acts optimally at pH 8.0-8.5 and temperature 50 °C. The proteolytic activity of
religiosin is strongly inhibited by PMSF and chymostatin indicating that the enzyme is a serine
protease. The extinction coefficient (ε 280) of religiosin is 29.47 M-1 cm-1with 16 tryptophan,
1%
2
6 tyrosine, and 11 cysteine residues per molecule. The enzyme shows broad substrate specificity
against natural as well as synthetic substrates with an apparent Km of 0.066 mM and 6.25 mM using
casein and Leu-pNA, respectively. MS/MS analysis confirms the novelty of the enzyme. Religiosin is
highly stable against denaturants, metal ions, and detergents as well as over a wide range of pH
and temperature. In addition, the enzyme exhibits milk-clotting as well as detergent activity.
KEYWORDS: Activated charcoal; Ficus religiosa; milk-clotting enzyme; religiosin; serine protease
INTRODUCTION
specificities and activities over a wide range of pH, temperature,
and other denaturing conditions (4). High stability is generally
considered as an economic advantage because of reduced enzyme
turnover. In addition, stable enzymes permit the use of high
process temperatures, which usually have a beneficial effect on
reaction rate, reactant solubility, and the risk of microbial
contaminations (5, 6). In the food industry, proteases are also
used for the coagulation of milk in the cheese industry. However,
the calf rennet used for cheese production is a relatively expensive
enzyme due to the limited availability and ethical considerations
associated with its use; therefore, the search for new enzymes
from other sources still continues to make it industrially applic-
able and cost-effective (7). In the detergent industry, proteases are
used to improve the cleaning efficiency of laundry detergents.
Nonenzymatic detergents are sometimes inefficient in removing
protein-based stains because of their formulation for degrading
mainly oil and grease. The addition of proteases leads to the
distinctly better cleansing power of detergents through hydrolytic
degradation of proteinaceous stains such as blood, egg-yolk,
chocolate, etc.
Plant latex is a complex emulsion of proteins, alkaloids,
starches, sugars, oils, tannins, resins and gums etc. In most of
the plants, the latex is white, but some have yellow, orange, or
scarlet latex due to the presence of some organic compounds.
Some melanin-like compounds present in the latex of the genus
Ficus impart brown color to the latex. The melanin-like com-
pounds bind to the proteins and prevent the adsorption of the
proteins to the matrix of the ion exchanger, thus posing problems
during the purification process. Besides, the retained color after
purification hinders the precise absorbance measurements of pure
protein. The removal of colored compounds by solvent partition-
ing, dialysis, and diafiltration is not always practical; these
methods are often tedious, time-consuming, and expensive (1).
Activated charcoal, the most widely used adsorbent for organic
compounds, has many applications in isolation and purification
of biomolecules from crude fermentation broths (2). In this view,
a simple and inexpensive process for the decolorization of the
crude latex of Ficus religiosa was optimized using activated
charcoal.
Plant lattices yielded a number of industrially important
proteases such as papain, bromelain, ficin, and calotropins (8,9).
Recently, the feasibility of using chemically modified papain as a
proteolytic enzyme component in detergents was explored and
proved to be an inexpensive alternative to the microbial alkaline
proteases used in the detergent industry (10).
Proteolytic enzymes regulate protein processing and intracel-
lular protein levels by removing abnormal and damaged proteins
from the cell as well as play a defensive role against herbivores,
insects, and pests (3). In addition to their biological roles,
proteases have also been exploited commercially in food, leather,
textile, and detergent industries because of their broad substrate
Serine proteases are one of the largest groups of proteolytic
enzymes involved in various physiological and regulatory pro-
cesses. In recent years, several serine proteases have been purified
*
Corresponding author. Phone: þ91-542-2367936. Fax: þ91-542-
2
37568. E-mail: jvm@bhu.ac.in.
©
2010 American Chemical Society
Published on Web 06/18/2010
pubs.acs.org/JAFC

8028 J. Agric. Food Chem., Vol. 58, No. 13, 2010
Kumari et al.
and characterized from different parts of the plants including
seeds, latex, and fruits (11). Unlike cysteine proteases, serine
proteases do not require oxidizing and chelating agents for their
function. Therefore, serine proteases could bemoreadvantageous
than cysteine proteases for industrial applications (12).
The Langmuir Equation. The equation for the adsorption isotherm is:
a ¼ ABcꢁ=1 þ Acꢁ
ð2Þ
where a is the amount of solute adsorbed per unit weight of adsorbent
= v(co-c*)] (from eq 1); A and B are the constants depending on the
[
F. religiosa, commonly known as pipal, is a large glabrous
evergreen tree that belongs to the family Moraceae. It is widely
distributed in the tropical areas of the world. F. religiosa is a
medicinal plant, and the different parts of the plant have been
used for years in the preparation of traditional and ayurvedic
medicines. The bark of F. religiosa shows antitumor and anti-
microbial activities and helps in the treatment of asthma, di-
arrhea, gonorrhea, ulcers, scabies, hiccups, and vomiting (13). In
view of the several medicinal applications of F. religiosa, explora-
tion of biochemical constituents of the plant is worthwhile and
was attempted in our laboratory. In the course of screening for
biochemical constituents of the plant, high proteolytic activity
was found in the latex; in view of this, a serine protease was
purified and named as religiosin, following the nomenclature of
proteases.
properties of the adsorbent and the solute being adsorbed. A plot of 1/c*
versus1/a is a straight line.
Purification of the Enzyme. All of the experiments of purification
were carried out at 4 °C unless stated otherwise. The decolorized crude
latex obtained from the previous step was subjected to anion exchange
chromatography on a DEAE-Sepharose fast flow column pre-equilibrated
with 0.01 M Tris-HCl buffer at pH 8.0. The column was washed
thoroughly with the same buffer until no protein was seen in the eluate.
The bound proteins were eluted with a linear salt gradient of 0-0.6 M
NaCl. The eluted fractions were monitored for protein content by
absorbance at 280 nm and assayed for proteolytic activity with casein as
a substrate. SDS-PAGE was used for the assessment of homogeneity and
the intactness of all of the fractions.
Protein Concentration. Protein concentration was determined by
absorbance at 280 nm as well as by Bradford’s method (15).
Electrophoresis and Zymography. Homogeneity, intactness, and
r
molecular mass (M ) of the purified enzyme were determined by 15%
SDS-PAGE under reducing and nonreducing conditions as described by
Laemmli (16). After electrophoresis, proteins in the gel were stained by
coomassie R-250. Zymography was performed to confirm the proteolytic
activity of religiosin in the gel using the protocol of Tomar et al (17). After
electrophoresis, protein in the gel was stained by coomassie G-250.
Activity Measurements. The proteolytic activity of religiosin was
monitored using natural substrates casein and hemoglobin. Ten micro-
grams of the enzyme equilibrated in 0.5 mL of 0.05 M Tris-HCl buffer, pH
MATERIALS AND METHODS
Acetonitrile, acrylamide, activated charcoal, bovine serum albumin,
BSA, casein, chymostatin, coomassie brilliant blue, DEAE-Sepharose
fast flow, DTNB, DTT, EDTA, EGTA, glycerol, GuHCl, hemoglobin,
2
hen egg white lysozyme, HgCl , IAA, N,N-methylene bis-acrylamide,
o-phenanthroline, papain, PMSF, rennin, ribonuclease A, SBTI, TFA,
trypsin, urea, β-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.
Decolorization of Crude Latex by Activated Charcoal. All of the
experiments of decolorization were carried out at 30 °C unless stated
otherwise.
8
.0, at 37 °C for 10 min, was added to 0.5 mL of 1% substrate in the same
buffer, and the reaction was allowed to proceed at 37 °C for 30 min. The
reaction was terminated by adding 0.5 mL of 10% TCA and kept for
1
0 min. The resultant precipitate was removed by centrifugation, and the
TCA-soluble peptides were measured by absorbance at 280 nm. A control
assay without enzyme in the reaction mixture was used as a blank. One unit
of enzyme activity is defined as the amount of enzyme that gives rise to an
increase in one unit of absorbency at 280 nm per min of casein digestion,
under the standard assay conditions. The specific activity is the number of
units of activity per milligram of protein.
Isoelectric Focusing. The isoelectric point (pI) of the purified enzyme
was determined by isoelectric focusing on polyacrylamide disc gel as
described by Kunduet al (18). The electrophoretic run was carried out with
ampholine carrier ampholytes in the pH range 3-6, 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.
Step 1: Gum Removal. Fresh latex of Ficus religiosa was collected from
stems in 0.01 M Tris-HCl buffer at pH 8.0 and frozen at -20 °C for 48 h.
The frozen latex was thawed to room temperature and centrifuged at
2
4000 ꢀ g for 10 min to remove any insoluble debris. The resulting clear
supernatant was termed crude latex and used for decolorization.
Step 2: Decolorization. One milliliter of the crude latex (c is equal to
.32) was mixed with 20 mg of activated charcoal and kept on a shaker for
o
1
1
0 min, followed by centrifugation at 7200 ꢀ g for 10 min. The color intensity
of the clear supernatant was measured by absorbance at 470 nm (14). The
protein content and activity of the enzyme werealso analyzed. The remain-
ing color intensity after decolorization was calculated using the following
formula:
Glycostaining and Carbohydrate Content. The glycostaining was
performed to confirm the presence of the carbohydrate moiety of the
protein molecule in the gel using Schiff’s reagent. After SDS-PAGE, the
gel was fixed in the mixture of 40% methanol and 20% acetic acid followed
by reswelling in 7% acetic acid. Furthermore, the gel was kept in an
oxidizing mixture of 1% periodic acid in 7% acetic acid in the dark for 1 h.
Subsequently, the gel was washed in 7% acetic acid with several changes
for 24 h. Finally, the gel was stained with Schiff’s reagent at4 °C in the dark
for 1 h and differentiated by 1% sodium metabisulfite in 0.1 M HCl. The
amount of carbohydrate content in the enzyme molecule was estimated by
the phenol sulfuric acid method as described by Sharma et al (12).
Substrate Specificity. The amidolytic activities of the enzyme were
Remaining color intensityð%Þ ¼ ðcꢁ=c
o
Þ ꢀ 100
where c
o
and c* are the absorbance of crude and decolorized latex,
respectively. (c is the initial solute concentration before adsorption, and
o
c* is the equilibrium solute concentration after adsorption. Here, the
solute is the color.)
Optimization of Conditions for Decolorization. Decolorization of crude
latex was studied as a function of initial color intensity (c , 2.38 to 0.89),
o
amount of charcoal (5 to 50 mg/mL), contact time (5 to 40 min), and
temperature (20 to 50 °C). All of the experiments were carried out as
described in the decolorization step.
studied using NR-benzoyl-DL-arginine-p-nitroanilide (BAPNA),
p-nitroanilide, -alanine alanine-p-nitroanilide, -leucine-p-nitroanilide,
N-succinyl- -phenylalanine-p-nitroanilide, and -γ-glutamyl-p-nitroani-
lide by the method of Arnon (19) with some modifications. The synthetic
substrates (5-20 mM) were prepared by dissolving the required amount
in a minimum volume of DMSO and making up the final volume with
L-alanine-
Models. The Freundlich and Langmuir models were tested for decolor-
ization to validate the adsorption by activated charcoal.
Freundlich Equation. The Freundlich equation, for the adsorption of
solutes from dilute solution is:
L
L
L
L
n
cꢁ ¼ k½vðco - cꢁÞꢂ
ð1Þ
0
.05 M Tris-HCl buffer, pH 8.0, at 30 °C. The enzyme was incubated with
where v is the latex volume to charcoal weight ratio; [v(co-c*)] is the
apparent adsorption per unit weight of adsorbent; k and n are the
constants. The value of n is the indicator of adsorption characteristics.
A plot of ln c* versus ln [v(co-c*)] is a straight line.
the assay buffer and synthetic substrate at 37 °C for 30 min. The reaction
was terminated by the addition of 0.2 mL of 30% acetic acid, and the
liberated p-nitroaniline (ε = 8800 M- cm at 410 nm) was monitored by
1
-1
absorbance at 410 nm, against a reaction blank without the enzyme (20).

Article
J. Agric. Food Chem., Vol. 58, No. 13, 2010 8029
pH and Temperature Optima. The activity of religiosin was mea-
sured at different pH values and temperatures. The optimum pH of the
enzyme was determined by measuring casein-hydrolyzing activity from pH
1.0 to 12.0. Hemoglobin was used as a substrate below pH 4.0 due to the
insolubility of casein at lower pH value. A control assay at the same pH
without enzyme in the reaction mixture was used as a blank. The effect of
temperature on the activity of purified enzyme was also investigated. The
enzyme was incubated at the desired temperature (20-90 °C) for 15 min,
and the activity was measured at the same temperature value.
Stability. The stability of an enzyme dictates its applicability; there-
fore, the effect of pH (1.0-12.0), temperature (20-90 °C), as well as
different concentrations of denaturants such as organic solutes, organic
solvents, detergents, and metal ions on the proteolytic activity of religiosin
was examined. The enzyme was incubated under the specified condition of
pH, organic solutes, 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.
Effect of Inhibitors. In order to classify the enzyme, the effect of
protease inhibitors on the activity of religiosin was studied. The inhibitors
2
used were PMSF, chymostatin, SBTI, HgCl , IAA, leupeptin, EDTA,
EGTA, and o-phenanthroline. The enzyme was incubated with increasing
concentrations of a specific inhibitor (0-50 mM) at 37 °C for 30 min, and
the residual proteolytic activity was assayed as described above. A control
assay without inhibitor was performed, and the activity was considered
as 100%.
Effect of Substrate Concentration. The effect of increasing substrate
concentration on the velocity of the enzyme-catalyzed reaction was studied
using natural as well as synthetic substrates at pH 8.0 and 37 °C. The
concentrations of the natural substrate (casein) and the synthetic substrate
(L-leucine-p-nitroanilide) were studied in the range of 20-450 μM and
5-20 mM, respectively. The enzyme activity was measured under the
standard assay conditions as described above. A Lineweaver-Burk plot
m
was drawn, and the value of the Michaelis constant, K , was calculated as
described by Segel (21).
Estimation of Specific Amino Acid Contents and Extinction
Coefficient. Total numbers of the tryptophan and tyrosine residues in
religiosin were measured spectrophotometrically as described by Goodwin
and Morton (22). The free and total cysteine residues of the enzyme were
estimated by Ellman’s method (23). For the free cysteine content estima-
tion, the enzyme was reduced with 0.01 M β-ME, whereas for the of total
cysteine content, estimation the enzyme was denatured in 6 M GuHCl and
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 the 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. The extinction
coefficient of the enzyme was determined by the spectrophotometric
method as described by Aitken and Learmonth (24).
Peptide Mass Fingerprinting/Mass Spectrometric Sequencing.
Tryptic digestion of religiosin was done using the protocol as described by
Shevchenko et al (25) with minor modifications. After SDS-PAGE, gel
Figure 1. Decolorization of the crude latex. (A) Effect of the amount of
activated charcoal on remaining color intensity (b) and protease recovery
(O). (B) Fitting of the decolorization data to the Freundlich equation.
(C) Fitting of the decolorization data to the Langmuir equation. Values in all
of the figures are represented as the mean ( SD (n = 3).
pieces were excised, destained, washed, dehydrated in CH
in a vacuum centrifuge. The gel pieces were cooled on ice and soaked
in digestion buffer containing 50 mM NH HCO , 5 mM CaCl , and
2.5 ng/μL of trypsin (Promega, sequencing grade) overnight at 37 °C. The
3
CN, and dried
4
3
2
1
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 R-cyano-4-
hydroxy-cinnamic acid in 0.1% TFA and 50% acetonitrile) and spotted
on a 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).
Milk-Clotting Activity and Proteinaceous Stain Removal Study.
The milk-clotting activity of religiosin was determined according to the
method described by Arima et al (26). One milk-clotting unit was defined
as the amount of enzyme that coagulates 10 mL of the substrate in 40 min.
For the proteinaceous stain removal study, clean cotton cloth pieces
The soiled cloth pieces were incubated with the 1% detergent and 0.02%
enzyme solution for 5 h. Aliquots of 5 mL of washing liquid were taken out
periodically, and the color intensity was measured by absorbance at
2
80 nm until the change in the color intensity leveled off. Stained-cloths
of the same size soaked in 1% detergent without enzyme were used as the
control. Commercial detergents Surf Excel (Hindustan Lever, India) and
Ariel (Procter and Gamble, India) were used as references for this study.
RESULTS AND DISCUSSION
Decolorization of Crude Latex by Activated Charcoal. The rate
(10 cm ꢀ10 cm) were soiled with blood and chocolate and allowed to dry.
of decolorization depends on the degree of adsorption, temperature,

8030 J. Agric. Food Chem., Vol. 58, No. 13, 2010
Kumari et al.
Table 1. Purification of Religiosin from the Latex of F. religiosa
a
total activity (units)
steps
total protein (mg)
specific activity (units/mg)
yield (%)
purification fold
crude latex
decolorized latex
DEAE- Sepharose
120
80
3000
2406
1440
25
100
80.2
48
1.00
1.20
2.3
30.07
57.6
25
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.
the protein are judged by SDS-PAGE under reducing and
nonreducing conditions as shown in Figure 3A. The single band
on SDS-PAGE suggests the monomeric nature of the enzyme.
The results of purification are summarized in Table 1.
Physical Properties of Religiosin. The molecular mass (M ) of
r
the purified enzyme is 43 kDa by both SDS-PAGE and MAL-
DI-TOF (Figure 3A and B). The molecular mass (M ) of the
r
majority of plant serine proteases lies in the range of 60-80 kDa,
and the molecular mass of religiosin falls below the range (11).
Gelatin zymography confirms the proteolytic nature of the
enzyme in the gel (Figure 3C). Religiosin shows a single band in
isoelectric focusing with an approximate isoelectric point (pI) of
pH 3.8 (Figure 3D). Most of the plant serine proteases have
isoelectric points in the range of pH 4.0-7.0 and rarely have highly
acidic pI values (11). The pI value of religiosin is lower than those
of the other plant proteases as compared in Table 2. Religiosin is a
glycoprotein with 12% carbohydrate content. Furthermore,
Schiff’s staining confirms the presence of the carbohydrate
moiety of the protein molecule in the gel (Figure 3E). The protein
band seems diffused and wider in SDS-PAGE gel due to the
intense differential glycosylation, a characteristic feature of
glycoproteins. Similar observations were reported in the case of
benghalensin (12). The deglycosylation of religiosin by TFMS
results in the complete loss of proteolytic activity as no band
appeared in the zymogram and reflects that the carbohydrate
moiety may be the part of the functional architecture of the
enzyme. The biochemical properties of religiosin are compared
with those of the other known serine proteases in Table 2.
Substrate Specificity. The enzyme hydrolyzes denatured na-
tural substrates such as casein and hemoglobin. Religiosin
exhibits significant hydrolyzing activity against synthetic sub-
Figure 2. Elution of the crude latex on anion exchange chromatography.
DEAE-Sepharose fast flow, pre-equilibrated with 10 mM Tris buffer, pH
8.0. The bound proteins were eluted with a linear salt gradient of 0-0.6 M
NaCl. Fractions of 5 mL volume at a flow rate of 5 mL/min were collected
and assayed for protein concentration (O) and proteolytic activity (b). The
fractions of the ascending limb of pool II (75-95) were pooled as indicated
by the horizontal line.
contact time, and amount of charcoal used, as shown in
Figure 1A. The optimum amount of charcoal is found to be
2
0 mg/mL of the crude latex (c is equal to 1.32) that helps to
o
achieve 90% decolorization and 80% of protease recovery. The
optimum contact time and temperature for decolorization are
found to be 10 min and 30 °C, respectively, without appreciable
loss of the enzymatic activity. The rate of adsorption of colored
solutes increases with increasing contact time as well as tempera-
ture and attains a constant value when equilibrium is established.
Similar results were also observed in the case of yellow zein (27).
In addition to decolorization, activated carbon helps in partial
purification of religiosin indicated by increased specific activity
and fold purification as presented in Table 1.
strates such as
troanilide, and
NR-benzoyl-DL-arginine-p-nitroanilide, N-succinyl-
nine-p-nitroanilide, and
L
-alanine-p-nitroanilide,
-leucine-p-nitroanilide while failing to hydrolyze
-phenylala-
-γ-glutamyl-p-nitroanilide. Thus, reli-
L
-alanine-alanine-p-ni-
L
L
L
giosin shows broad substrate specificity, giving preferences to
small and nonpolar amino acids at the P position, whereas the
1
activity of enzyme over polar and bulky hydrophobic groups at
Models. From Figure 1, v, c*, and v (c c*) were calculated.
position P is not detectable. Thus, the P specificity of religiosin
1 1
o-
The linear plot of ln c* versus ln [v(c c*)] testifies that the
adsorption characteristics with the Freundlich constant n equal to
is somewhat similar to that of cucumisin and differs from that of
subtilisin, trypsin, and R-chymotrypsin, confirming distinct sub-
strate specificity for religiosin (11, 28).
pH and Temperature Optima. Religiosin retains at least 50% of
the proteolytic activity from pH 6.0-10.0 and in the temperature
range of 35-65 °C. The pH and temperature optima for the
activity of religiosin are pH 8.0-8.5 and 50 °C, respectively
(Figure 4A and B). The pH and temperature optima of religiosin
are compared with those of other serine proteases and shown in
Table 2.
Stability. Religiosin retains more than 80% of the activity
from pH 5.5-10.0 and in the temperature range of 20-65 °C
(Figure 4A and B). Religiosin shows an increase in proteolytic
activity at lower concentrations of GuHCl up to 2.0 M. The
increase of proteolytic activity of the enzyme is about 2-fold at
2.0 M GuHCl, which is not found when the enzyme is treated with
urea. Religiosin retains full activity at higher concentrations of
o-
2
.6 ( 0.012 validates the adsorption by activated charcoal as
shown in Figure 1B. Also, the linear plot of 1/c* versus 1/v (c c*)
o-
confirms the validity of the Langmuir model (Figure 1C).
Purification of the Enzyme. A new serine protease is purified to
homogeneity from the decolorized latex of Ficus religiosa using
anion exchange chromatography. The protein elution profile
resolves into three peaks (Figure 2). The proteolytic activity is
observed only in one peak (peak II). The ascending limb of peak
II (fractions 75-95) is found to be homogeneous and resulted in a
single band by SDS-PAGE. The pure enzyme is named as
religiosin according to protease nomenclature. The enzyme is
purified up to 2.3-fold with 48% yield and a specific activity of
57.6 U/mg. The purification protocol is simple and highly
reproducible in terms of yield and specific activity of the enzyme
within the experimental errors. Homogeneity and intactness of

Article
J. Agric. Food Chem., Vol. 58, No. 13, 2010 8031
Figure 3. (A) Assessment of homogeneity and molecular weight of religiosin by 15% SDS-PAGE. Gel electrophorized lanes 1-4 represent the marker,
religiosin (30 μg), under reducing and nonreducing conditions and the crude latex, respectively. (B) Religiosin (10 ng) was used for MALDI-TOF analysis.
(
C) Zymogram (in-gel activity) of religiosin. The clear region in the gel (indicated by the double head arrow) shows the hydrolysis of gelatin by the enzyme.
D) Glycostaining by Schiff’s reagent. The magenta color (indicated by the double head arrow) confirms the presence of the carbohydrate moiety in the protein
(
molecule. (E) Isoelectric focusing was performed by 5% polyacrylamide disk gel electrophoresis with the ampholine carrier ampholyte, pH 3.0-6.0, at a
constant current of 5 mA. The isoelectric point of the purified protein is indicated by an arrow.
Table 2. Comparison of Biochemical Properties of Religiosin with Other Known Plant Serine Proteases
enzyme
plant
mol. mass (kDa)
pH optimum
temperature optimum (°C)
isoelecric point (pI )
religiosin
benghalensin
F. religiosa
F. benghalensis
C. melo L
43.3
47
8.0-8.5
8.0
7.1
50
3.8
4.4
a
50
b
i
cucumisin
c
54.0
59.0
67.0
50.5
134.3
80.24
72.0
70
NR
RSIP
taraxilisin
Z. mays L
6.0-6.5
8.0
NR
40
4.55
4.5
6.0
4.8
5.6
4.6
d
T. officinale
C. buchanani
M. indica
e
cryptolepain
f
indicain
8.0-10.5
8.5
6.5
70-75
80
g
carnein
h
KLSP
I. carnea
P. vulgaris
65
9.9
60
a
b
c
d
e
f
g
h
Ref 12. Ref 31. Ref 32. Ref 33. Ref 34. Ref 35. Ref 36. Ref 37. NR represents the data not reported.
i
denaturants, up to 3.5 M GuHCl and 7 M urea. The enhanced
activity of religiosin might be due to the denaturant-induced
alteration of tertiary structure and the conformational stability of
the active site. By contrast, religiosin shows a considerable loss of
proteolytic activity at low concentrations of organic solvents,
which may be due to the profound effects of organic solvents on
the structural integrity and catalytic activity of the enzyme as
summarized in Table 3. Besides, religiosin can also be stored for a
longer time at neutral conditions and low temperatures with the
þ
þ
2þ
retention of full activity. Metal ions, such as Na , K , Mg , and
2þ
Ca , do not show any considerable effect on the activity of
religiosin up to 10 mM, as shown in the Table 3. Similar observa-
tions have been reported for other serine proteases, wrightin (17)
and dubiumin (29). The high stability of religiosin, without per-
turbing the enzymatic activity by various agents, could make it
possible to work under mild denaturing conditions.

8032 J. Agric. Food Chem., Vol. 58, No. 13, 2010
Kumari et al.
Table 4. Effect of Inhibitors on the Activity of Religiosin
a
inhibitor
concentration (mM)
residual activity (%)
PMSF
chymostatin
SBTI
1
2
5
5
5
5
5
5
5
6.45 ( 0.17
10.21 ( 0.18
100.02 ( 1.3
100.01 ( 0.04
99.5 ( 0.35
100.05 ( 0.24
98.20 ( 0.11
96.21 ( 0.53
99.09 ( 0.16
HgCl
IAA
2
leupeptin
EDTA
EGTA
o-phenanthroline
a
Residual activities are shown as the mean ( SD (n = 3).
Figure 4. Effect of pH (A) and temperature (B) on the activity (b) and
stability (O) of religiosin. To see the effect of pH on activity, 10 μg of
religiosin was taken at required pH, and the activity was measured, using
casein as substrate at the same pH. For pH stability, the enzyme was
incubated overnight at the required pH, and the residual activity was
measured at 37 °C and pH 8.0. Similarly, for temperature optima, 10 μg of
religiosin was used for the activity assay at the required temperature. For
temperature stability, the enzyme was incubated at the required tempera-
ture for 15 min, and the activity was measured at 37 °C and pH 8.0. Values
in both figures are represented as the mean ( SD (n = 3).
Table 3. Stability of Religiosin under Different Conditions
a
condition
concentration residual activity (%)
pH
pH 6.5-9.5
25-60 °C
3.5 M
7 M
98.57 ( 1.51
96.5 ( 2.90
temperature
GuHCl
urea
Figure 5. Effect of the substrate concentration on the reaction velocity (b)
of religiosin with natural (A) and synthetic (B) substrates. (A) Line-
weaver-Burk plot. The enzyme (10 μg) incubated in 0.5 mL of 0.5 M
Tris-HCl buffer, pH 8.0, was added to 0.5 mL of casein in a concentration
99.32 ( 0.64
100.02 ( 0.34
79.07 ( 0.05
80.03 ( 0.08
70.44 ( 0.12
100.01 ( 0.03
100.02 ( 0.05
methanol
acetonitrile
DMSO
30%
20%
50%
10 mM
1%
m
range of 20-410 μM. Inset: Michaelis-Menten curve. K was calculated
þ
þ
2þ
2þ
)
metal ions (Na , K , Mg , and Ca
detergent (Surf-excel and Arial)
according to the Lineweaver-Burk plot. (B) Lineweaver-Burk plot. The
enzyme (10 μg) incubated in 0.5 mL of 0.5 M Tris-HCl buffer, pH 8.0, was
added to0.5 mL ofLeu-pNAinaconcentrationrangeof 0.25-7.0mM. The
activity was determined under the standard assay conditions, as described
a
Residual activities are shown as the mean ( SD (n = 3).
in the text. Inset: Michaelis-Menten curve. K
the Lineweaver-Burk plot.
m
was calculated according to
Effect of Inhibitors. PMSF and chymostatin inhibit more than
0% of the proteolytic activity of religiosin, while leupeptin,
9
HgCl , IAA, o-phenanthroline, EDTA, and EGTA do not inhibit
enzymatic activity (Table 4) and confirm that religiosin belongs to
serine proteases while failing to inhibit plant serine proteases such
as benghalensin and milin (12, 30).
2
the serine protease class. However, soybean trypsin inhibitor
Effect of Substrate Concentration. The enzyme obeys the
Michaelis-Menten kinetics, and the nature of kinetics is typically
hyperbolic with increasing substrate concentration, natural as
(SBTI) does not alter the proteolytic activity of religiosin. Gen-
erally, SBTI effectively inhibits the activity of bacterial or animal

Article
J. Agric. Food Chem., Vol. 58, No. 13, 2010 8033
Table 5. Ratio of Milk-Clotting Activity to Proteolytic Activity of Religiosin and
Other Proteases
inhibitor; SDS, sodium dodecyl sulfate; TCA, trichloroacetic
acid; TFMS, trifluoromethanesulfonic acid; Tris-HCl, tris-
(hydroxymethyl) aminomethane hydrochloride.
a
milk-clotting activity
(units/mL)
proteolytic activity
(OD 660 nm)
ratio
(units/OD 660 nm)
protease
SAFETY
religiosin
rennin
papain
trypsin
ficin
211 ( 0.21
249 ( 0.500
216 ( 0.14
1.6 ( 0.003
267 ( 0.15
0.54 ( 0.02
0.05 ( 0.001
0.59 ( 0.01
0.44 ( 0.002
0.68 ( 0.012
387 ( 12.67
4989 ( 109.771
367 ( 5.90
3.6 ( 0.025
393 ( 6.12
Acrylamide is a potent neurotoxin and carcinogen, and it was
handled with safety gloves. The handling of phenol and TCA was
done carefully, because of their highly corrosive nature to skin.
All other experiments were carried out with the utmost precau-
tion and care.
a
All data are shown as the mean ( SD (n = 3).
well as synthetic substrates (insets of Figure 5A and B). The value
ACKNOWLEDGMENT
of K , obtained from the Lineweaver-Burk plot, is 0.066 (
m
We thank Dr. Kalpana Bhargava (Peptide and Proteomics
Lab, DIPAS, DRDO, Delhi, India) for assistance with the
MALDI-TOF experiment and fruitful discussions.
0
.02 mM and 6.25 ( 0.10 mM, with casein and Leu-pNA as
substrates, respectively (Figure 5A and B).
Specific Amino Acid Residues and Extinction Coefficients. The
total cysteine residues in the protein are found to be 11 (measured
value 11.34 ( 0.05), out of which 10 (measured value 9.89 ( 0.09)
residues are involved in making five disulfide bridges, and one is
free. The total numbers of tryptophan and tyrosine residues in the
enzyme molecule are estimated to be 16 (measured value 16.42 (
LITERATURE CITED
(
(
(
1) Kumar, C. G.; Parrack, P. Activated charcoal: a versatile decolor-
ization agent for the recovery and purification of alkaline protease.
World J. Microbiol. Biotechnol. 2003, 19, 243–246.
2) Baker, F. S.; Miller, C. E.; Repik, A. J.; Tolles, E. D. Activated
Carbons. In Encyclopedia of Separation Technology; Ruthven, D. M.,
Ed.; Wiley-Interscience: New York, 1997; Vol. 1, pp 72-93.
3) Konno, K.; Hirayama, C.; Nakamura, M.; Tateishi, K.; Tamura, Y.;
Hattori, M.; et al. Papain protects papaya trees from herbivorous
insects: role of cysteine proteases in latex. Plant J. 2004, 37, 370–378.
0.01) and 26 (measured value 26.09 ( 0.10), respectively. The
-1 -1
extinction coefficient of the protein is 29.47 M cm
.
Peptide Mass Fingerprinting Analysis. Attempts were made to
characterize religiosin by peptide mass finger printing. Tryptic-
digested religiosin resolved into a number of peptide fragments.
The peptide masses with high relative intensities were selected for
further mass spectrometry using peptide mass fingerprinting.
Two major peptides with m/z of 1619.82 and 1186.5 were
sequenced by mass spectrometry. The sequences obtained were
submitted to BLAST analysis, and no significant hits were
detected. However, a good score of 54 was obtained with
sequence similarities to ATP dependent protease. This MS/MS
result provides evidence that the purified protease is a new
protein.
(4) Patel, B. K.; Jagannadham, M. V. A high cysteine containing thiol
proteinase from the latex of Ervatamia heyneana: purification and
comparison with ervatamin B and C from Ervatamia coronaria.
J. Agric. Food Chem. 2003, 51, 6326–6334.
(
5) Haki, G. D.; Rakshit, S. K. Developments in industrially important
thermostable enzymes: a review. Bioresour. Technol. 2003, 89, 17–34.
6) Choudhury, D.; Biswas, S.; Roy, S.; Dattagupta, J. K. Improving
thermostability of papain through structure-based protein engineer-
ing. Protein Eng. Des. Sel. 2010, 23, 457–467.
(
(
7) Tavaria, F. K.; Sousa, M. J.; Domingos, A.; Malcata, F. X.;
Bordelius, P.; Clemente, A.; Paris, M. S. Degradation of caseins
from milk of different species by extracts of Centaurea calcitrapa.
J. Agric. Food Chem. 1997, 45, 3760–3765.
Milk-Clotting Activity and Proteinaceous Stain Removal Study.
The enzyme coagulates skimmed milk and forms a white and firm
curd. Moreover, the ratio of milk-clotting activity to proteolytic
activity of religiosin is determined to be 390 ( 12.67 U/OD
(
8) Caffini, N. O.; Lopez, L. M. I.; Natalucci, C. L.; Priolo, N. S.
Proteases of higher plants. General features, physiological roles and
applications. Acta Farm. Bonaerense 1988, 7, 195–213.
660 nm and compared with those of other proteases and shown in
Table 5. The capacity of religiosin to produce milk curds together
with its high ratio of milk clotting to proteolytic activity could
make it useful as a new milk coagulant, although more studies
about the quality of both milk curds and the cheese formed
should be carried out in the future to confirm its usefulness in the
dairy industry.
(
9) Wang, J.; Zhao, M.; Zhao, Q.; Jiang, Y. Antioxidant properties of
papain hydrolysates of wheat gluten in different oxidation systems.
Food Chem. 2007, 101 (4), 1658–1663.
(
10) Khaparde, S. S.; Singhal, R. S. Chemically modified papain for
applications in detergent formulations. Bioresour. Technol. 2001,
78, 1–4.
The enzyme is highly stable in the presence of detergents shown
in Table 3. The addition of religiosin to the nonenzymatic
detergents helps in the removal of proteinaceous stains better
than the detergent alone. Thus, religiosin might be a good choice
for the detergent industry, though it includes a deeper under-
standing of the structure-function relationship of the enzyme as
well as factors influencing cleaning performance.
(11) Antao, C. M.; Malcata, F. X. Plant serine proteases: biochemical,
physiological and molecular features. Plant Physiol. Biochem. 2005,
43, 637–650.
(
12) Sharma, A.; Kumari, M.; Jagannadham, M. V. Benghalensin, a
highly stable serine protease from the latex of medicinal plant Ficus
benghalensis. J. Agric. Food Chem. 2009, 57, 11120–11126.
13) Kapoor, L. D. Handbook of Ayurvedic Medicinal Plants; CRC Press:
Boca Raton, FL, 1990; pp 188.
14) Gutcho, S. J. Microbial Enzyme Production. In Fungal Enzymes;
Gutcho, S. J., Ed.; Noyes Data Corporation: Park Ridge, NJ, 1974;
pp 116-136.
(
(
ABBREVIATIONS
BSA, bovine serum albumin; DEAE, diethylaminoethyle;
DFP, diisopropylfluorophosphate; DMSO, dimethyl sulfoxide;
DTNB, 5,5-μ-dithiobis (2-nitrobenzoic acid); DTT, dithiothrei-
tol; EDTA, ethylene diammine tetraacetic acid; EGTA, ethylene
glycol-bis (R-amino ethyl ether) tetraacetic acid; GuHCl, guani-
dine hydrochloride; HgCl2, mercuric chloride; IAA, iodoacetic
acid; MALDI-TOF, matrix-assisted laser desorption/ioniza-
tion time-of-flight; PAGE, polyacrylamide gel electrophoresis;
PMSF, phenyl-methanesulfonyl fluoride; SBTI, soybean trypsin
(
15) Bradford, M. M. A rapid and sensitive method for quantitation
of microgram quantities of protein utilizing principle of protein-
dye-binding. Anal. Biochem. 1976, 72, 248–254.
(
16) Laemmli, U. K. Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 1970, 227, 680–685.
(17) Tomar, R.; Kumar, R.; Jagannadham, M. V. A stable serine
protease, wrightin, from the latex of the plant Wrightia tinctoria
(Roxb.) R. Br.: purification and biochemical properties. J. Agric.
Food Chem. 2008, 56, 1479–1487.

8034 J. Agric. Food Chem., Vol. 58, No. 13, 2010
Kumari et al.
(
18) Kundu, S.; Sundd, M.; Jagannadham, M. V. Purification and
characterization of a stable cysteine protease ervatamin B, with
two disulfide bridges, from the latex of Ervatamia coronaria. J. Agric.
Food Chem. 2000, 48, 171–179.
(30) Yadav, S. C.; Pande, M.; Jagannadham, M. V. Highly stable
glycosylated serine protease from the medicinal plant Euphorbia
milii. Phytochemistry 2006, 67 (14), 1414–1426.
(31) Yamagata, H.; Ueno, S.; Iwasaki, T. Isolation and characterization of a
possible native cucumisin from developing melon fruits and its limited
autolysis to cucumisin. Agric. Biol. Chem. 1989, 53, 1009–1017.
(32) James, F.; Brouquisse, R.; Suire, C.; Pradet, A.; Raymond, P.
Purification and biochemical characterization of a vacuolar serine
endopeptidase induced by glucose starvation in maize roots. Bio-
chem. J. 1996, 320, 283–292.
(
19) Arnon, R. Papain. Methods Enzymol. 1970, 19, 226–244.
20) Erlanger, B. F.; Kokowsky, N.; Cohen, N. The preparation and
properties of two new chromogenic substrates of trypsin. Arch.
Biochem. Biophys. 1961, 95, 271–278.
(
(
21) Segel, I. H. Enzymes. In Biochemical Calculations, 2nd ed.; John Wiley
and Sons: New York, 1976; pp 208-323.
22) Goodwin, W.; Morton, R. A. The spectrophotometric determi-
(
33) Rudenskaya, G. N.; Bogacheva, A. M.; Preusser, A.; Kuznetsova,
A. V.; Dunaevsky, YaE.; Golovkin, B. N.; et al. Taraxalisin: A serine
proteinase from dandelion Taraxacum officinale Webb s.l. FEBS
Lett. 1998, 437, 237–240.
(
nation of tyrosine and tryptophan in proteins. Biochem. J. 1946,
4
0, 628–632.
23) Ellman, L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959,
2, 70–77.
(
(
(
34) Pande, M.; Dubey, V. K.; Yadav, S. C.; Jagannadham, M. V. A
novel serine protease cryptolepain from Cryptolepis buchanani:
purification and biochemical characterization. J. Agric. Food Chem.
8
24) Aitken, A.; Learmonth, M. Protein Determination by UV Absorp-
tion. In The Protein Protocol Handbook; Walker, J. M., Ed.; Humana
Press: Totowa, NJ, 1997; pp 3-6.
25) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric
sequencing of proteins from silver-stained polyacrylamide gels.
Anal. Chem. 1996, 68, 850–858.
2006, 54, 10141–10150.
(
(
(
35) Singh, V.; Patel, A. K.; Moir, A. J.; Jagannadham, M. V. Indicain, a
(
(
(
dimeric serine protease from Morus indica cv. K2. Phytochemistry
2008, 69, 2110–2119.
36) Patel, A. K.; Singh, V. K.; Jagannadham, M. V. Carnein, a serine
protease from noxious plant weed Ipomoea carnea (morning glory).
J. Agric. Food Chem. 2007, 55, 5809–5818.
37) Popovic, T.; Puizdar, V.; Brzin, J. A novel subtilase from common
bean leaves. FEBS Lett. 2002, 530, 163–168.
26) Arima, K.; Yu, J.; Iwasaki, S. Milk-Clotting Enzyme from Mucor
pusillus var. Lindt. In Methods in Enzymology; Pearlman, E. G.,
Lorand, L., Eds.; Academic Press: New York, 1970; pp 446-459.
27) Sessa, D. J.; Palmquist, D. E. Effect of heat on the adsorption
capacity of an activated carbon for decolorizing/deodorizing yellow
zein. Bioresour. Technol. 2008, 99, 6360–6364.
Received for review March 17, 2010. Revised manuscript received
June 2, 2010. Accepted June 7, 2010. Financial assistance to MK
and AS in the form of a research fellowship from CSIR (Council of
Scientific & Industrial Research), Government of India is gratefully
acknowledged.
(
28) Perona, J. J.; Craik, C. S. Structural basis of substrate specificity in
the serine proteases. Protein Sci. 1995, 4, 337–360.
(
29) Ahmad, I. A. M.; Morishima, I.; Babiker, E. E.; Mori, N. Dubiumin,
a chymotrypsin-like serine protease from the seeds of Solanum
dubium Fresen. Phytochemistry 2009, 70, 483–491.