Appl Biochem Biotechnol (2015) 175:1732–1744

DOI 10.1007/s12010-014-1376-2

A Cysteine Protease Isolated from the Latex of Ficus

microcarpa: Purification and Biochemical
Characterization

Ibtissem Hamza Mnif & Rayda Siala & Rim Nasri &
Samiha Mhamdi & Moncef Nasri & Alya Sellami Kamoun

Received: 6 May 2014 / Accepted: 10 November 2014 /

Published online: 26 November 2014
# Springer Science+Business Media New York 2014

Abstract A plant protease named microcarpain was purified from the latex of Ficus
microcarpa by acetonic (20–40 % saturation) precipitation, Sephadex G-75 filtration, and
Mono Q-Sefinose FF chromatography. The protease was purified with a yield of 9.25 % and a
purification factor of 8. The molecular weight of the microcarpain was estimated to be 20 kDa
by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified
enzyme showed maximum activity at pH 8.0 and at a temperature of 70 °C. Proteolytic
activity was strongly inhibited by dithio-bis-nitrobenzoic acid (DTNB), Hg2+, and Cu2+. The
N-terminal amino acid sequence of the purified microcarpain “VPETVDWRSKGAV” showed
high homology with a protease from Arabidopsis thaliana. Inhibition studies and N-terminal
sequence classified the enzyme as a member of the cysteine peptidases family.

Keywords Ficus microcarpa latex . Cysteine protease . Purification . Microcarpain

Introduction

Proteases refer to a group of enzymes which catalyze the cleavage of peptide bonds in peptides
and proteins. They are also the single class of enzymes that occupy a pivotal position with
respect to their applications in both physiological and commercial fields. They represent one of
the most important groups of industrial enzymes, accounting for about 60 % of the total
enzyme market [1]. The global market enzymes was estimated at around $344 million in 2007
and expected to reach $727 million in 2015 [2]. Proteolytic enzymes could be obtained from
microorganisms, animals, and plants.
Proteases play important roles in plants, such as stress response, mobilization of storage
proteins during germination, induction of effective defense response, senescence, and initiation
of cell death [3]. Plant proteases have been isolated from various parts of plants, including
seeds, stems, fruit, and latex [4]. Although the biological roles of plant latex proteases still

I. H. Mnif (*) : R. Siala : R. Nasri : S. Mhamdi : M. Nasri : A. S. Kamoun

Laboratory of Enzyme Engineering and Microbiology, National School of Engineering of Sfax, University
of Sfax, 1173-3038 Sfax, Tunisia
e-mail: ibtissemhamzamnif@yahoo.com
Appl Biochem Biotechnol (2015) 175:1732–1744 1733

remain speculative, they have been found to participate in defense mechanisms by protecting
ripening fruits against plant pathogens like fungi and insects [5]. Many proteolytic enzymes
have been identified and studied from the latices of several plant families such as
Asclepiadaceae, Apocynaceae, Cariacaceae, Euphorbiaceae, and Moraceae [6].
Most of the plant-derived proteases have been classified as cysteine proteases and more
rarely belong to aspartic proteases [4]. Cysteine proteinases are proteolytic enzymes found in
all eukaryotic organisms. They shall be the first ones to be discussed, since much of the initial
work on plant proteases focused on this class of enzymes, and cysteine proteases are actually
believed to be the predominant class of proteases in plants. The physiological functions
attributed to cysteine proteinases include the buildup and breakdown of storage proteins
during seed germination, organ senescence, and programmed cell death. Proteases also appear
to play key roles in the recognition of pathogens and pests and in the induction of effective
defense responses [7, 8]. Plant latex is a rich source of proteases belonging mainly to the serine
or cysteine family [9, 10]. Latex is a milky fluid composed of a liquid serum, aqueous
suspension or solution, and a complex mixture of molecules [11] like proteins, lipids,
amino acids, vitamins, carbohydrates, terpenes, and alkaloids. The presence of proteases
in the latex suggests that they help plants in the defense against parasites, herbivores, and
pathogens by executing the attack on the invading organisms once the plant cell is lysed
[12]. When plants are injured, latex exudates transiently until a protein clot is formed
around the wounded area. The coagulation process is vital in creating a physical barrier
against predator attack. The latices exhibited several pharmacological properties and are
exploited in folk medicine [13]. Latex from several plant species has been shown to be
involved in hemostatis [14, 15], wound healing, and pain-killing effects [16]. The
screening of the latex of some plants showed very high proteolytic activity. Over 110
latices of different plant families are known to contain at least one proteolytic enzyme.
Most of them belong to the cysteine or serine endopeptidases family and only one to the
aspartic endopeptidases family [17].
Ficus microcarpa is a Moraceae tree, with common names Chinese or Malayan banyan, is a
popular ornamental woody tree grown in many tropical regions of the world. It is native from
Ceylon to India. F. microcarpa is an evergreen tree a height of 15 m or more, with a rounded
dense crown, smooth gray bark, dangling aerial roots, alternate leaves, small size of fruits, tiny
flowers, and milky sap [18].
The objective of this investigation was to purify and characterize a protease from the latex
of F. microcarpa.

Materials and Methods

Reagents

Casein sodium salt from bovine milk, bovine serum albumin, ovalbumin, gelatin, ethylenedi-
aminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), benzamidine, dithio-
bis-nitrobenzoic acid (DTNB), trichloroacetic acid (TCA), and protein markers were pur-
chased from Sigma Chemical Co. (St. Louis, MO). Soybean trypsin inhibitor (SBTI) was
obtained from Fluka Biochemica (USA). Sodium dodecyl sulfate (SDS), acrylamide, and
Coomassie brilliant blue R-250 were from Bio-Rad Laboratories (Mexico City, Mexico).
Sephadex G-75 was from Pharmacia Biotech (Uppsala, Sweden) and Mono Q-Sefinose FF
(Bio Basic). Polyvinylidene difluoride membrane (PVDF) was purchased from Applied
Biosystems (Roissy, France). All other reagents were of analytical grade.
1734 Appl Biochem Biotechnol (2015) 175:1732–1744

Plant Material

The fresh latex was collected by superficial incisions on the trunk of F. microcarpa plants. The
latex was centrifuged at 13,000 rpm for 30 min to remove the gum and other insoluble
materials. The resulting clear liquid phase, termed as the “crude latex,” was conserved at 4 °C
and used for enzyme purification.

Purification of the Crude Latex

All purification steps were carried out at 4 °C to minimize any complication due to possible
autodigestion or unfolding of the enzyme at higher temperature.

Acetone Precipitation

The crude latex was first subjected to acetone precipitation. Acetone fractions of 0–20, 20–40,
40–60, and 60–80 % (v/v) were collected by centrifugation at 13,000 rpm, and the precipitate
obtained in each fraction was air-dried and resuspended in a minimal volume of 100 mM Tris–
HCl buffer (pH 8.0).

Sephadex G-75 Gel Filtration

The acetonic fraction (20–40 %), which showed the highest proteolytic activity, was subjected to gel
filtration on a Sephadex G-75 column (2.5 cm×90 cm) equilibrated with buffer A (25 mM Tris–
HCl, pH 8.0, containing Triton X-100 at 0.5‰). Fractions of 4.5 ml were collected at a flow rate of
30 ml/h with the same buffer. Protein content (absorbance at 280 nm) and proteolytic activity in all
fractions were determined. Fractions showing protease activity were pooled.

Mono Q-Sefinose FF Anion-Exchange Chromatography

The active fractions from Sephadex G-75 gel filtration were pooled and applied to a Mono Q-
Sefinose FF column (2 cm×10 cm) previously equilibrated with buffer B (25 mM Tris–HCl,
pH 8.0). After being washed with the same buffer, bound proteins were eluted with a linear
gradient of NaCl in the range of 0–1 M in buffer B. Fractions of 4.5 ml were collected at a flow
rate of 60 ml/h and analyzed for protease activity and protein concentration. The fractions
showing protease activities were pooled and stored at −20 °C for further analyses.

Protein Concentration

Protein concentration was determined by the method of Bradford [19], using bovine serum
albumin as a standard and during the course of enzyme purification by measuring the
absorbance at 280 nm.

Assay for Proteolytic Activity

Protease activity was measured by the method of Kembhavi et al. [20] using casein as a substrate. A
0.5-ml aliquot of the enzyme, suitably diluted, was mixed with 0.5 ml of 100 mM Tris–HCl buffer
(pH 8.0) containing 1 % (w/v) casein and incubated for 15 min at 70 °C. The reaction was stopped
by the addition of 0.5 ml of TCA (20 %, w/v). The mixture was allowed to stand at room temperature
for 10 min and then centrifuged at 13,000 rpm for 15 min to remove the precipitate. A blank was
Appl Biochem Biotechnol (2015) 175:1732–1744 1735

conducted in the same manner, except that 0.5 ml of 100 mM Tris–HCl buffer (pH 8.0) was used
instead of the enzyme. The absorbance of the supernatant was measured at 280 nm. A standard
curve was generated using solutions of 0–50 mg/l tyrosine. One unit of protease activity was defined
as the amount of enzyme required to liberate 1 μg of tyrosine per minute under the experimental
conditions used. Protease activities represent the means of at least two determinations carried out in
duplicate. The difference between values did not exceed 5 %.

Polyacrylamide Gel Electrophoresis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out for
the control of the purity and the determination of the molecular weight of the purified enzyme,
as described by Laemmli [21], using 5 % (w/v) stacking and 12 % (w/v) separating gels.
Samples were prepared by mixing the purified enzyme at 1:5 (v/v) ratio with the SDS-PAGE
sample buffer (10 mM Tris–HCl (pH 8.0), 2.5 % SDS, 10 % glycerol, 5 % β-mercaptoethanol,
and 0.002 % bromophenol blue). The samples were heated at 100 °C for 5 min before loading
onto the gel. After electrophoresis, the gel was stained with 0.25 % Coomassie brilliant blue R-
250 in 45 % ethanol and 10 % acetic acid, and destained with 5 % ethanol and 7.5 % acetic
acid. The molecular weight of the enzyme was estimated using a low molecular mass
calibration kit as marker. The molecular mass marker consists of phosphorylase b
(97.4 kDa), bovine serum albumin (66.0 kDa), ovalbumin (45.0 kDa), carbonic anhydrase
(29.0 kDa), soybean trypsine inhibitor (20.1 kDa), and lysozyme (14.3 kDa).

Detection of Protease Activity by Zymography

Zymography was performed according to the method of Garcia-Carreno et al. [22]. The sample was
not heated in order to keep the enzyme active. Briefly, after electrophoresis, the gel was submerged
in 100 mM Tris–HCl buffer (pH 8.0) containing Triton X-100 at 2.5 % for 30 min with shaking at
constant agitation to remove SDS. Triton X-100 was then removed by washing the gel three times
with 100 mM Tris–HCl buffer (pH 8.0). The gel was then immersed in 50 ml of 1 % (w/v) casein in
100 mM Tris–HCl buffer (pH 8.0) for 10 min at 4 °C to allow the substrate to penetrate the gel and
then incubated at 60 °C for 20 min with constant agitation to develop the activity zone. Finally, the
gel was stained with 0.25 % Coomassie brilliant blue R-250 in 45 % ethanol and 10 % acetic acid,
and destained with 5 % ethanol and 7.5 % acetic acid. The appearance of clear zones on the blue
background of the gel indicated the presence of protease activity.

Determination of the N-terminal Amino Acid Sequence of the Purified Protease

The purified enzyme, from Mono Q-Sefinose FF anion-exchange chromatography, was

applied to SDS-PAGE and then transferred to a PVDF membrane. The PVDF band corre-
sponding to the protease was excised, and the N-terminal amino acid sequence was determined
by the Edman degradation method on an automated ABI Procise 494 protein sequencer
(Applied Biosystems, Foster City, CA).

Biochemical Properties of the Purified Protease

Effect of pH on Activity and Stability

The optimum pH for the hydrolysis of casein by the purified enzyme was studied over a broad
range of pH from 5.0 to 12.0, at 60 °C for 15 min. To check pH stability, the purified enzyme
1736 Appl Biochem Biotechnol (2015) 175:1732–1744

was incubated for 60 min at 4 °C in different buffers and then the residual proteolytic activities
were determined under standard assay conditions. For this study, the following buffers were
used at 100 mM each: acetate buffer for pH 5.0–6.0, Tris–HCl buffer for pH 7.0–8.0, glycine–
NaOH buffer for pH 9.0–11.0, and Na2HPO4–NaOH buffer for pH 12.0.

Effect of Temperature on the Activity and Stability

The effect of temperature on the protease activity was tested at different temperatures ranging from
30 to 90 °C for 15 min at pH 8.0, using casein as a substrate. The thermal stability was tested by
incubating the enzyme at different temperatures for 1 h, and then the residual activity was measured
under standard assay conditions. The non-heated enzyme was considered as the 100 % control.

Effects of Cysteine and Enzyme Inhibitors

The effects of the addition of cysteine or enzyme inhibitors to the reaction mixture on protease
activity were studied. The inhibitors used were PMSF, benzamidine, β-mercaptoethanol,
DTNB, EDTA, and SBTI. The purified enzyme was preincubated with each inhibitor, at
5 mM (with the exception of 5 mg/ml for SBTI) for 30 min at room temperature, and then the
remaining protease activity was estimated using casein as a substrate. A control assay of the
enzyme activity was done without inhibitors, and the resulting activity measured at pH 8.0 and
70 °C was taken as 100 %.

Effects of Metal Ions on Enzyme Activity

The effects of various metal ions (5 mM) on protease activity were investigated by adding the
monovalent (K+ or Na+) and divalent (Ca2+, Mn2+, Zn2+, Cu2+, Ba2+, Mg2+, or Hg2+) metal
ions to the reaction mixture. The proteolytic activity of the purified enzyme without any
additives was considered as 100 %.

Hydrolysis Specificity

The proteolytic activity was tested using various proteins as substrates (casein, ovalbumin,
BSA, and gelatin). The relative protease activity toward casein was taken as 100 % control.

Results and Discussion

Purification of the Enzyme

A new protease from the latex of F. microcarpa was extracted and purified successively by the
three-step procedure described in the “Materials and Methods” section. The enzyme was
named microcarpain. The results of the purification procedure are summarized in Table 1.
In the first step, the crude enzyme extract was precipitated. The precipitate obtained at 20–
40 % (v/v) acetone showed the highest specific activity (16,491.36 U/mg). At this stage of
purification, the enzyme was purified by 5.57-fold with a recovery of 88.06 % (Table 1). The
precipitate was resuspended in 100 mM Tris–HCl buffer (pH 8.0) and then subjected to
Sephadex G-75 gel filtration. This procedure yielded one peak of protease activity as shown
in Fig. 1a. Active fractions of this peak were pooled and then loaded on a Mono Q-Sefinose FF
anion-exchange chromatography column equilibrated with buffer B. Bound proteins were
Appl Biochem Biotechnol (2015) 175:1732–1744 1737

Table 1 Summary of the purification of the microcarpain

Purification steps Total activity Total protein Specific activity Recovery Purification
(UT) (mg) (U/mg) (%) fold

Crude extract 255,816.33 86.51 2957.07 100 1

Precipitate 20–40 % 225,272 13.66 16,491.36 88.06 5.57
Sephadex G-75 56,504 3.66 15,438.25 25.08 5.22
Mono Q-Sefinose FF 23,676.3 1 23,676.3 9.25 8

All operations were carried out at 4 °C. Proteolytic activity was assayed at pH 8.0 and 70 °C for 15 min using
casein as a substrate

eluted with a linear gradient of NaCl concentrations from 0 to 1.0 M. Protease activity
appeared in a single peak (Fig. 1b). At the final purification step, the microcarpain was

Fig. 1 a Purification profile of the plant protease from the latex of F. microcarpa by gel filtration on Sephadex
G-75 column. The 20–40 % acetone precipitate was resuspended in 100 mM Tris–HCl buffer (pH 8.0) and
applied to a 2.6 cm×90 cm column, equilibrated and eluted with buffer A at a flow rate of 30 ml/h. Fractions
collected from the column were assayed for protein content at 280 nm and protease activity. b Elution profile of
the microcarpain from Mono Q-Sefinose FF column. Active fractions from gel filtration G-75 were collected and
applied to a Mono Q-Sefinose column, equilibrated with buffer B. The enzyme was eluted with a linear gradient
of NaCl (0–1 M) in buffer B at a rate of 70 ml/h
1738 Appl Biochem Biotechnol (2015) 175:1732–1744

purified eightfold, with a recovery of 9.25 % and a specific activity of 23,676.3 U/mg, using
casein as substrate.

Molecular Weight Determination

The purified microcarpain gave a single band on SDS-PAGE confirming that the enzyme is
homogenous. The purified enzyme has a molecular weight of approximately 20 kDa, corre-
sponding to that estimated by Mono Q-Sefinose FF column (Fig. 2a). The purity of the enzyme
was also evaluated using zymogram activity staining. Figure 2b shows a unique clear band of
casein hydrolysis, indicating the purity and the homogeneity of the purified enzyme.
The molecular weight of 20 kDa for the microcarpain was lower than those from other plant
species, such as pergularain e I (23.356 kDa) from the latex of Pergularia extensa [23];
asclepain cII (23.590 kDa), a papain-like protease isolated from the latex of Asclepias
curassavica L. [24]; procerain B (25.7 kDa) from the latex of Calotropis procera [25];
quercifoliain I (26 kDa) from the latex of Vasconcellea quercifolia [26]; and the ginger
protease (36 kDa) from ginger rhizome [27].

N-terminal Amino Acid Sequence of Microcarpain

The N-terminal amino acid sequence of the purified enzyme was determined by the automated
Edman’s method after SDS-PAGE and electroblotting. The sequence of the first 13 residues of
the microcarpain was determined to be VPETVDWRSKGAV. This sequence showed unifor-
mity, indicating that it was isolated in a purified form. The alignment of the N-terminal amino
acid sequence of microcarpain with those of other plant proteases showed the conservation of
the consensus sequences VDWR and KGAV, as well as the proline residue located in position
2 of the N-terminal sequence (Table 2).
The N-terminal sequence of microcarpain was compared with those of some sequences in the
database, and it shared 92 % identity with the cysteine proteinase 1 from Arabidopsis thaliana

Fig. 2 a SDS-PAGE of the puri-

fied microcarpain from the latex of
(a) MM (b)
F. microcarpa. Lane 1, standard (kDa) 1 2
protein marker of different molec-
ular weights; lane 2, purified en-
zyme from Mono Q-Sefinose FF. b
Zymogram detection of proteolytic 66
activity of the purified enzyme
from the latex of F. microcarpa 45
29

20.1

14.2
Appl Biochem Biotechnol (2015) 175:1732–1744 1739

Table 2 Alignment of the N-terminal amino acid sequence of microcarpain, the purified protease from the latex
of F. microcarpa with the sequences of other plant proteases

Conserved amino acids are shown as white characters on a black background

(NCBI accession no. NP 195406.2) and cathepsin L-like protease from Earias vitella (NCBI
accession no. AFP 50672.1). Otherwise, the N-terminal amino acid sequence of microcarpain
showed 76.92 % identity with papaya proteinase omega from Carica papaya [28]; 61.54 % identity
with proteases isolated from Morrenia brachystephana Griseb [29], Funastrum clausum [30], and
Asclepias curassavica L. [24]; 53.85 % identity with endopeptidases from the latex of Morrenia
odorata [31]; and 46.15 % identity with CMS1MS1-B from latex of Carica candamarcensis [32].
These results indicated that the purified microcarpain is a novel protease.

Biochemical Characterization

Effect of pH on Enzyme Activity and Stability

The effect of pH on the activity of the purified microcarpain was studied using casein as a substrate
over a pH range of 5.0–12.0 at 60 °C (Fig. 3a). The purified protease was highly active in the pH
range of 6.0–9.0, with an optimum activity at around pH 8.0. The relative activities at pH 6.0, 7.0,
9.0, and 10.0 were about 81, 94, 83, and 70 %, respectively, of that measured at pH 8.0. Protease
activity decreased significantly at pH 12.0 and was only 21 % of the maximum enzyme activity. The
optimum pH of microcarpain was similar to that reported for eumiliin from Euphorbia milii var.
hislopii latex [10]. Singh et al. [25] reported an optimum activity in a broad range of pH 6.5–8.5 for
procerain B, a protease extracted from the latex of Calotropis procera. The optimum pH of
microcarpain was higher than that of ginger rhizome, which showed maximum activity at pH 5.5
[27], while it was lower than that reported for asclepain cII, a cysteine peptidase of the Asclepias
curassavica latex, which showed maximum activity within the range of pH 9.4–10.2 [24]. The
cysteine peptidase VQ-VII, isolated from the latex of V. quercifolia, had an optimum pH of 8.5 [33].
The pH stability profile of microcarpain reported in Fig. 3b showed that after 1 h of incubation,
the protease retained 51, 45, and 43 % of its original activity at pH 8.0, 7.0, and 9.0, respectively.
However, at pH 6.0 and 12.0, the enzyme retained only 26 and 31 %, respectively.
1740 Appl Biochem Biotechnol (2015) 175:1732–1744

Fig. 3 Effect of pH on activity (a)

and stability (b) of the purified
(a)
microcarpain. The protease activity
was assayed in the pH range of 120
5.0–12.0 at 60 °C. The maximum
100

Relative activity (%)

activity obtained at pH 8.0 was
considered as 100 % activity. The 80
pH stability of the enzyme was 60
determined by incubating the en-
zyme in different buffers for 40
60 min at 4 °C, and the residual 20
activity was measured at pH 8.0
0
and 70 °C. The proteolytic activity
5 6 7 8 9 10 11 12 13
before incubation was taken as pH
100 %. Buffer solutions used for
pH activity and stability are pre-
sented in “Materials and Methods”
(b)
60
Remaining activity (%)

50
40
30
20
10
0
6 7 8 9 10 11 12 13
pH

Effect of Temperature on Enzyme Activity and Stability

The relative activities at various temperatures using casein, as a substrate, are reported in
Fig. 4a. The optimal temperature for the activity of the purified protease was 70 °C, when
incubated for 15 min at pH 8.0. Afterward, the activity declined radically with further increase
in temperature due to thermal denaturation. The relative activities at 60 and 80 °C were about
80 and 40 %, respectively, compared to that at 70 °C. The optimum temperature for
F. microcarpa latex protease was similar to that of Carica papaya L. [34] and higher than
those of capparin from Capparis spinosa [35], plumerin-R from the latex of Plumeria rubra
[36], and procerain, a stable cysteine protease from the latex of Calotropis procera [37], which
had respective optimum temperatures at 60, 55, and 55–60 °C.
To check the effect of temperature on the stability, the protease was preincubated at various
temperatures (40, 50, 60, 70, and 80 °C) for 60 min prior to substrate addition. As shown in
Fig. 4b, the purified microcarpain is highly stable at temperatures below 40 °C, retaining 90 %
of its original activity after 60 min of incubation, while it lost about 60 and 35 % of its initial
activity at 50 and 60 °C, respectively. However, the enzyme activity was highly affected at
higher temperatures. The thermal stability profile of microcarpain was similar to that of the
cysteine protease from wheat, Triticum aestivum (cv. Giza 164) [38].

Effect of Cysteine and Various Inhibitors on the Protease Activity

In order to determine the class of this protease, proteolytic activity was measured after
incubation for 30 min in the presence of cysteine or enzyme inhibitors (Table 3). The activity
of the purified enzyme was completely inhibited by the thiol reagent (DTNB), indicating that
Appl Biochem Biotechnol (2015) 175:1732–1744 1741

Fig. 4 Effect of temperature on

activity (a) and stability (b) of the (a)
purified microcarpain. The tem- 120
perature profile was determined by

Relative activity (%)

assaying protease activity at tem- 100
peratures between 30 and 90 °C. 80
The activity of the enzyme at
70 °C was taken as 100 %. The 60
temperature stability was deter- 40
mined by incubating the purified
20
enzyme at temperature from 40 to
80 °C for 60 min. The residual 0
proteolytic activity was assayed 30 50 70 90 110
under the standard conditions as- Temperature C
say. The non-heated enzyme was
considered as control (100 %) (b)
120 40 °C
Remaining activity (%)

100 50 °C
60 °C
80
70 °C
60
80 °C
40

20

0
0 20 40 60 80
Time (min)

the purified enzyme is a cysteine peptidase. This result was confirmed by the activation of the
proteolytic activity by the addition of β-mercaptoethanol and cysteine. In fact, the purified
microcarpain was activated at a level of 171 %, by the addition of 5 mM cysteine to the
reaction mixture. The activation of proteolytic activity by β-mercaptoethanol and cysteine is in
accordance with several works [39, 38]. Further, the purified enzyme was slightly inhibited by
EDTA, the metalloprotease inhibitor. EDTA inhibited the protease activity by 18 %,

Table 3 Effects of cysteine and various inhibitors on microcarpain activity

Chemical Concentration Residual activity (%)

Control – 100±0
Cysteine 5 mM 171.25±0.92
PMSF 5 mM 25.64±0.34
β-Mercaptoethanol 5 mM 146.93±0.97
EDTA 5 mM 82.08±0.28
Benzamidine 5 mM 62.4±0.7
SBTI 5 mg/ml 71.38±1.95
DTNB 5 mM 0

The residual proteolytic activity was measured after the preincubation of microcarpain with each inhibitor at
room temperature for 60 min. Proteolytic activity measured in the absence of any inhibitor was taken as 100 %
control. All given values are means of three determinations
1742 Appl Biochem Biotechnol (2015) 175:1732–1744

Table 4 Effects of various metal

ions (5 mM) on the purified Chemicals Relative activity (%)
microcarpain
None 100±0
Ca2+ 58.445±0.51
Ba2+ 81.23±0.08
Zn2+ 8.305±0.1
Cu2+ 0
Mg2+ 20.86±0.98
The activity of the purified pro-
tease was determined after incu- Hg2+ 0
bating microcarpain in the pres- Mn2+ 15.125±1.06
ence of various metal ions at Na+ 60.48±0.14
5 mM, for 15 min at 70 °C and K+ 80.115±0.66
pH 8.0

indicating the importance of some metal ions in enzyme stabilization or activity. However,
benzamidine and PMSF (serine inhibitors) inhibited the enzyme by 39 and 85 %,
respectively. Further, a small decrease in activity by 28.6 % was observed in the presence
of soybean trypsin inhibitor (SBTI).

Effect of Metal Ions on the Microcarpain Activity

The effect of some metal ions at a concentration of 5 mM on the purified microcarpain activity
was studied at pH 8.0 and 70 °C by adding salts containing monovalent ions (Na+ or K+) or
divalent ions (Ca2+, Mn2+, Zn2+, Cu2+, Ba2+, Mg2+, or Hg2+) to the reaction mixture. As shown
in Table 4, the protease activity was slightly affected by the addition of Ba2+ and K+ and
moderately inhibited in the presence of Na+ and Ca2+. However, the addition of Mg2+ and
Mn2+ decreased the proteolytic activity by 79 and 85 %, respectively. The activity of the
protease was potentially abolished by the presence of Zn2+, while Cu2+ and Hg2+ inhibited
completely the activity of the enzyme. It is known that Hg2+ is among ions that act on
sulfhydryl residues in proteins [40]. The inhibition of the enzyme by Hg2+ suggested the
existence of sulfhydryl residues for the catalytic action of most proteases [41]. Similar findings
were reported for other cysteine proteases such as a milk-coagulating protease purified from
ginger rhizome [27], cysteine protease from capsules of caper [35], and the protease extracted
from ripe fruit of Bromeliad antiacantha Bertol [39].

Table 5 Relative proteolytic activities of microcarpain toward different proteins as substrates

Substrate Relative activity (%)

Casein 100±0
Gelatin 47.15±0.57
Ovalbumin 42.2±0.49
BSA 1.41±0.17

Proteolytic activity was assayed by mixing 0.5 ml of the purified microcarpain and 0.5 ml of buffer (Tris–HCl
100 mM, pH 8) containing substrate at 1 % (w/v). After 15 min of incubation at 70 °C, the reaction was stopped
by adding 0.5 ml TCA 20 % (w/v) and allowed to stand at room temperature for 15 min. The increase in
absorbance due to the hydrolysis and release of peptides was measured at 280 nm. The relative protease activity
on casein was taken as a control (100 %)
Appl Biochem Biotechnol (2015) 175:1732–1744 1743

Hydrolysis Specificity

A study of substrate specificity for microcarpain was made by using different proteins at
1 mg/ml (Table 5). In the present study, hydrolytic capability of enzyme to casein was higher
than that of other substrates investigated. The affinity of the enzyme for the substrate decreased
in the order of casein, gelatin, ovalbumin, and bovine serum albumin (BSA). Siala et al. [42]
also reported that casein was the preferable substrate for the alkaline metalloprotease of
Arthrobacter arilaitensis Re117.

Conclusion

The present study describes the purification and the characterization of microcarpain,
a new vegetal cysteine protease, from the latex of F. microcarpa. The purification to
homogeneity of the enzyme was achieved by acetone precipitation (20–40 %), gel
filtration G-75, and anion-exchange chromatography on Mono Q-Sefinose FF. After
the final purification step, the enzyme was purified eightfold with a specific activity
of 23,676.3 U/mg and 9.25 % recovery. The purified microcarpain appeared homog-
enous on SDS-PAGE, and its molecular weight was estimated to be 20 kDa. The
optimum temperature and pH for microcarpain were 70 °C and 8.0, respectively. The
N-terminal amino acid sequence showed high homology with that from Arabidopsis
thaliana and other plant cysteine proteases. Further studies will be needed to identify
the responsible gene of the purified microcarpain.

Acknowledgments This work was funded by the Ministry of Higher Education and Scientific Research,
Tunisia.

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