Antimicrobial activity of a trypsin inhibitor from the seeds of Abelmoschus moschatus .L

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Original Research Article
Antimicrobial activity of a trypsin inhibitor from the seeds of
Abelmoschus moschatus .
L
Muni Kumar Dokka and Siva Prasad Davuluri*
Department of Biochemistry, Andhra University, Visakhapatnam
530 003,
Andhra Pradesh
, India.
*
Corresponding author
A B S T R A C T
Introduction
Protease inhibitors, a class of proteins
which reversibly, stoichiometrically and
competitively inhibit the catalytic activity
of proteolytic
enzymes
, are
widespread
in
plants, animals and microorganisms. They
are
abundant
in the reproductive and
storage organs and vegetative tissues of
most plant families (Ryan, 1990).
These proteins, apart from regulating
proteolytic processes, are also involved in
defense mechanisms against insects and
other pathogenic microorganisms (Kim
et
al.,
2006). A positive correlation existed
between plants with higher levels
of
trypsin and chymotrypsin inhibitors
and
their resistance
to
wards
pathogen
s.
Sev
eral
ISSN: 2319
-7706
Volume
3
Number
5
(201
4
) pp.
184
-
199
http://
www.ijcmas.com
Ke ywords
Trypsin
inhibitor,
Abelmoschus
moschatus
,
antibacterial,
antifungal,
protein
purification.
A novel trypsin inhibitor (AMTI-II), with both antibacterial and antifungal
activities, was purified to homogeneity following conventional methods of protein
purification from the seeds of Abelmoschus moschatus. The trypsin inhibitor has
been found to be homogenous by the criteria of native PAGE and gel filtration and
its molecular weight was determined to be 21.2 kDa by SDS-PAGE and Sephadex
G-200 gel filtration. AMTI-II exerted strong inhibition towards bovine pancreatic
trypsin and it showed moderate inhibition towards elastase. The inhibitor was
found to be stable under conditions of extreme of pH (3.0 to 12.0), at high
temperatures and in the presence of denaturing agents, urea and SDS and it was
devoid of free thiol groups. AMTI-II exhibited potent antibacterial activity towards
Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Bacillus subtilis,
Streptococcus pneumoniae, Bacillus cereus and it was moderately active against
Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas syringae and
Streptococcus pyogenes. The trypsin inhibitor also moderately affected the growth
of fungal species, Candida albicans, Candida tropicalis, Asperigillus flavus,
Saccharomyces cerevisiae, Candida glabrata and Asperigellus niger. Results
obtained
in the present study suggest that AMTI-II may serve as an antimicrobial
agent active against pathogenic microbes.

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studies on protease inhibitors were
published with the aim of investigating
enzyme mechanisms of controlling disease
and pathological processes using genes
encoding protease inhibitors (Brogden,
2005).
In recent years, appearance of new mutant
strai
ns of microorganisms resistant to
commonly used antibiotics have
stimulated a systematic analysis of natural
products for bactericidal and fungicidal
properties having therapeutic applications.
Recently, protease inhibitors have also
been recognized as potential drugs for
controlling retroviral infections.
Many phytopathogenic bacteria and
fungi
are known to produce
extracellular
proteinases
(
Kalashnikova
et al., 2003)
which may play an active role in the
development
of diseases caused by them
(Sara and Heale, 1990). In response to
such
attack by proteinases, plants
synthesize
inhibitory polypeptides that can
suppress the enzyme
activitie
s.
This
phenomenon was first recorded in
tomatoes infected with Phytophthora
infestans
(
Woloshuk
, 1991). In this case,
increased levels of trypsin and
chymotrypsin inhibitors correlated with
the plant resistance to the
pathogen.
Some of the serpins, cystatins, pepstatins
and metallo protease inhibitors have been
reported to possess antimicrobial activities
(Kim
et al., 2009). Trypsin inhibitors from
the seeds of chinese white cabbage and
bottle gourd are reported to possess
antibacterial activities (
Ngai
and Ng,
2004;
Shee Chandan et al.,
2009).
D
ouble
-
headed inhibitors from broad b
eans
and
potato tubers showed
antifu
ngal activity
(
Ye
, et al., 2001;
Kim
et al., 2005
).
Prote
in
ase inhibitors, Mungoin from mung
bean and Potide G from potato tubers
,
exhibited
both antifungal and antibacterial
activities
(Wang et al., 2006; Kim et al.,
2006).
Proteinase inhibitors have al
so
been
studied as model systems for elucidating
proteinase inhibition mechanisms, as well
as
protein
-
protein
asso
ciations (Oliva and
Sampaio, 2009
).
In pharmacological and
medical fields, investigations have been
ma
de into the potential of these inhibit
ors
as therapeutic agents in the treatment of
wide range of disorders associated with
enhanced proteolytic activities like
pancreatitis, shock, allergy, inflammation
etc.
(Richardson
, 1977). They also
find application in HIV therapy (Martin
et
al.,
1995) and cancer (Kim
et al.,
2009).
Abelmoschus moschatus (L.) Medic,
family
Malvaceae
, is an aromatic and
medicinal plant popularly known as
Mushkdana
/ Kasturi bhendi. The seeds
are rich in protease inhibitors and they are
used to check excessive thirst, cure for
stomatitis, dyspepsia, urinary discharge,
gonorrhea, leucoderma and itchiness.
Not much work has been done on protease
inhibitors from these seeds including their
influence on the growth of bacterial and
fungal
strains.
This paper, theref
ore, deals
with the isolation, purification of a trypsin
inhibitor from the seeds of
Abelmoschus
moschatus
and to examine its effects on
the growth
of
selected bacterial and fungal
strains.
Materials and Methods
Source
Abelmoschus moschatus plants bearing
pods
of uniform size were selected in and
around Visakhapatnam district. Pods were
collected at the ripening stage and seeds
removed from the
pods
were used for the

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isolation and purification of
trypsin
inhibitor.
C
hemicals
Bovine pancreatic trypsin (1 x cr
ystallized,
DCC
-treated, type XI), bovine serum
albumin (BSA), porcine pancreatic
elastase type I, chymotrypsinogen A,
ovalbumin, lysozyme, catalase,
phosphorylase b, soybean trypsin inhibitor
(type I-S) were purchased from Sigma
Chemical Company, St. Louis, Missouri,
U.S.A.
N-
acetyl
-DL-
phenylalanyl
- -
naphthylester
(APNE),
-N-
benzoyl
-
DL
-
argin
ine
-p-
nitroanilide
HCl (BAPNA), blue dextran,
DEAE
-
ce
llulose,
N,
N-
dimethylsulfoxide,
N,N -methylene bis acrylamide, sod
ium
dodecyl sulfate (SDS) were also from
Sigma Chemical company, St. Louis,
Missouri, U.S.A.
Sephadex G-100 and Sephadex G-
200
were purchased from Pharmacia Fine
Chemicals, Uppsala, Sweden. 2-
mercaptoethanol was purchased from
Fluka, Switzerland.
Guani
dine hydrochloride and trichloro
acetic acid (TCA) were purchased from
Riedel, Germany. Acrylamide was
purchased from J.T. Baker Chemical
Company, Phillipsburg, N.J., U.S.A.
N,
N,
N , N
tetramethylene 1,2 diaminoethane
(TEMED) was purchased from B.D.H.
Chemical Ltd., Poole, England.
Casein
was purchased from E.Merck, Darmstadt,
Germany.
Nutrien
t agar, Peptone, Beef extract, Agar
-
agar, Potato dextrose agar (PDA)
were
purchased from Himedia Pvt Ltd
,
Mumbai, India. All other chemicals used
were of analyt
ical grade.
Purification of Abelmoschus moschatus
Trypsin Inhibitor (AATI
-
II)
A procedure has been established for the
purification of proteinase inhibitor from
the seeds of
Abelmoschus moschatus. 25
g
of the seeds were homogenized with 150
ml of 0.1
M
sodium phosphate buffer,
pH
7.6 and then made up to 250 ml with the
same buffer. The extract was then
centrifuged at 5,600 rpm for 15 min at
4°C. The supernatant (250 ml) was
dialyzed against the buffer for 24h in the
cold
and rapidly heated to 70°C and
maintained at this temperature for 10 min.
The extract was quickly cooled in ice and
then centrifuged at 5,600
rpm
for 15 min
at 4°C. To the supernatant, solid
ammonium sulfate was added to 60%
saturation with constant stirring at 4°C.
The mixture was kept overnight at 4°C.
The precipitate was collected by
centrifugation at 3,000 rpm for 10 min at
4-6°C, dissolved in 0.1 M sodium
phosphate buffer, pH 7.6 and dialyzed
against the same buffer. The dialyzed
sample was loaded on a DEAE-
cellulose
column (2.2 x 34 cm) and the elution was
performed
with 0.1- 1.0
M NaCl in the buffer. Fractions of 8 ml
were collected at a flow rate of 60 ml/h
and were
assayed for protein by measuring
their absorbance at 280 nm as well as the
inhibitory
activity against trypsin using
BAPNA
as the substrate.
Protein from the previous step was loaded
on Sephadex G-100 column (1.9 x 63 cm)
and eluted with the same buffer. Fractions
(2 ml) were collected at a flow rate of 12
ml/ h and the protein was monitored by
measuring the absorbance at 280 nm. The
trypsin inhibitory activities of the fractions
were assayed using BAPNA as the
substrate. Fractions containing the trypsin
inhibitory activities were pooled, dialyzed

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against distilled water at 4-6°C and then
l
yophilized.
Protein estimation
Protein was estimated by the method of
Lowry
et al., (1951) using bovine serum
albumin as the standard.
Determination of molecular weight
Molecular weight of the inhibitor was
determined by SDS-PAGE using the
method of L
aemmli
(1970)
and also by gel
filtration on Sephadex G-
200 column.
Measurement of Trypsin and Trypsin
Inhibitory Activity
The inhibition of trypsin activity by the
inhibitor
was established by first assaying
the proteinase activity of the enzyme on an
a
ppropriate substrate and then incubating a
fixed amount of the enzyme with various
amounts of the inhibitor and assaying the
residual enzyme activity. Trypsin activity
was assayed by the method of Kakade
et
al.,
(
1969)
using BAPNA as the substrate.
Trypsin
(30 g) in 2 ml water was
incubated with 7 ml of substrate solution
at 37°C for 10 min. The reaction was
stopped by adding 1 ml of 30%(v/v) acetic
acid. The absorbance of the solution was
measured at 410 nm against an incubated
blank containing 2 ml of water instead of
trypsin solution.
To determine the inhibitory activities,
suitable aliquots of the inhibitor solutions
were included in the assay medium to
obtain 30-70% inhibition. One enzyme
unit is defined as an increase in 0.01
absorbance unit at 410 nm for trypsin
under the assay conditions. One enzyme
inhibitory unit is defined as the number of
enzyme units inhibited under these
conditions.
Assay of
other proteases
Elastase was assayed using elastin congo
red as the substrate by the method of
Naught
on and Sanger (1961),
Staphylococcus aureus protease and
Aspergillus oryzae protease activities were
assayed using casein as the substrate
following the methods of
Drapeau
(1976)
and Birk (1976)
respectively.
Effect of Protein
denaturing
conditions/agents
Temperature
Three
ml samples of 100
g/ml solution of
AMTI
-
II
in 0.1 M sodium phosphate
buffer,
pH 7.6, were separately incubated
in a water bath at different temperatures
for 10 min. After the heat treatment, the
solutions were quickly cooled in ice and
appropriate aliquots were used for the
assay of inhibitory activity against trypsin
using BAPNA as the substrate.
pH
In order to determine the pH stability of
the inhibitor, 1 mg/ml solution of AMTI-
II
in an appropriate buffer (10 mM) was kept
at 5°C for 24 h. The buffers used were 10
mM glycine
-
HCl (pH 3.0), sodium citrate
(pH 5.0), sodium
phosphate (pH 7.0),
Tris
-
HCl (pH 9.0) and glycine-NaOH (pH
12.0). Aliquots of the inhibitor were
diluted with phosphate buffer, pH 7.6 and
assayed as described above for trypsin
inhibitory activities using BAPNA as the
substrate.
Urea
To determine the stability of AMTI-
II
in 8
M urea, 1 g/ml solution of the inhibitor
was prepared in 0.1 M sodium phosphate
buffer, pH 7.6 containing 8 M urea, and

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was incubated 5°C for 24 h. The
solutions were diluted to 1 M in urea with
0.1 M phosphate buffer, pH 7.6 and the
appropriate aliquot of this diluted solution
was used for the assay of trypsin inhibitory
activity. The control assay mixture had
the same amount of urea as was present in
the diluted inhibitor solution.
Sodium dodecyl sulfate
One mg per ml solution of the inhibitor
was prepared in 0.1M sodium phosphate
buffer containing 1% SDS and was kept at
room temperature for 24 h. The solution
was dialyzed ex
tensively against the buffer
and aliquots were used for the assay of
trypsin inhibitory activity using BAPNA
as the substrate.
Guanidine hydrochloride
To determine the stability of the inhibitor
in 6M guanidine hydrochloride, 1 mg/ml
solution of the inhibitor was prepared in
0.1 M sodium phosphate buffer, pH 7.6
containing 6M guanidine
hydrochloride and was incubated at room
temperature for 24 h. The solution was
diluted to 1 M in guanidine hydrochloride
with the phosphate buffer and the
appropri
ate aliquot of this diluted solution
was used for the assay of trypsin inhibitory
activity. The control assay mixture had
the same amount of guanidine
hydrochloride as was present in the diluted
inhibitor solution.
Test organisms
The microbial strains, Bacillus subtilis
(MTCC 121), Bacillus cereus (MTCC
430),
Escherichia coli (MTCC 118 ),
Proteus vulgaris (MTCC 426),
Staphylococcus aureus (MTCC 96),
Klebsiella pneumoniae (MTCC 2405),
Streptococcus pneumoniae (MTCC 2672),
Streptococcus pyogenes (MTCC 1923 ),
Pseudomonas aeruginosa (MTCC 424 ),
Pseudomonas syringae (MTCC 1604 ),
Asperigellus niger (MTCC 2723),
Asperigillus flavus (MTCC 4633),
Fusarium oxysporum (MTCC 1755),
Alternaria alternata (MTCC 1362),
Candida albicans (MTCC 227),
Candida
glabrata
(MTCC 3016 ),
Candida
tropicalis
(MTCC 184 ), Mucor indicus
(MTCC 6333), Penicillium chrysogenum
(MTCC 161) and Saccharomyces
cerevisiae
(MTCC 2918) were collected
from Microbial Type Culture Collection
(MTCC), Institute of Microbial
Technology, Chandigar
h.
Determination of antimicrobial activity
Active cultures were generated by
inoculating a
loop
ful of culture in separate
100 ml nutrient/potato dextrose broths
and
incubating on a shaker at 37oC overnight.
The cells were
har
vested by centrifuging
at 4000 rpm for 5 min, washed with
normal
saline,
spun
at 4000 rpm for 5 min
again and diluted in normal saline to
obtain
5 x 10
5
cfu/ml
.
Antibacterial activity
AMTI-II was subjected to anti
bacterial
assay using the agar well diffusion method
of Murray et al., (
1995)
as modified by
Olurinola (
1996
).
Nutrient agar (20
ml
)
was dispensed into
sterile universal bottles,
these
were then
inoculated with 0.2 ml of cultures, mixed
gently and poured into sterile petri dishes.
After setting, a number 3-cup borer (6mm
diameter
) was properly sterilized b
y
flaming and used to make four uniform
wells in each petri dish. The wells were
filled
with
buffer containing 25 µg - 100

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µg of inhibitor and allowed for diffusion
of
the inhibitors for 45 min. The plates
were incubated at 37°C for 24 h for
bacteria.
Rifampicin, Benzyl Penicillin
and Tetracycline were included in the
positive control.
The inhibition
zones
were
measured with antibiotic zone scale in mm
and the experiment was carried out
in
triplicates.
Antifungal activity
The trypsin inhibitor was also subjected to
antifungal assay using the agar well
diffusion method of
Perez
et al., 1990.The
cultures of 48 h old grown on potato
dextrose agar (PDA) were used for
inoculation of fungal strains on PDA
plates. An aliquot (0.2 ml) of inoculum
was introduced to molten PDA and poured
into a petri dish by pour plate technique.
After solidification, the appropriate wells
were made and t
he
y
were
filled with the
buffe
r containing 50 - 100 µg of inhibitor
and allowed for diffusion of the inhibitor
for 45 min. The plates were incubated at
25°C for 48 h. The fungicides,
Flucanazole and Ketoconazole replaced
the inhibitor in the positive control. The
zones of inhibition were measured
as
described earlier.
Minimum inhibitory concentrati
on
(MIC) assays
Minimum
inhibitory concentrations
(M
IC)
of AMTI-II was determined according to
the method of Elizabeth (1999). A series
of two fold dilution of each inhibitor,
ranging from 50-2000 µg/ml, was
prepared. After sterilization, the medi
um
was inoculated
with
the
aliquots of culture
containing approximately
5×10
5
CFU/ml
of
each organism of 24 h slant culture in
aseptic condition and transferred
into
sterile 6 inch diameter petri dishes and
allowed to set at room temperature for
about 10 min and then kept in a
refrigerator
for 30 min. After the media
was solidified, wells were made and
d
ifferent
concentrations of inhibitor
ranging from 50-2000 µg/ml were added
to the wells of each petri dish. T
he
blank
plates
were
without
inhibitors
. Inhibi
tion
of
the
growth
of the organism in the plates
containing
inhibitor
was judged
by
comparison with
the
growth in
the
control
plates. The MICs were determined as the
lowest concentration of the AMTI-
II
inhibiting visible
grow
th of each organism
on the agar
plate.
MICs for fungal strains were also
determined in a similar manner by using
spores/cultures and incubating for 48 h.
Results and Discussion
Isolation and purification
The major trypsin inhibitor from
Abelmoschus moschatus seeds
was
purified by ammonium sulphate
fractionation and chromatography on
DEAE
-cellulose and Sephadex G-100.
By
employing
DEAE
- cellulose
chromatography,
four
protein
peaks
have
been resolved by linear NaCl gradient
(01M
1M NaCl in buffer).
Protein
eluted with 0.1 M NaCl on DEAE-
cellulose
column showed higher
antitryptic activity and seems to be the
major potent trypsin inhibitor obtained
when compared to the other protein peaks.
The inhibitor was designated as
Abelmoschus moschatus trypsin inhibitor,
AMTI-II, in the order of their elution from
DEAE
-cellulose column. When the
lyophilized active fractions
we
re subjected
to Sephadex G-100 column
chromatography, the inhibitor eluted out
as a single peak with corresponding
trypsin inhibitory activity (
Fig. 1
).

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Recoveries and relative purification at
each step fo
r a typical purification from 25
g seeds are shown in Table 1. By this
procedure, about 52.4 mg of the inhibitor
was obtained with a final yield of
about
17
%.
The molecular weight of AMTI-II as
determined by SDS-PAGE (F
ig
-
2)
was
found to be 21.2 kDa which was close to
that obtained with gel filtration on
Sephadex G-200 (
Fig
-
3).
The inhibitor
gave a single sharp band on SDS-
PAGE
even in the presence of 2-
mercaptoethanol
supporting the monomeric nature of the
protein.
The inhibitory specificity of the inhibitor
was tested against various serine proteases
of bacterial, fungal and mammalian origin.
AMTI-II was strongly active against
bovine pancreatic trypsin with an IC 50
value of 7.5 µg. It, however, moderately
inhibited porcine elastase with an IC50
value of 20.1 µg. Staphylocccus aureus
protease and Aspergillus oryzae protease
were weakly inhibited by AMTI
-
II.
The purified inhibito
r (AMTI
-
II) was quite
stable up to 80°C for 10 min. When th
e
incubation was for 10 min at 900C, there
was about 25% loss of its trypsin
inhibitory activity. Further incubation of
the inhibitor for 20 min at 90°C resulted in
the loss of 40
%
trypsin inhibitory activity.
When kept in a boiling water bath, there
was a 7
0%
loss of its trypsin inhib
itory
activity in 10 min and 85% loss in 20 min
.
Incubation of
inhibitor
for 30 min at
boiling temperature caused the total loss of
trypsin
inhibitory activity (Table-
2).
Table
-3 shows that trypsin
inhibitory
activity
of AMTI-II was not affected at
alkal
ine as well as acidic conditions under
the conditions tested. However, when kept
at room temperature with 6 M guanidine
hydrochlorid
e for 24 h, AMTI-II had lost
35% of trypsin inhibitory activity.
AMTI-II was devoid of free thiol groups
and was found to be stable in the presence
of denaturing agents, urea and SDS.
Antimicrobial activity
Antimicrobial
assay of the purified trypsin
inhibitor was examined against various
bacterial and fungal strains by assessing
the minimum inhibitory concentrations
and the microbicidal effect of AMTI-
II
was further visualized as inhibition zone
by treating the test organisms with
inhibitor and then spreading the cells on
agar plates. AMTI-II strongly affected the
growth of Staphylococcus aureus followed
by Escherichia coli, Proteus vulgaris
,
Bacillus subtilis,
Streptococcus
pneumoniae,
Bacillus cereus
and with
zone
s of inhibition recorded as 28m
m,
27mm, 26mm, 25mm, 25mm and 24mm
at
a concentration of 50
µg
of the inhibitor.
On the other hand, the growth of
Klebsiella
pneumoniae, Pseudomonas
aeruginosa,
Pseudomonas syringae and
Streptococcus pyogenes was
affected
moderately by the inhibitor with zones of
inhibition recorded as17mm, 16mm,
16mm and 15mm at a concentration of 100
µg AMTI
-
II respectively (
Fig
-4
).
Minimum inhibitory concentrations of
AMTI-
II
for
its antibacterial activity were
presented in
Table
- 4. The minimum
inhibitory concentrations (MIC) for
Staphylococcus aureus, Escherichia coli,
Proteus vulgaris
,
Bacillus subtilis,
Streptococcus
pneumoniae was
found to
be
62.5
µg
/ml, for
Bacillus cereus,
Klebsiella
pneumonia, the MIC value was
125 µg/ml
and
for the last three bacterial
strains namely Pseudomonas aeruginosa,
Pseudomonas syringae and Streptococcus

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pyogenes was found to be 250 µg/ml of
the inhibitor. From the results obtained, it
is clear that AMTI-II exerted a significant
inhibitory effect on the growth of selected
bacterial strains at a concentration of 50 -
100
µg
of the inhibitor.
Antifungal activity
The trypsin inhibitor was also tested for its
antifun
gal activity against Asperigellus
niger,
Asperigillus flavus,
Fusarium
oxysporum, Alternaria alternate,
Candida
albicans
, Candida glabrata, Candida
tropicalis,
Mucor indicus,
Penicillium
chrysogenum and Saccharomyces
cerevisiae
in the range 500-2000 µg/
ml
along with the positive control containing
the fungicides, Flucanazole and
Ketoconazole. AMTI-II moderately
affected the growth of Candida albicans
,
Candida tropicalis, Asperigillus flavus,
Saccharomyces cerevisiae, Candida
glabrata and Asperigellus niger
with
zones of inhibition recorded as 21 mm, 21
mm, 19 mm, 19 mm, 20 mm and 21 mm at
a concentration of 100
µg
of the inhibitor
respectively (
Fig
-5
).
The inhibitor did not
show any inhibitory effect on the growth
of
other
fungal strains tested.
Minimum
inhibitory concentrations of
AMTI-
II
for anti
fungal
activity was
presented in
Table
- 5.
Except
for
Saccharomyces cerevisiae,
the
MIC of
AMTI-II for other fungal strains was
found to be 250
µg/
ml of the inhibitor.
Protease inhibitors are ubiquitous in
plants
generally acting as storage proteins and
wound
-induced defensive agents against
herbivores and pathogens (Basir et al.,
2000). The role of protease inhibitors in
plant
protection against insects is studied
relatively well. On the other hand, data on
the role of protease inhibitors against
fungal or bacterial infections are very few
and nee
d to be provided more.
In the present study, a novel trypsin
inhibitor from Abelmoschus moschatus
seeds have been isolated and purified to
homogeneity following c
onventional
methods of protein purification. The
observation that trypsin inhibitory activity
in the crude extracts of the seeds is stable
at 70oC for 10 min has led to the use of
this treatment as the first step in the
purification of the inhibitor. About
52
% of
proteins present in the crude extract were
removed by this step. When the
ammonium sulphate fraction was
subjected to DEAE-cellulose column
chromatography, trypsin inhibitory
activity was found to be associated with
protein present in the void volume and
also with the proteins bound to the matrix.
A weakly bound protein eluted by 0.1 M
NaCl showed strong trypsin inhibitory
activity and moderately and firmly bound
proteins eluted with 0.25 M NaCl and 0.5
M NaCl exhibited both trypsin and
chymo
trypsin
inhibitory activities.
The
protein
obtained with 0.1 M NaCl also
eluted out as a single protein with
corresponding trypsin inhibitory activity
when subjected to Sephadex G-100 gel
filtration.
The final yield of the inhibitor was about
17
%. AMTI
-II was found to be
homogenous by native PAGE and gel
filtration on Sephadex G-200 column. The
coomassie blue stainable protein band
corresponded to the specific staining band
for the visualization of the trypsin
inhibitory activity. The molecular mass of
AMTI-II was found to be 21.2 kDa as
determined by SDS-PAGE and was close
to the mass that obtained with gel filtration
of Sephadex G
-
200 column.

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The trypsin inhibitor from the seeds of
Abelmoschus moschatus was found to be
stable under conditions of extreme pH,
temperature and in the presence of
denaturants, 8 M urea and 1% SDS. The
unusual stability of the inhibitor could be
due to intramolecular disulphide bridges
and strong hydrophobic interactions
forming an inner core in the protein. In
this respect, it resembles proteinase
inhibitors from other plant sources such as
soybe
an (Edelhoch and Steiner, 1963),
Italian millet (Udupa and Pattabiraman,
1987),
Archidendron ellipticum
(Bhattacharyya
et al., 2006),
Calliandra
selloi
(Yoshizaki et al., 2007) and
Inga
laurina
(Macedo
et al
., 2007).
It is well known that some plant proteinase
inhibitors possessed in vitro antibacterial
and antifungal activities. In the present
study, AMTI-II exhibited antibacterial and
antifungal activities with varying degrees
in a dose dependent manner. It did not
differentiate Gram positive bacteria fr
om
Gram negative bacteria in its anti
bacterial
activity.
Among all the bacterial strains tested,
Staphylococcus aureus, Escherichia coli,
Proteus vulgaris
,
Bacillus subtilis,
Streptococcus
pneumoniae,
Bacillus
cereus
were found to be more sensitive
and
Klebsiella
pneumoniae, Pseudomonas
aeruginosa,
Pseudomonas syringae and
Streptococcus pyogenes
were
found to be
less sensitive. However, AMTI-II showed
a moderate effect on the growth of funga
l
species,
Candida albicans, Candida
tropicalis,
Asperigillus flavus,
Saccharomyces cerevisiae, Candida
glabrata and Asperigellus niger and it had
no inhibitory effect on the growth of fungi
- Fusarium oxysporum, Alternaria
alternat
a, Mucor indicus and Pen
icillium
chrysogenum
tested.
AMTI-II was similar to napin from
chinese white cabbage (
Brassica
chinensis
) and trypsin inhibitor
from b
ottle
gourd
(Lagenaria siceraria) in posses
sing
antibacterial activity
towards
Pseudomonas aeruginos
a,
Bacillus subtilis
and
Bacillus
cereus
(Ngai and Ng, 2004
and
Shee Chandan
et al.,
2009)
.
It was also
close to
prote
in
ase inhibitors from broad
beans
(Vicia faba) and buckwheat
(Fagopyrum esculentum) seeds in
its
antifungal activity (Ye et al., 2001
;
Dunaevsky
et al.
,
2001).
Several kunitz
proteinase
inhibitors have
shown potential antimicrobial activity
against Gram
positive
and Gram-
negative
bacteria and fungi. Inhibitors possessing
bactericidal a
ctivity
include those from the
corms of Xanthosoma blandum, active
against
Staphylocccus aureus, Salmonella
typhimurium
, and Escherichia coli (Lima
et al., 2011) and seeds of
Achyranthes
aspera
(AATI) active against
Proteus
vulgaris
,
Bacillus subtilis
,
Staphylocccus
aureus,
Escherichia coli and
Klebsiella
pneumonia
(Geeth
a
et al
., 2012).
Microbes are known to elaborate proteases
into extracellular medium for gaining
entry into the host and protease inhibitors
by binding to such extracellular proteases
could exert antimicrobial effect.
Possibility of protease inhibitors en
tering
into microbial cells and interfering with
the function of intracellular proteases
cannot be ruled out for their antimicrobial
activity. Bactericidal proteins are reported
to form a channel on cell membrane and
cell dies as a result of the out flowing of
the cellular contents through a mechanism
different from that of antibiotics. Whether
protease inhibitors form such a channel is
yet to be established. The growth of
inhibition of fungi cannot be fully

Int.J.Curr.Microbiol.App.Sci
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-
199
193
Fig.1
Gel filtration of AMTI
-
II on Sephadex G
-1
00 of the DEAE
-
cellulose preparation
58.8 mg of AMTI-II, was applied
on
to the column (1.9 x 63) in 0.1 M phosphate buffer, pH
7.6 and eluted with the
same buffer.
Fractions, each 2 ml, were collected at a flow rate of
12
ml/h.
(
------
--
--
)
Prot
ein was monitored by absorbance at 280 nm
(
---------
) Trypsin inhibitory activity
Fig.2
Molecular weight determination of AMTI
-II by SDE-
PAGE on
5 -
20% gradient slab gel
1 2
1.
Standard proteins
(a
) Phosphorylase b, 97kDa
(b
) Bovine serum albumin, 67kDa
(c) Ovalbumin, 45
kDa
(d
) Chymotrypsinogen A, 25kDa
(e
) Soybean trypsin inhibitor, 20.1 kDa
(f
) Lysozyme, 14kDa
2. Purified AMTI
-
II

Int.J.Curr.Microbiol.App.Sci
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):
184
-
199
194
Fig.3
Molecular weight determination
of AMTI
-
II
by gel filtration on Sephadex G
-200
Plot of elution volume against log molecular weight of
s
tandard
proteins (
) and AM
TI
-
II
(
).
Fig.4
Antibacterial activity of AMTI
-
II
PC
-
Positive control (Benzyl penicillin)
S.a- Staphylococcus aureus; B.s- Bacillus subtilis; B.c- Bacillus cereus; E.c-
Escherichia
coli
; P.v - Proteus vulgaris; S.p-
Str
eptococcus
pneumonia; K.p -
Klebsiella
pneumonia;
P.a
-
Pseudomonas aeruginosa
; P.s
-
Pseudomonas s
y
ringae
; S.py
-
Streptococcus pyogenes
Bacterial strains were spread on agar plates. Different amounts of AMTI-II (50 µg for the
first six bacterial strains
and
100
µg
for the remaining strains) were placed in the wells.
Control
contained
Benzyl Penicillin
(20
µg) in place of inhibitor. The incubation period was
24 h at37
0
C.
Zone of inhibition was measured.

Int.J.Curr.Microbiol.App.Sci
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-
199
195
Fig.5
Antifungal activity o
f AMTI
-
II
PC
-
Positive control (Flucanazole)
A.n
-
Asperigillus niger
; A.f
-
Asperigillus flavus; C.a
-
Candida albicans; C.g
-
Candida
glabrata
; C.t
-
Candida tropicalis
; S.c
- Saccharomyces cerevisiae
Fungal strains were spread on potato dextrose agar plates. AMTI-II (100 µg) was placed in
the wells and allowed for diffusion. Control contained Flucanazole (20 µg) in place of
inhibitor. The incubation period was 48 h at 250C. Zone of inhibition was measured and
minimum inhibitory concentration of eac
h inhibitor was determined.
Table
-1
Summary of purification of AMTI
-
II from seeds of
Abelmoschus moschatus
*Yield and fold purification were calculated on the basis of TIU and TIA respectively.
TIU
-
Trypsin inhibitory units
TIA
-
Trypsin inhibitory activity
Total
activity
units
Specific
activity
Units/mg
protein
Preparation
Volum
e
(ml)
Total
protein
(mg)
TIU×10
3
TIA×1
02
Yield (%)
Fold
purificatio
n
Crude extract
250 2087.5 788.4 3.77 100 1.00
Heat treatment
215 1016.4 626.4 6.16 79.45 1.63
Ammonium
sulphate (60%)
Fractionation
60 424.8 482.8 11.36 61.24 3.01
DEAE
-
Cellulose
0.1M NaCl
elution
216 58.8 136.8 23.26 17.35 6.17
Se
phadex
-G-100
fraction
50 52.4 132.6 25.30 16.81 6.71

Int.J.Curr.Microbiol.App.Sci
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196
Table
-2
Effect of heat on AMTI
-
II
AMTI
-
II
Temperature
°C
Time
(min)
TIU/mg of
AMTI
-II
x 10²
25
37
50
60
70
80
90
90
100
100
100
10
10
10
10
10
10
10
20
10
20
30
25.28
25.34
25.22
25.16
25.18
25.36
22.56
19.84
7.68
3.86
0
*
Inhibitory activity at 25°C was taken as 100%
TIU
Trypsin inhibitory units
Table
-3
Effect of pH on AMTI
-
II
TIU
Trypsin inhibitory units
AMTI
-
II was incubated for 24 h at 4°C in the respective buffers and assayed for Trypsin inhibito
ry
activity using BAPNA as the substrate.
Table
.4
Minimum Inhibitory concentrations (MIC) of AMTI
-
II on bacterial growth
Minimum Inhibitory
Concentration (µg/ml)
Name of the bacterial strain
AMTI
-
II
Staphylococcus aureus
62.5
Bacillus subtilis
62.5
Bacillus cereus
125
Escherichia coli
62.5
Proteus vulgaris
62.5
Streptococcus pneumoniae
62.5
Klebsiella pneumoniae
125
Pseudomonas aeruginosa
250
Pseudomonas syringae
250
Streptococcus pyogenes
250
Bacterial strains were spread on agar plates. Different concentrations of AMTI-II (0.025-2 mg/ml) were placed
in the wells. Controls contained Tetracycline, Rifampicin and Benzyl Penicillin (20µg) in place of isoinhibitors.
The incubation period was 24 h at 370C. Zone of inhibition was measured and minimum inhibitory
concentration of inhibitor was determined.
AMTI
-
II
pH
Name of the Buffer
TIU/mg of
AMTI
-
II
x 10²
3
5
7
9
12
Glycine
-
HCl
Sodium citrate
Sodium Phosphate
Tris
-
HCl
Glycine
-
NaOH
25.20
25.38
25.44
25.24
25.18

Int.J.Curr.Microbiol.App.Sci
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197
Table
.5
Minimum Inhibitory concentrations (MIC) of AMTI
-
II on fungal growth
Minimum Inhibitory
Concentration
(µg/ml)
Name of the
fungal strain
AMTI
-
II
Asperigillus niger
250
Asperigill
us flavus
250
Fusarium oxysporum
-
Alternaria alternate
-
Candida albicans
250
Candida glabrata 250
Candida tropicalis
250
Mucor indicus -
Penicillium chrysogenum
-
Saccharomyces cerevisiae 500
Fungal strains were spread on potato dextrose agar plates Different amounts of the inhibitor
(0.05
-2mg/ml) were placed in the wells and allowed for diffusion. Controls contained
Flucanazole (20 µg) and Ketoconazole (20 µg) in place of inhibitor. The incubation period
was 48 h at 250C. Zone of inhibition was measured and minimum inhibitory concentration of
inhibitor was determined.
explained by trypsin inhibition alone. The
antifungal role of trypsin inhibitors has
also been attributed to their ability to
interfere with chitin biosynthetic process
durin
g fungal cell wall development by
inhibiting the proteolytic activation of
chitin synthase zymogen
(Adams
et al.,
1993).
Some proteinase inhibitors have shown
both antibacterial and antifungal activities.
Kim
et al., (2006) demonstrated that
inhibito
rs from potato tubers
strongly
inhibited
the growth of a wide variety of
bacteria, including Staphylococcus aureus,
Listeria monocytogenes, Clavibacter
michiganense
, and Escherichia coli, and
fungi such as Candida albicans and
Rhizoctonia solani
.
In conclusion, the purified trypsin
inhibitor from the seeds of
Abelmoschus
moschatus
is found to be active against
selected bacterial and fungal strains with
varying efficiencies. AMTI-II can find
application in the medical front as
therapeutic agent for infections caused by
specific
bacteria
l and fungal strains and it
can be explored in the agricultural front
for developing transgenics after carrying
out extensive in vitro studies
against
midgut proteases of insect pests.
Acknowledgement
The financial assistance provided to
D.
Muni Kumar through UGC - Rajiv Gandhi
National Fellowship (RGNF) is greatly
acknowledged

Int.J.Curr.Microbiol.App.Sci
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