antimicrobial activity of protease inhibitor from leaves of...

Indian Journal of Experimental Biology

Vol. 49, May 2011, pp. 366-374

Antimicrobial activity of protease inhibitor from leaves of

Coccinia grandis (L.) Voigt.

L Shilpa Satheesh & K Murugan*

Plant Biochemistry and Molecular Biology Lab, Department of Botany

University College, Thiruvananthapuram 695 034, Kerala, India

Received 14 July 2010; revised 28 December 2010

Antimicrobial activity of protease inhibitor isolated from Coccinia grandis (L.) Voigt. has been reported. A 14.3 kDa protease

inhibitor (PI) was isolated and purified to homogeneity by ammonium sulfate precipitation (20-85% saturation), sephadex G-75,

DEAE sepharose column and trypsin-sepharose affinity chromatography from the leaves of C. grandis. The purity was checked by

reverse phase high performance liquid chromatography. PI exhibited marked growth inhibitory effects on colon cell lines in a dose-

dependent manner. PI was thermostable and showed antimicrobial activity without hemolytic activity. PI strongly inhibited

pathogenic microbial strains, including Staphylococcus aureus, Klebsiella pneumoniae, Proteus vulgaris, Eschershia coli, Bacillus

subtilis and pathogenic fungus Candida albicans, Mucor indicus, Penicillium notatum, Aspergillus flavus and Cryptococcus

neoformans. Examination by bright field microscopy showed inhibition of mycelial growth and sporulation. Morphologically, PI

treated fungus showed a significant shrinkage of hyphal tips. Reduced PI completely lost its activity indicating that disulfide bridge

is essential for its protease inhibitory and antifungal activity. Results reported in this study suggested that PI may be an excellent

candidate for development of novel oral or other anti-infective agents.

Keywords: Antimicrobial activity, Chromatography, Coccinia grandis, Cytotoxicity, Protease inhibitors, Purification.

Protease inhibitors (PIs) play essential role in

biological systems regulating proteolytic processes

and in defense mechanisms against insects, and other

pathogenic microorganisms1. In recent years

appearance of new mutant strains of microorganisms

resistant to commonly used antibiotics have

stimulated a systematic analysis of natural products

for bactericidal and fungicidal properties having

therapeutic applications. Several studies on PIs were

published with the aim of investigating enzyme

mechanisms of controlling disease and pathological

processes using genes encoding protease inhibitors2.

Recent studies have revealed that protease inhibitors

as new drugs in highly active antiretroviral

combination therapy, increasing life expectancy in

HIV-positive patients3. Antimicrobial peptides

(AMPs) are important effector molecules of innate

immunity. Such peptides provide protection against

bacteria, fungi and viruses by acting on cell

membranes of pathogens. Recently, several

antimicrobial plant proteins and peptides that inhibit

growth of agronomically important pathogens have

been isolated from various plant sources4.

Antimicrobial peptides and proteins with a

spectacular diversity of structures, includes

thaumatin-like proteins, embryo-abundant proteins,

allergen like peptides, cyclophilin-like proteins,

cysteine-rich small proteins, arginine and glutamate

rich proteins, lectins, thionins, peroxidases, ribosome

inactivating proteins, lipid-transfer proteins, chitin

binding proteins, ribonucleases, deoxyribonucleases

and protease inhibitors. These proteins can inhibit a

wide variety of phytopathogens, but display different

potencies according to target microorganisms3.

Coccinia grandis (L.) Voigt. is a wild

cucurbitaceous medicinal plant with many

pharmaceutical applications. As an ethnic tribal plant,

it has potential therapeutic values as anti-diabetic,

anti-ulcer, anti-inflammatory, antioxidant and

antitumor properties5. In this study, antimicrobial

activity of a protease inhibitor (PI) purified from

leaves of C. grandis has been reported.

Materials and Methods

Materials Fresh leaves from Coccinia grandis

(L.)Voigt. were collected from the department garden,

University College, Trivandrum, Kerala and stored

at -20°C.

______________

*Correspondent author

Telephone: 0471- 2320049

E-mail: [email protected]

SATHEESH & MURUGAN: ANTIMICROBIAL ACTIVITY OF PROTEASE INHIBITOR FROM COCCINIA LEAVES

367

Isolation and purification of PIAll the

purification steps were carried out at 4°C as described

by Macedo et al 6. A crude inhibitor preparation was

obtained by extraction of fresh leaves from C. grandis

with 100 mM phosphate buffer, pH 7.6 (1:10, w/v) for

2 h with subsequent centrifugation at 7500 g for 30

min and the supernatant was collected. The crude

inhibitor was fractionated with solid ammonium

sulfate (20-85% saturation) and the precipitate was

recovered by centrifugation at 10, 000 × g for 20 min,

redissolved in 0.01 M phosphate buffer (pH 7.6) and

dialyzed against distilled water for 24 h and

lyophilized. Lyophilized PI was dissolved in 100 mM

phosphate buffer, pH 7.6, containing 100 mM NaCl,

and applied to a Sephadex G-75 column equilibrated

with the same buffer. The fraction with inhibitory

activity was further fractionated by ion-exchange

chromatography on a DEAE-Sepharose column

equilibrated with 50 mM Tris–HCl buffer, pH 8.0, and

eluted with a linear gradient of NaCl (0–1 M) in the

same buffer. The fraction eluted with 0.4 to 0.5 M

NaCl showing highest PI activity was applied to a

trypsin-Sepharose affinity column equilibrated with

100 mM phosphate buffer, pH 7.6, containing

100 mM NaCl. The adsorbed PI was eluted with

0.01 N HCl. The purity was further checked by

reverse phase HPLC (C18 column) at a flow rate of

1.0 ml/min with 100% solvent A (0.1% trifluoroacetic

acid (TFA) in water) for 10 min and a linear gradient

(0–100%) of solvent B (0.08% TFA in 80%

acetonitrile) over 55 min. Proteins were detected by

monitoring the absorbance at 280 nm.

Apparent molecular mass was obtained by

Sephadex G-75 gel filtration column (0.1 M

phosphate buffer, pH 7.6) calibrated with proteins

of known molecular mass. The molecular mass and

homogeneity of PI was determined by SDS–PAGE

as described by Schagger and von Jagow7.

Assay of trypsin and chymotrypsin inhibitory

activityTrypsin or chymotrypsin inhibitory activity

was determined by using an appropriate volume of

purified PI as per the methodology of Macedo et al.6

that results in a 40–60% decrease in corresponding

enzyme activity. Assay mixture consists of inhibitor

in assay buffer 50 mM Tris–HCl containing 20 mM

CaCl2 either at pH 8.2 for trypsin or pH 7.8 for

chymotrypsin. Trypsin (10 µg) or chymotrypsin

(80 µg) was added to the assay mixture and incubated

for 15 min at 37°C. Residual trypsin or chymotrypsin

activity in the above assay mixture was determined

after incubating for 45 min at 37°C with synthetic

substrates: 1 mM BAPNA (N-benzoyl-L-arginine-p-

nitroanilide) or 1 mM BTPNA (N-benzoyl-L-tyrosyl-

p-nitroanilide)5, respectively. The reaction was

terminated by adding 30% acetic acid. The resulting

absorbance at 410 nm was recorded. One inhibitor

unit was defined as the amount of inhibitor required to

inhibit 50% of the corresponding enzyme activity per

unit time.

Test microorganismsSelected pathogenic

bacteria; Staphylococcus aureus (MTCC 740),

Klebsiella pneumoniae (MTCC109), Escherichia coli

(MTCC 42) Bacillus subtilis (MTCC 47), Proteus

vulgaris (MTCC 426) and fungi like Cryptococcus

neoformans, Candida albicans, Mucor indicus,

Penicillium notatum and Aspergillus flavus were

obtained from culture collection of the Department of

Applied Botany and Biotechnology, University of

Mysore, Mysore, India. All the test bacterial stock

cultures were maintained at 4oC on nutrient agar

medium (HiMedia). Active cultures for experiments

were prepared by transferring a loopful of culture to

10 ml of nutrient broth (HiMedia) and incubated at

37°C for bacterial proliferation. After 24 h incubation,

the bacterial suspension was centrifuged at 10, 000 g

for 15 min. The pellet was resuspended in sterile

distilled water and the concentration was adjusted to

1 × 108 cfu/ml by reading the OD of the solution

(A610 nm) and used for further studies. The fungal

colonies were inoculated into Sabauroud Dextrose

Agar (SDA) and incubated at 35 ± 2°C for 4 h. The

turbidity of the resulting suspensions was diluted with

SBCB to obtain a transmittance of 25.0 % at 580 nm.

That percentage was found spectrophotometrically

comparable to McFarland turbidity standard. This

level of turbidity is equivalent to approximately

1 × 108 CFU/ml.

Antimicrobial assayAntimicrobial assay was

performed in 96 well, sterile, flat bottom microtiter

plates, based on broth microdilution assay, which is

an automated colorimetric method, uses the

absorbance (optical density) of cultures in a microtiter

plate8. Each well of microtiterplates was filled with

50 µl of test organism and 200 µl different

concentrations PI extracts. For bacteria and fungi the

microtiterplates were incubated at 37±2°C and

30±2°C for 24 h and 48 h respectively. After the

incubation period the plates were read at 620 nm

using ELISA reader. Minimum inhibitory

concentration (MIC) was determined as the lowest

INDIAN J EXP BIOL, APRIL 2011

368

concentration of PI inhibiting the growth of the

organism, based on the readings.

To determine minimum killing concentration, 50 µl

broth was taken from each well and inoculated in

200 µl nutrient broth for 24 h at 37° C for bacteria and

48 h at 30°C for the fungal cells. The minimum

killing concentration is defined as the lowest

concentration of the PI at which inoculated

microorganism was completely killed. Each test was

performed in triplicate and repeated twice.

Levofloxacin was used as a positive control.

Preparation of human red blood cells and assay of

hemolytic activityHuman red blood cells were

centrifuged and washed three times with phosphate-

buffered saline (PBS; 35 mM phosphate buffer with

0.15M NaCl, pH 7.0). The hemolytic activity of the

potamin-1 and melittin (positive control) were

evaluated by measuring the release of hemoglobin

from fresh human erythrocytes. Aliquots (100 µl) of

an 8% suspension of red blood cells were transferred

to 96-well plates, and hemolysis was determined by

measuring the absorbance at 414 nm using the Emax

plate reader. No hemolysis (0%) and full hemolysis

(100%) were determined in the presence of PBS and

0.1% Triton X-100, respectively. The percent

hemolysis was calculated using the following

equation: % hemolysis = [(A414 nm with TPI solution

- A414 nm in PBS)/(A414 nm with 0.1% Triton

X-100 - A414 nm in PBS) × 100]9.

MTT assayThis assay detects the reduction of MTT

[3-(4, 5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium

bromide] by mitochondrial dehydrogenase, to blue

formazan product, which reflects the normal functioning

of mitochondrial and cell viability. HeLa cells were

seeded in 96 well plates at a density of 1 × 104 followed

by incubation for 24 h. Different concentrations of the PI

were then added. After 72 h, 20 µl of a 5-mg/ml solution

of MTT was spiked into each well. After 4 h, the plates

were centrifuged at 324 g for 5 min. The supernatant

was removed and 150 µl of dimethyl sulfoxide was

added to dissolve the MTT-formazan at the bottom of

the wells. After 10 min, the absorbance at 570 nm was

measured using a microplate ELISA reader.

Statistical analysis All the experiments were

designed with six replications. The data were

statistically analyzed for a student t-test.

Results

C. grandis protease inhibitor (PI) was purified to

homogeneity in four steps by ammonium sulfate

precipitation (20-85% saturation), sephadex G-75,

DEAE sepharose column and trypsin-sepharose

affinity chromatography. The crude PI was

fractionated with (NH4)2SO4 and dialyzed. The

fractions showing maximum PI activity was applied

to gel-filtration column (Sephadex G-75) and eluted

with 100 mM phosphate buffer (pH 7.6) containing

100 mM NaCl. The active fractions with PI activity

(fraction number 66 to 70) were pooled, dialyzed and

loaded to a DEAE-sepharose column, pre-equilibrated

with 50 mM Tris–HCl, pH 8.0. The bound PI was

eluted with a gradient of irrigating buffer (0 to 1.0 M

NaCl in the same buffer) at a flow rate of 30 ml/h.

The fractions showing PI activity (0.4 to 0.5 M NaCl

elution) were pooled, dialyzed against 50 mM Tris-

HCl buffer (pH 8.0). The pooled PI fractions were

loaded onto a Trypsin sepharose affinity column, pre-

equilibrated with 100 mM Tris–HCl, pH 7.6. The

bound protein was eluted with 0.01 N HCl at a flow

rate of 30 ml/h and subsequently the fractions (1.0 ml)

were neutralized with 2 M Tris. The fractions with PI

activity were pooled and concentrated using Amicon

filters for further use. It proved to be a convenient

step for isolating PI, although the possibility of

limited digestion of the inhibitor by the immobilized

trypsin during purification cannot be excluded. After

affinity purification high inhibitory activity and

purification fold observed in the present study

suggests its functional integrity was retained even

after binding with trypsin. The fractions showing PI

activity were pooled and subjected to further

analysis.

Thus, the protocol yielded a purified PI with

specific activity 377.9 U/mg; with a low protein

content of 1.4 mg. Overall, the specific activity

increased about 114.5-fold with 12 % yield of activity

Table 1. This is significantly higher than that obtained

for Arachidendron ellipticum10

and Brassica

campestris 11

.

The purified PI was further subjected to RP-HPLC

showed a single peak with a retention time of 8.2 min

in 50 mM Tris–HCl buffer, pH 8.0 and coincided with

the protein peak. The purity of the protein was

analyzed by SDS–PAGE showed a single thick

polypeptide band with a molecular mass of

approximately 14.3 kDa (Fig. 1), which is lower than

those reported for other cucumisin-like proteases and

PI of legumes11-13

. The molecular mass estimated by

size elution chromatography (14.3 kDa) was agreed

fairly well with the SDS-PAGE results.


369

Antimicrobial activity Antimicrobial assay of the

purified PI was examined against various bacterial

and fungal cells by assessing the minimum inhibition

concentration and minimum killing concentration. As

shown in Table 2, PI had exhibited different degree of

growth inhibition against tested bacterial and fungal

strains such as S. aureus, K. pneumoniae, P. vulgaris,

E. coli, B. subtilis, C. albicans, M. indicus,

P. notatum, A. flavus and C. neoformans. The

microbicidal effect of PI was further visualized as

inhibition zone by treating the pathogens with PI and

then spreading the cells on agar plates. Among the all

pathogens tested K. pneumoniae and A. flavus were

the most sensitive and S. aureus, B. subtilis,

C. albicans and C. neoformans were the most

resistant strains.

The MTT assay was used to examine the effects of

PI on HeLa cells and their proliferation. HeLa cells

were incubated with varying concentrations of

PI (5-50 µM) for 72 h, the colour intensity was

measured using an ELISA plate reader at

570 nm. Half maximal inhibitory concentration (IC50)

determined by dose-response curve was 25 µM

(P < 0.01). The results of the apoptotic cell death

indicate that PI is toxic to HeLa cells (Fig. 2). In this

aspect it resembles Buckwheat antifungal peptide

suppresses proliferation in a variety of tumor cells,

including hepatoma, leukemia, and breast cancer cells14

.

Table 1 Purification profile of Coccinia grandis PI

Purification steps Total activity

(U/g tissue)

Yield (%)

Total protein

(mg/g tissue)

Specific activity

(U/mg protein)

Fold purification

Crude inhibitor 4359 100 1316 3.3 0

Ammonium sulfate (85%) 2763 63.4 388 7.1 2.2

Sephadex G-75 1425 32.7 13 109.6 33.2

DEAE-Sepharose 874 20 3.2 273 82.7

Trypsin-Sepharose 529 12 1.4 377.9 114.5

One unit is defined as 1µmol of substrate hydrolyzed/min of reaction. One inhibition unit is defined as unit of enzyme inhibited

Fig. 1Chromatogram and SDS-PAGE analysis of purified PI.

C-18 reversed phase chromatography (RP-HPLC) elution,

acetonitrile gradient (0 ± 80%) in 0.1% TFA, flow rate: 1 ml/min.

Lanes 1 protein from trypsin sepharose affinity column fraction.

Protein bands were stained with coomassie blue R-250.

Table 2 Antimicrobial activity, MIC and MBC of PI

against tested bacteria and fungi

Pathogens MIC

(mg /ml)

MBC/MFC

(mg/ml)

Staphylococcus aureus (+) 1 1.2

Bacillus subtilis (+) 1 1.25

Klebsiella pneumoniae (-) 0.01 0.5

Escherichia coli (-) 0.63 1

Proteus vulgaris (-) 0.2 0.5

Cryptococcus neoformans 0.63 1

Aspergillus flavus 0.08 0.5

Candida albicans 0.63 0.7

Penicillium notatum 0.2 1

Mucor indicus 0.2 0.5

Gram-positive (+) and Gram-negative (-) bacteria.

MBC/MFC, minimal bactericide/fungicide concentration

Fig. 2Inhibition of HeLa cancer cell line by PI [Values are

mean ± SE of 3 assays]


370

Cytotoxicity of PI was checked against mammalian

cells by measuring the lysis of human erythrocytes. PI

had no hemolytic activity; melittin was used as a

positive control showing strong hemolytic activity

(Table 3). These results demonstrated that PI has a

remarkable antimicrobial activity against various

microbial cells but no hemolytic activity.

PI showed inhibitory activity against trypsin and

chymotrypsin, a characteristic feature of Bowman-

Birk inhibitors type (BBI) PIs. This inhibitory activity

of PI was more pronounced against trypsin, when

compared with chymotrypsin. Most of the BBIs were

known to inhibit both trypsin and chymotrypsin due

to the presence of two different reactive sites15

.

However, with higher affinity towards trypsin

compared with chymotrypsin. The stoichiometry of PI

with trypsin was found to be 1:2, i.e., one molecule of

PI will exhibit TI activity by binding with two

molecules of trypsin (Fig. 3). On the other hand, PI

showed no obvious stoichiometry with chymotrypsin,

which is evident from the titration pattern of its

inhibitory activity (Fig. 3). While the PIs isolated from

Apios americana tubers16

showed a similar pattern of

stoichiometry to that shown by PI with respect to

trypsin, PIs isolated from Peltophorum dubium seeds17

and A. americana tubers16

showed a similar pattern of

stoichiometry to that of chymotrypsin.

To study the importance of the disulfide bonds in

PI, it was treated with the reducing agent,

dithiothreitol (DTT) to visualize the fungal cell

growth inhibition zone. The purified PI and reduced

PI treated paper disk was tested on the spread fungal

agar plate. As shown in Fig. 4, purified PI had strong

antifungal activity, whereas reduced PI had no

antifungal activity suggesting the presence of

disulfide bonds in PIs functional integrity.

The synthesis of fungal cell wall at hyphal apex

consists of a complex assembly sequence and it

protects the organism against a hostile environment

and relays signals for invasion and infection of a

likely plant, animal or human host. Several classes of

antimicrobial proteins involve inhibition of the

synthesis of the fungal cell wall or disrupt cell wall

structure and/or function; others perturb fungal

membrane structure, resulting in fungal cell lysis18

.

Examination by bright field microscopy showed

inhibition of mycelial growth and sporulation.

Morphologically, in PI treated fungus, a dramatic

shrinkage of hyphal tips was observed (Figs 5 b, d, f,

h, and j). Similar results were observed with Bavistin

Table 3 Hemolytic activities of antimicrobial protease inhibitors

PI conc.[(Hemolysis (%)]

40 20 10 5 2.5 1.25 0.62 0.31

PI 0 0 0 0 0 0 0 0

Melittin 100 100 100 96 75 38 18 0

Fig. 3Titration curves of trypsin and chymotrypsin inhibition by

PI [Values are mean ± SE of 3 assays]

Fig. 5Differences in morphology of fungal hyphae (control treated with sterile Tris-HCl buffer; and test with CGPI). Shrinkage of

hyphal tip (indicated by arrow) was observed in (a, b)-Candida albicans; (c, d)-Mucor indicus; (e, f)-Aspergillus flavus;

(g, h)- Penicillium notatum; (i, j)-Cryptococcus neoformans; and (k)-Candida albicans treated with bavistin, 40 ×.

Fig. 4 Antifungal activity of reduced PI [(a) purified PI + DTT

(0.5 mM); (b) purified PI (100 µM); and (c) PI + DTT (1 mM)]


371


372

at a concentration of 0.2% (W/V) (Fig. 5 k). In

contrast, the hyphae growing under the influence of

sterile Tris-HCl buffer did not show any obvious

growth aberrations (Figs 5 a, c, e, g and i). Fungal

growth was routinely checked microscopically to

confirm the micro spectrophotometric data. Major

morphological difference in the hyphae was apparent

between fungi harvested from inhibited zone and

those with control. PI produces the complete

inhibition of K. pneumoniae and A. flavus spore

germination at a concentration of 0.5 mg/ml while at

0.01 mg/ml, a reduction of hyphal growth is observed.

S. aureus, B. subtilis, C. albicans and

C. neoformans, were also inhibited but appears to be

less sensitive than K. pneumoniae and A. flavus; at a

concentration of 0.5 mg/ml only shortened hyphae

could be observed (Figs. 5). PIs can exert their

activity by inhibiting the serine proteases involved in

growth or differentiation of the microbial cells.

Discussion PIs are ubiquitous in plants; generally act as

storage proteins and wound-induced defense response

of plants against herbivores and pathogens19

.

Recently, protease inhibitors are reported as potent

anticarcinogens in a wide variety of in vivo and in

vitro systems. In addition, serine protease inhibitors

can increase the level of cholecystokinin due to the

inhibition of trypsin, they may be useful for reducing

food intake in human16

. PIs for use in humans must be

non-toxic and capable of inhibiting each of the major

intestinal proteases, including pancreatic trypsin, α-

chymotrypsin, and elastase. Several potentially non-

toxic protease inhibitors, mostly of bacterial or plant

origin, for example from barley seeds, cabbage

leaves, and Streptomyces, have been purified and are

commercially available for preventing protease-

induced peri-anal dermatitis. Although the

bactericidal mechanism of action of the PIs has not

yet been elucidated in detail, the presented data

confirm the in vitro antibacterial activity of PI against

pathogenic bacteria. It has been proposed that the

proteins with antibacterial action form a channel on

cell membrane and the cell dies as a result of the out

flowing of cellular contents, being this mechanism

different from that of antibiotics. Cucurbits proteins,

efficiently inhibit human fecal proteases, they could

also be useful in the treatment of peri-anal

dermatitis13

. The biological role of PIs may be

considered antinutritional by decreasing the activity

of proteases produced by the pathogens suggesting

that the inhibitor might interfere in the development

of fungi or bacteria by decreasing the endogenous

protease activity.

The growth inhibition of fungi cannot be fully

explained by trypsin inhibition alone. In fact a later

antifungal role has been reported for trypsin

inhibitors, due to the ability of these proteins to

interfere with the chitin biosynthetic process during

fungal cell wall development by inhibiting the

proteolytic activation of the chitin synthase

zymogen20-22

. The trypsin inhibitors belonging to the

Bowman Birk family are usually accumulating only in

developing seeds23

. During germination these

inhibitors are rapidly released from the seeds. Thus,

the role of PI may be associated with the protection of

seeds, which lack an active defense system, and

during early germination, when the tissues are

particularly exposed to potential attack by soil borne

pathogens. It is, therefore, possible that PIs acts

synergistically with other defense proteins expressed

in plants contributing to limiting invasion by

pathogens.

Various pathogenic fungi that are protease deficient

have been characterized as non-pathogenic, suggesting

a function for the proteases secreted during the process

of infection, colonization, and pathogenesis of

susceptible plants23

. Several studies confirmed that

protease secretion by the fungus is a determining factor

for pathogenicity, and its inhibition significantly

reduces the fungal infection24

. Protease inhibitors are

widely distributed within the plant kingdom. Some

inhibitors are constitutively expressed in seeds and

storage organs while others are induced on wounding

in plants. The protease inhibitors play an important role

in the protection of plant tissues from pest and

pathogen attack by virtue of an antinutritional

interaction13,14

. Thus, the PI can act directly on the

proteases produced by pathogenic fungi and reduce

their pathogenicity by decreasing their germination.

Molecular mechanism of PI related to

antiproliferative activity in HeLa cells remains

unclear but, probably may transinactivate epidermal

growth factor receptor (EGFR) tyrosine kinase

activity with marked reduction in phosphotyrosyl

level of cellular proteins or may dampened the

phosphorylation of focal adhesion kinase (FAK) and

the secreted matrix metalloproteinase (MMP) that

may lead to the suppression of invasive potential and

cell migration in vitro25

.


373

The antiproliferative nature of PI on HeLa cell

lines and no hemolytic nature may be because of their

potent activity against a specific cell line, but they

have not shown a wider range of action. Similar

antitumor and antiproliferative actions are well

documented in leguminous trypsin inhibitors8,26

.

Some trypsin inhibitors manifest antifungal activity,

e.g. those from the broad bean, lima bean, and the

yellow soybean. However, trypsin inhibitor from

Japanese large black soybean is devoid of antifungal

activity but have HIV-1 reverse transcriptase

inhibitory potency of this trypsin inhibitor than that

reported for Kunitz-type trypsin inhibitor from

soybean24,27

.

Amino acid composition, amphipathicity, charge

and size of TPi allow them to attach to and insert into

membrane bilayers to form pores by ‘barrel-stave’,

‘carpet’ or ‘toroidal-pore’ mechanisms. Although

these models are helpful for defining mechanisms of

antimicrobial peptide activity, their relevance to how

peptides damage and kill microorganisms still need to

be clarified. Brogden2 showed that transmembrane

pore formation is not the only mechanism of

microbial killing but also the translocated peptides

can alter cytoplasmic membrane septum formation;

inhibit cell-wall synthesis, nucleic-acid synthesis and

protein synthesis or enzymatic activity.

The purification of a protease inhibitor by gel, ion

exchange and affinity chromatography is described.

The apparent molecular mass is 14.3 kDa. This PI has

a potent antimicrobial activity as demonstrated by the

growth inhibition (in vitro) of important pathogenic

bacteria and fungi. These results indicate that future

finding of PI applications obtained from medicinal

plants can be of great importance for clinical

microbiology and possible therapeutic applications.

Acknowledgment This work was supported by grant from the Kerala

State Council of Science Technology and

Environment (KSCSTE) Govt. of Kerala, India.

References 1 Kim K M, Lee B Y, Kim Y T, Choi S G C, Lee J, Cho S Y &

Choi W S, Development of antimicrobial edible film

incorporated with green tea extract, Food Sci Biotechnol, 15

(2006) 478.

2 Brogden K A, Antimicrobial peptides: Pore formers or

metabolic inhibitors in bacteria, Nature Reviews, 3 (2005) 238.

3 Bobbarala V, Vadlapudi V R & Naidu C K, Antimicrobial

potentialities of mangrove plant Avicennia marina, J Pharm

Res, 2(6) (2009) 1019.

4 Chen Z Y, Brown R L, Lax A R, Cleveland T E & Russin J

S, Inhibition of plant-pathogenic fungi by a corn trypsin

inhibitor over expressed in Escherichia coli, Appl Environ

Microbiol, 65 (1999) 1320.

5 Linney G, Coccinia grandis (L.) Voight: A new

cucurbitaceous weed in Hawaii, Hawaii Bot Soc Newslett,

25(1) (1986) 3.

6 Macedo M L R, Mello G C, Freire M G M, Novello J C,

Marangoni S & Matos D G G, Effect of a trypsin inhibitor

from Dimorphandra mollis seeds on the development of

Callosobruchus maculates, Plant Physiol Biochem, 40

(2002) 891.

7 Schagger H & von Jagow G, Tricine-sodium dodecyl sulfate-

polyacrylamide gel electrophoresis for the separation of

proteins in the range from 1 to 100 kDa, Anal Biochem, 166

(1987) 368.

8 Suffredini J B, Sader H S, Goncalves A G, Reis A O, Gales A

C, Varella A D & Younes R N, Screening of antimicrobial

extracts from plants native to the Brazilian Amazon rainforest

and Atlantic forest, Braz J Med Biol Res, 37 (2004) 379.

9 Park Y, Park S N, Park S C, Park J Y, Park Y H, Hahm J S &

Hahm K S, Antibiotic activity and synergistic effect of

antimicrobial peptide against pathogens from a patient with

gallstones, Biochem Biophys Res Commun, 321 (2004) 631.

10 Bhattacharyya A, Mazumdar S, Leighton S M & Babu CR, A

Kunitz proteinase inhibitor from Archidendrone ellipticum

seeds: Purification, characterization, and kinetic properties,

Phytochemistry, 67 (2006) 232.

11 Hung C H, Huang C C, Tsai W S, Wang H L & Chen Y L,

Purification and characterization of a trypsin inhibitor from

Brassica campestris seeds, J Yuanpei Univ Sci Tech, 10

(2003) 13.

12 Gomes C E M, Barbosa A E D, Macedo L LP, Pitanga J C

M, Moura F T, Oliveira A S, Moura R M, Queiroz A F S,

Macedo F P, Andrade L B S, Vidal M S & Sales M P, Effect

of trypsin inhibitor from Crotalaria pallida seeds on

Callosobruchus maculates (cowpea weevil) and Ceratitis

capitata (fruit fly), Plant Physiol and Biochem, 43 (2005)

1095.

13 Mufti Asif-Ullah, Key-Sun Kim & Yeon Gyu Yu, Purification

and characterization of a serine protease from Cucumis

trigonus Roxburghi, Phytochemistry, 67 (2006) 870.

14 Edwin H W Leung & NG T B, A relatively stable antifungal

peptide from buckwheat seeds with antiproliferative activity

toward cancer cells, J Pep Sci, 13 (2007) 762.

15 Singh R R & Appu Rao A G, Reductive unfolding and

oxidative refolding of a Bowman-Birk inhibitor from

horsegram seeds (Dolichos biflorus): Evidence for ‘hyper

reactive’ disulfide bonds and rate-limiting nature of disulfide

isomerization in folding, Biochem Biophys Acta, 1597 (2002)

280.

16 Zhang Y, Kouzuma Y, Miyaji T & Yonekura M,

Purification, characterization, and cDNA cloning of a

Bowman-Birk type trypsin inhibitor from Apios americana

Medikus tubers, Biosci Biotechnol Biochem, 72 (2008) 171.

17 Macedo MLR, Freire MGM, Cabrini EC, Toyama MH,

Novello JC & Marangoni S, A trypsin inhibitor from

Peltophorum dubium seeds active against pest proteases and

its effect on the survival of Anagasta kuehniella

(Lepidoptera:Pyralidae), Biochm Biophys Acta, 1621 (2003),

170.


374

18 Selitrennikoff C L, Antifungal proteins, Appl Environ

Microbiol, 67 (2001) 2883.

19 Basir Y J, Knoop F C, Dulka J & Conlon J M, Multiple

antimicrobial peptides and peptides related to bradykinin and

neuromedin N isolated from the skin secretions of the North

American pickerel frog, Rana palustris, Biochim Biophys

Acta, 1543 (2000) 95.

20 Mignogna G, Pascarella S, Wechselberger C,

Hinterleitner C, Mollay C, Amiconi G, Barra D & Kreil

G, BSTI, a trypsin inhibitor from skin secretions of

Bombina bombina related to protease inhibitors of

nematodes, Protein Sci, 5 (1996) 357.

21 Conlon J M & Kim J B, A protease inhibitor of the Kunitz

family from skin secretions of the tomato frog, Dyscophus

guineti (Microhylidae), Biochem Biophys Res Commun, 279

(2000) 961.

22 Adams D J B E, Causier K J, Mellor V, Keer R, Milling J &

Dada, Regulation of chitin synthase and chitinase in fungi. In

Muzzarelli R A A (ed.,), Chitin Enzy, (1993) 15.

23 Garcia-Olmedo F, Molina A, Sefura A & Moreno M, The

defensive role of non-specific lipid transfer proteins in

plants, Trends Microbiol, 3 (1995) 72.

24 Soares-Costa A, Beltramini L M, Thiemann O H &

Henrique-Silva F, A sugarcane cystatin: Recombinant

expression, purification, and antifungal activity, Biochem

Biophys Res Commun, 296 (2002) 1194.

25 Aramburu J, Yaffe M B, Lopez-Rodriguez C, Cantley L C,

Hogan P G & Rao A, Affinity-driven peptide selection of an

NFAT inhibitor more selective than cyclosporine A, Science,

285 (1999) 2129.

26 Ye X Y, Ng T B & Rao P F, A Bowman-Birk type trypsin-

chymotrypsin inhibitor from broad beans, Biochem Biophys

Res Commun, 289 (2001) 91.

27 Oliveira M D L, Andrade C A S, Santos-Magalhaes NS,

Coelho L C B B, Teixeira J A, Carneiro-da-Cunha M G &

Correia M T S, Purification of a lectin from Eugenia uniflora

L. seeds and its potential antibacterial activity, Lett Appl

Microbiol, 46 (2008) 371.

antimicrobial activity of protease inhibitor from leaves of...

Documents