J. Plant Biochemistry & Biotechnology Vol.

19(1), 103-105, January 2010

Short Communication

Studies on Trypsin Inhibitor in Bean (Phaseolus vulgaris L)

Cultivars of Himalayan Region

Raj Deepika1* and Amarjit Kaur Nath

Department of Biotechnology, Dr Y S Parmar University of Horticulture and Forestry, Solan 173 230, Himachal Pradesh, India

Eight Phaseolus vulgaris L cultivars of Himalayan region were analyzed for trypsin inhibitor activity and inhibition of gut
trypsin enzyme extracted from Spodoptera littoralis larvae. Trypsin unit inhibited per gram seed weight was maximum in
local yellow cultivar. The trypsin inhibitor was purified to 65.9-fold with 55.8% recovery from seeds of selected cultivar. The
purified protein had a molecular weight of 14,130 Daltons and was found to be a monomer by SDS-PAGE. It was heat stable
at 100C for 10 minutes and had a pH optimum of 7.5. Hence, the purified inhibitor appears to be of Bowman-Birk type. It lost
its activity on exposure to 0.2M 2-mercaptoethanol. The inhibition pattern was of non-competitive type and the K i value was
0.8M. The KM value of trypsin enzyme for the substrate BApNA was 2.2mM.

Key words: Phaseolus vulgaris L, Trypsin inhibitor.

Protease inhibitors are ubiquitous molecules, found in
animals, plants and microbes. These protease inhibitors
inhibit the proteolytic activity of certain enzymes by forming
complexes with them. They are usually present in seeds
and reserve organs (1,2) but are also expressed in the
aerial parts of plants upon insect attack or mechanical
wounding (3,4). Their activity on gut proteases attenuates
amino acid assimilation and slows the growth of feeding
insects. Owing to the direct defensive properties of protease
inhibitors several transgenic plants have been produced
expressing specific inhibitors and tested for enhanced
resistance against phytophagous insects. Genetic
engineering of crops using insecticidal protein gene is
one of the major areas of research in plant biotechnology.
It could reduce the cost, time and efforts spent on protecting
crops from insects and also contribute to environment
friendly protection system. The protease inhibitors have
been purified and characterized from a wide variety of
legume seeds (5-8). However, the information on isolation,
purification and characterization of trypsin inhibitor from
native beans of Himalayan region is still lacking.
*Corresponding author. E-mail: rajdeepika@gmail.com
1
Present address: Department of Genetics, Faculty of Natural and
Agricultural Sciences, University of Pretoria, Pretoria 0002, South
Africa
Abbreviations: DEAE diethyl amino ethyl; BSA - bovine serum
albumin; TUI tripsin unit inhibited
Seeds of eight Phaseolus vulgaris L cultivars namely
local yellow, panth anupma, luxmi, contender, EC-108101,
EC-8392, PDR-21 and BLD-14 were procured from
Vegetable Research Station, Kalpa, HP, India and
Department of Vegetable crops, Dr Y S Parmar University
of Horticulture and Forestry, Solan (HP) India. The protein
was extracted from seeds following the protocols given by
Hajela et al (8) and Maggo et al (5). Seeds were ground to
make a fine powder, flour was defatted with acetone (1:10w/
v) and air dried for 3-4 times. One gram of defatted flour
was shaken with 10ml (1:10w/v) of the extraction media
(distilled water, 0.1M Sodium phosphate buffer; pH 7.5 and
0.2% NaCl) in shaking water bath for 4 h at room
temperature, separately. The suspensions obtained were
centrifuged at 10,000xg for 30 min at 4C. The results of
trypsin inhibition assay showed that the three extraction
media could extract the trypsin inhibitor equally well and
distilled water was selected as extraction medium. For
trypsin inhibition studies, 1.0mg of bovine trypsin was
dissolved in 10ml of buffer-II (prepared fresh by mixing
5.0ml 0.1M phosphate buffer pH7.5, 0.5ml 0.1M CaCl2,
0.036ml 3M NaCl and 4.464ml distilled water). Alternately,
the midgut enzymes from actively growing lab cultured
fourth instar Spodoptera littoralis larvae was extracted
according to the protocol standardized by Raj Deepika et
al (4). One ml of larval midgut extract or trypsin enzyme
solution was mixed with 0.1ml of crude extract of P. vulgaris
cultivars and pre-incubated in a metabolic shaking water
104 J Plant Biochem Biotech

bath at 37C for 10 min followed by addition of 0.6ml of 5,01,187 Dalton), Gamma globulin (mol wt 1,58,489),
buffer-I (prepared fresh by mixing 10ml 0.1M phosphate Bovine serum albumin (mol wt 66,000) and Myoglobulin
buffer; pH7.5, 4.0ml 0.1M CaCl2 and 6.0ml distilled water) (mol wt 15,848). The molecular weight was also determined
and 0.3ml of BApNA (4.0mg of D-Benzoyl-DL-arginine-p- by SDS-PAGE using mixture of molecular weight marker
nitroanilide was dissolved in 0.1ml of dimethyl sulphoxide proteins viz., Bovine serum albumin (mol wt 67,000),
by vortexing and final volume was made 1.0ml by adding Ovalbumin (mol wt 43,000) and Lactalbumin (mol wt
0.9ml buffer-I). The total volume of the reaction mixture 14,400).
was 2ml. The reaction mixture was incubated at 37C in
To study the thermal stability of purified product,
shaking water bath for 10 min and reaction was stopped
reaction was carried out at different incubation
by adding 0.5ml of 30% acetic acid. In blank, same
temperatures (30-100C). The pH stability was determined
procedure was adopted except for the fact that BApNA
using different buffers viz. acetate buffer (pH 4.0 to 6.0),
was not added to the reaction mixture. In control, buffer-I
phosphate buffer (pH 6.0 to 8.0) and Tris buffer (pH 8.0 to
was added in place of trypsin inhibitor to the reaction
9.0). Lineweaver-Burk plot (10) was plotted using different
mixture. The optical density of test and control mixtures
concentrations of substrate at two fixed inhibitor
was measured at 410nm against the blank using UV/VIS
concentrations (12.2g and 53.3g) and K M (Michaelis-
spectrophotometer to measure trypsin inhibitor activity.
Menten constant) value was determined. Dixons plot (11)
For purification, the crude extract was prepared from was plotted using different concentrations of inhibitor at
10g defatted flour of selected bean cultivar following the two fixed concentrations of substrate (2.3mM and 4.6mM)
method described previously. The crude extract was to determine the Ki (Inhibitor constant) value. The effect of
saturated to 20% followed by 70% with ammonium 8M urea and reducing agent 0.2M 2-mercaptoethanol on
sulphate and allowed to precipitate overnight at 4C. The the stability of purified trypsin inhibitor was studied. The
precipitated protein was collected by centrifugation at inhibitor was incubated with 8M urea and 0.2M 2-
10,000xg for 15 min and the pellet was resuspended in a mercaptoethanol and trypsin inhibition assay was
small volume of distilled water. The precipitates were performed after 1h and 24h of incubation.
dialysed for 24h against distilled water. The dialysed (20-
Considerable variation was observed among the
70%) ammonium sulphate fraction was subjected to
cultivars for trypsin inhibitor activity and for the inhibition of
Sephadex G-100 column (60x2.5cm; flow rate 12ml hr-1).
gut trypsin enzyme extracted from S. littoralis larvae. The
The column was eluted with distilled water and 3ml
trypsin unit inhibited (TUI) per g seed weight was maximum
fractions were collected. The most active fractions were
(818.3 and 892.7) in local yellow and minimum (37.7 and
pooled and subjected to DEAE(diethylaminoethyl)
55.7) in EC-108101 (Table 1). Hence, local yellow cultivar
Sephadex column (10x1.0cm; flow rate 12ml hr-1). The was selected for further studies using Sephadex G-100
column was eluted with distilled water to wash out the
unbound proteins. The bound proteins were eluted with
Table 1. Trypsin inhibitor activity in different Phaseolus vulgaris
linear salt gradient using 3 bed volumes of 0.1M, 0.2M,
cultivars
0.3M, 0.4M, and 0.5M KCl in distilled water. Fractions (2ml
Bean cultivar TUI per g TUI per g
each) were collected and monitored for protein content at seed weight seed weight
280nm and for trypsin inhibitor activity. The active fractions (using trypsin (using gut trypsin
enzyme) enzyme of
were pooled together and concentrated by freeze drying
S. littoralis larvae)
(-86C). The purity of trypsin inhibitor protein was judged
Local Yellow 818.3 1.202 892.7 0.333
by SDS-polyacrylamide gel electrophoresis using 9%
PDR-21 805.7 2.906 791.8 0.441
acrylamide gels and anionic system on vertical slab gel
Luxmi 741.0 1.893 715.5 0.289
electrophoresis. The soluble protein was estimated after
Panth anupma 681.5 0.289 702.2 1.302
each step of purification according to the method of Lowry
BLD-14 641.3 0.333 692.7 1.411
et al (9) using bovine serum albumin (BSA) as standard.
Contender 635.5 0.289 732.5 0.764
The molecular weight of purified inhibitor was estimated
EC-8392 159.0 0.289 495.7 0.441
by gel filtration through Sephadex G-100 column calibrated
EC-108101 37.7 0.441 55.7 0.436
with molecular weight markers namely, Apoferatin (mol wt

Table 2. Purification of trypsin inhibitor from Phaseolus vulgaris L cv Local Yellow
Step Total Total soluble
TUI protein (mg)
Crude extract 8160.1 2066.0
Ammonium sulphate precipitation 7614.1 1619.4
Gel filtration chromatography 4894.4
Ion exchange chromatography 4552.8
column and DEAE sephadex trypsin inhibitor was purified
to 65.86 fold with 55.8% recovery (Table 2). Maggo et al (5)
purified trypsin inhibitor to 80.2 fold with 28.7% recovery
from rice bean. SDS-PAGE of the isolated inhibitor showed
a single band proving it to be a monomer with the molecular
weight of 14,130Daltons (results not shown). Similar value
for molecular weight was obtained when determined by
gel filtration chromatography. Hajela et al (8) had purified
protease inhibitor from Phaseolus mungo to homogeneity
as evident from single band corresponding to molecular
weight of 14.2kD in SDS-PAGE. The purified trypsin
inhibitor was found to be stable over a wide range of
temperature (30C-100C) with maximum inhibitor activity
at 37 C. A double headed protease inhibitor having
molecular weight of 14,200 Daltons and with 50% loss of
inhibitory activity after heating at 100C for 20 min has
been purified from Phaseolus mungo (8).
The purified inhibitor appeared to have pH optimum
of 7.5 (results not shown). The nature of inhibition was
found to be non-competitive type as indicated by
Lineweaver-Burk plot and Dixons plot (results not shown).
The trypsin inhibitor of rice bean (5) also inhibited trypsin
non-competitively. The KM and K i value of the purified
inhibitor was found to be 2.2mM and 0.8M, respectively.
Low Ki value indicated high affinity of the inhibitor for trypsin.
The trypsin inhibitor from rice bean was found to have
high Ki value of 2.07mg per ml (5). The purified trypsin
inhibitor was also found to be susceptible to 0.2M 2-
mercaptoethanol. The inhibitor exhibited 21% and 11.74%
trypsin inhibitor activity after 1h and 24h of incubation,
respectively. However, if 0.2M 2-mercaptoethanol was
added to the inhibitor in presence of 8M urea, activity of
inhibitor was restored. This could be explained by the fact
that the three dimensional structure of inhibitor is stabilized
by disulphide bonds which gets reduced in the presence
of 2-mercaptoethanol. Thus, the inhibitor may become
incapable of inhibiting trypsin. Similar observations have
also been made earlier by Maggo et al (5) and Hajela et al
Short Communication 105
Specific activity Fold purification Percent yield
3.95 1.0 100.0
4.70 1.19 93.3
656.8 7.45 1.89 60.0
17.5 260.16 65.86 55.8
(8). The purified trypsin inhibitor showed thermal stability,
susceptibility to 2-mercaptoethanol and molecular weight
less than 20,000 Dalton indicating that the inhibitor could
be of Bowman-Birk type.
At present, there is a need to isolate more effective
trypsin inhibitor and identify novel insecticidal genes. The
inhibitor extracted from P. vulgaris was found to be effective
against midgut enzyme of S. littoralis. The purified trypsin
inhibitor was also found to be effective against Pieris
brassicae larvae as it showed marked effect on larval
survival and development (4). Thus, the present findings
and previous report points towards possibility of using this
trypsin inhibitor in crop protection against insect pests. In
future, combination of these insecticidal genes may be
used for conferring resistance against insect pests and
other plant pathogens in plants.
Received 2 February, 2009; accepted 11 November, 2009
Online published 31 December, 2009
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