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ORIGINAL PAPER
A kidney bean trypsin inhibitor with an insecticidal potentialagainst Helicoverpa armigera and Spodoptera litura
Anuradha Mittal • Rekha Kansal • Vinay Kalia •
Monika Tripathi • Vijay Kumar Gupta
Received: 8 April 2013 / Revised: 28 September 2013 / Accepted: 30 October 2013 / Published online: 15 November 2013
� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2013
Abstract In the present study, trypsin inhibitor extracts
of ten kidney bean seed (Phaseolus vulgaris) varieties
exhibiting trypsin and gut trypsin-like protease inhibitor
activity were tested on Helicoverpa armigera and Spo-
doptera litura. Trypsin inhibitor protein was isolated and
purified using multi-step strategy with a recovery
of *15 % and purification fold by *39.4. SDS-PAGE
revealed a single band corresponding to molecular mass
of *15 kDa and inhibitory activity was confirmed by
reverse zymogram analyses. The inhibitor retained its
inhibitory activity over a broad range of pH (3–11), tem-
perature (40–60 �C) and thermostability was promoted by
casein, CaCl2, BSA and sucrose. The purified inhibitor
inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies
showed that the protein is a competitive inhibitor with an
equilibrium dissociation constant of 1.85 lM. The purified
trypsin inhibitor protein was further incorporated in the
artificial diet and fed to second instar larvae. A maximum
of 91.7 % inhibition was obtained in H. armigera, while it
was moderate in S. litura (29 %) with slight varietal dif-
ferences. The insect bioassay showed 40 and 22 %
decrease in larval growth followed by 3 and 2 days delay in
pupation of H. armigera and S. litura, respectively. Some
of the adults emerged were deformed and not fully formed.
Trypsin inhibitor protein was more effective against
H. armigera as it showed 46.7 % mortality during larval
growth period compared to S. litura (13.3 %).
Keywords Kidney bean � Trypsin inhibitor �Helicoverpa armigera � Spodoptera litura �Artificial diet � Insect bioassay
Introduction
Plants are under constant attack by insect pests, and to
produce inhibitors against the insect’s gut proteases is one of
the potent plant defense responses (Ryan 1973). Several
studies have demonstrated that these inhibitor proteins are
specifically produced in the plant upon biotic stress, pro-
tecting the plant tissue from damage (Ryan 1990; Tatyana
et al. 1998). Some of these inhibitors include proteins such
as protease inhibitors (PIs), amylase inhibitors, lectins and
class of pathogenesis-related proteins (Ryan 1990; Tatyana
et al. 1998). Plant PIs have been well established to play a
potent defensive role against predators and pathogens.
Developing resistance to pesticides in Lepidopteran insect
pests is a significant economic, ecological and public health
issue. Agricultural industries develop alternative chemical
pesticides effective against these insect pests. Since the use
of these chemical pesticides has a deleterious effect on
human health, a recent trend is to use other safer strategies to
enhance the defense mechanism of crops. Bate and Roth-
stein (1998) proposed a ‘copy nature’ strategy for insect pest
Communicated by B. Barna.
Electronic supplementary material The online version of thisarticle (doi:10.1007/s11738-013-1433-4) contains supplementarymaterial, which is available to authorized users.
A. Mittal � V. K. Gupta (&)
Department of Biochemistry, Kurukshetra University,
Kurukshetra 136119, India
e-mail: vkgupta59@rediffmail.com
R. Kansal
NRC on Plant Biotechnology, Lal Bahadur Shastri Building,
PUSA Campus, New Delhi, India
V. Kalia � M. Tripathi
Division of Entomology, Indian Agricultural Research Institute,
New Delhi, India
123
Acta Physiol Plant (2014) 36:525–539
DOI 10.1007/s11738-013-1433-4
lndian J Agric Biochem26 (2),118-124,2013
Standardization of Trypsin Inhibitor Extraction from Kidney Beanand Gumulative Effect of Temperature, Incubation Time and pH onIts Activity
ANURADHA MITTAL1, SUSHIL NAGAR1, REKHA KANSAL', VUAY KUIVTAR GUPTAI'lDepartment of Biochemistry, Kurukshetra University, Kurukshetra-136119. lrdia2NRC on Plant Biotechnology, Lal Bahadur Shastri Building, PUSA Campus, New Delhi 11001 2, India
This work investigates the factors alfecting the extraction of trypsin inhibitor from kidney bean (Phaseolus vulgaris)seeds. Trypsin inhibitor extracted with 0.1 % NaCl rendered a higher recovery than other solvents. The extraction was alsoaffected by pH and extraction time. It took 5 h for complete recovery ot trypsin inhibitor activity. Response surfacemethodology was used to study the effect ot temperature and pH with respect to time on the extraction of trypsininhibitor. Statistical optimization studies revealed that trypsin inhibitor activity from kidney bean flour was stable to 60 tfor 52 min at neutral and acidic pH and labile under strong alkaline conditions.
Key words: Trypsin inhibitor activity, response surface methodology, optimizalion
Protease inhibitors (Pls) are small proteins that have
the ability to inhibit function of proteolytic enzymes. They
are widely distributed among different plant families andare found abundant (1-10% of total proteins) in storageorgans like seeds and tubers (1). Trypsin inhibitors (Tls),
i.e. protease inhibitors that inhibit trypsin, constitute 2-
5% of the total seed protein of the edible dry bean (2).
Due to th'eir ability to inhibit the enzymes involved in
digestive processes, they have been referred to as an'antinutritional factors'. Trypsin inhibitor differs from otherprotease inhibitor proteins in specificity and in potency
of inhibition. Some Tls of legume origin have potential
for insect enzyme inhibition, supporting a defense rolefor these proteins (3). Furthermore, it is also involved inprotecting seed proteins, either by preventing premature
hydrolysis of storage proteins or by acting as enzyme-stabilizing agents (4). Two main types of inhibitors areKunitz;type (KTl) and Bowman-Birk (BBl). The former
types of inhibitors strongly inhibit trypsin but have onlyweak effects on chymotrypsin (5). BBls are double-headed and can simultaneously inhibit bolh trypsin andchymotrypsin (5). They decrease lood intake by animals,reducing digestion and absorption ol dietary matter andlower retention of absorbed nitrogen, when included in
diets (6). However, in contrast to KTl, BBI does not
significantly impair food intake, food conversion efficiency
or growth of animals (6). Reducing the levels of these
anti-nutritional factors in beans would be expected toincrease the nutritive quality of the seed, and this indeed
appears to be the case with recently developed isolines
deficient in Kunitz protease inhibitor (7). However, sincePls are believed to be involved in protection of the seedfrom attack by specific bacteria, fungi or insects, anynut r i t iona l benef i t s a r is ing f rom th is reduc t ion in
antinutrient content (8) may be canceled by a higher
susceptibility of the crop to predators (5).
Protease inhibitors can be extracted by using alkalineextraction (9-11), aqueous salt solution with varying pH
(12, 13) or with water. Since the extraction of Tl may be
affected by several factors including the nature and pH
of extraction medium, duration of extraction etc, it was
considered worthwhile to optimize the Tl extraction from
wi th respec t to these fac to rs . Under op t im izedconditions, the yield of Tl protein is higher, which is often
desired for its purification and investigation of biologicalroles such as ant i fungal and ant i feedant potent ial .
Moreover. it is likely to give a better reflection of Tl levelin the plant t issue. Howe$er, invest igat ions on theextraction of Tl and the effect of temperature and pH
using response surface methodology (RSM) have been
"Author tor correspondence : Email : vkguptasg@rediffmail.com
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