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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