Arthropod-Plant Interactions DOI 10.

1007/s11829-011-9167-y

ORIGINAL PAPER

A proteinaceous thermo labile a-amylase inhibitor from Albizia lebbeck with inhibitory potential toward insect amylases
Neeta D. Kalve Purushottam R. Lomate Vandana K. Hivrale

Received: 16 July 2011 / Accepted: 9 November 2011 Springer Science+Business Media B.V. 2011

Abstract Insects feeding on stored grains cause considerable damage to harvested cereals and legumes every year. The use of a-amylase inhibitors to interfere with the pests digestion process has become an interesting alternative biocontrolling agent. In this study, we have studied the interactions of a-amylase inhibitors from Albizia lebbeck seeds with the amylases of coleopteran and lepidopteran insect pests. We isolated and puried the a-amylase inhibitor using acetone precipitation and gel ltration chromatography. Two prominent activity bands of a-amylase inhibitors were detected in electrophoretic analysis using 8% starch PAGE. We found that the a-amylase inhibitor, isolated as a monomer, had a molecular weight of 14.4 kDa. The a-amylase inhibitor was puried 36.15-fold with gel ltration chromatography. Its specic activity was determined at 14.4 U/mg/min. Feeding analysis of Tribolium confusum larvae on a diet containing puried a-amylase inhibitor from Albizia lebbeck revealed that survival of the larvae was severely affected, with the highest mortality rate occurring on the fth day of feeding. We found that the isolated a-amylase inhibitor inhibits T. confusum and Helicoverpa armigera a-amylases in electrophoretic analysis as well as in solution assays. The isolated a-amylase inhibitor was found to be resistant to commercial protease as well as T. confusum and

H. armigera digestive proteinases. The isolated a-amylase inhibitor was degraded by heating above 60C. Our results suggest that A. lebbeck a-amylase inhibitor could be a useful future biocontrolling agent. Keywords Albizia lebbeck a-amylase inhibitor Biocontrolling agent Insect amylases

Introduction Insect pests are responsible for severe crop losses. Worldwide, losses in agricultural production due to pest attack are around 40%. Several starch-dependent insect pests utilize a-amylase for the digestion of carbohydrate to obtain energy. To carry out efcient digestive processes, different forms of a-amylase can be found in insect species. These insects are totally dependent on a-amylase for their survival; therefore, a-amylases are good target candidates for bio-insecticides by using a-amylase inhibitors (Franco et al. 2002; Shivakumar et al. 2006). Plants contain secondary compounds that have a regulatory or defensive role. Proteinaceous inhibitors of enzymes are important due to their major role in plant defense (Ryan 1990). a-amylase inhibitors are extensively found in many plant seeds and tubers and predominantly abundant in cereals and legumes (Franco et al. 2002; Svensson et al. 2004; Giri and Kachole 1998; Hao et al. 2009; Wisessing et al. 2010). Since the amylase inhibitors are good candidates to control storage pests, this area has received increasing interest during past 40 years. Some a-amylase inhibitors show strict target enzyme specicity and recognize only one out of several closely related isozymes or enzymes from different species (Franco et al.

Handling Editor: Chen-Zhu Wang N. D. Kalve P. R. Lomate V. K. Hivrale (&) Department of Biochemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431004, Maharashtra, India e-mail: vkhbiochem@gmail.com

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2000; Svensson et al. 2004) Other inhibitors have high afnity for both mammalian and insect a-amylases that share around 35% sequence identity. Several examples, moreover, are found of inhibitors acting on insect but not on mammalian enzymes and vice versa (Franco et al. 2002). Proteinaceous a-amylase inhibitors are found in microorganisms, plants and animals (Franco et al. 2000; Silano 1987; Iulek et al. 2000). In plants, proteinaceous inhibitors are mainly present in cereals such as wheat Triticum aestivum (Franco et al. 2000; Petrucci et al. 1976; Feng et al. 1996) barley Hordeum vulgareum (Abe et al. 1993), sorghum Sorghum bicolor (Bloch and Richardson 1991), rye Secale cereale (Iulek et al. 2000; Garcia-Casado et al. 1994) and rice Oryza sativa (Yamagata et al. 1998) and also in leguminosae such as pigeonpea Cajanus cajan (Giri and Kachole 1998), cowpea Vigna unguiculata (Melo et al.1999), bean Phaseolus vulgaris (Grossi de Sa et al. 1997; Young et al. 1999) and mungbean (Wisessing et al. 2010). In recent years, a-amylase inhibitors are available as genetic sources for production of insect resistant transgenic crops (Gatehouse and Gatehouse 1998). Enzyme inhibitors could be an efcient strategy to control phytophagous and storage seed insect pests (Shivakumar et al. 2006). A study demonstrated the efciency of bean a-amylase inhibitor 1 against digestive amylases of the important Coleopteran pest pea weevil (Bruchus pisorum) especially when utilized in genetically engineered plants (Morton et al. 2000). Seeds of little millet (Panicum sumatrense) and nger millet (Eleusine coracanas) are observed to be practically free from insect attack after long-time storage, probably due to the presence of some toxic and antibiotic chemical compounds (Rajendran and Thayumanvan 2000). a-amylase inhibitors with insecticidal potential from these plants were identied, and their interactions with a-amylases from various economically important insect species were further studied (Shivakumar et al. 2006). Taking this into account, identication and characterization of novel potent amylase inhibitors with insecticidal potential are needed and it is also important to use the a-amylase inhibitors as genetic engineering candidates. The focus of the present study is the identication, purication and characterization of amylase inhibitors from Albizia lebbeck and their in vitro interactions with mammalian salivary amylase and several insect amylases. We screened several plants to nd potent amylase inhibitors. We found that the seeds of A. lebbeck possess excellent amylase inhibitory activity. Amylase inhibitors from A. lebbeck were identied, puried and characterized with respect to their biochemical properties and in vitro interaction with mammalian salivary amylase and several insect amylases.

Materials and method Materials Starch, dinitrosalicylic acid, iodine and chemicals for electrophoresis were obtained from Sisco research laboratories, Mumbai, India. Low molecular weight markers were purchased from Genei, Bangalore, India. All other chemicals used were of high analytical grade. Seeds of A. lebbeck were purchased from local market in Aurangabad, India. Larvae of Tribolium confusum were collected from rice grains, and larvae of Helicoverpa armigera were collected from chickpea elds. Extraction of inhibitors Seeds were nely ground and defatted. Defatting and depigmentation were carried out by Folchs mixture (chloroform: methanol = 1:2) and several washes of hexane (100 ml/wash). Defatted seed powder was dissolved in distilled water (1:6 w/v) containing 1% PVP and kept for overnight extraction at 4C. The extract was centrifuged at 10,000 rpm at 4C for 20 min, and the step was repeated 23 times to complete removal of debris. The supernatant was collected and divided into small aliquots and kept at -20C until use. The supernatant was used as crude inhibitor extract. Protein concentration in extract was estimated by Lowrys method (Lowry et al. 1951) using bovine serum albumin as standard. Detection of AI by starch PAGE Amylase inhibitors were detected by using the method of Giri and Kachole (1996). AI was electrophoretically separated on 8% polyacrylamide gel containing 0.5% soluble starch. Electrophoresis was carried out using Davis buffer system (Davis 1964) at constant current of 20 mA. After electrophoresis, the gel was washed twice with distilled water and equilibrated with 0.1 M phosphate buffer pH 6.9. Then the gel was placed in salivary amylase solution (1:10 dilution with 0.1 M phosphate buffer pH 6.9) for 1 h at 37C. Again the gel was washed twice with distilled water, placed in iodine solution (10 mM iodine in 14 mM potassium iodide) for 45 min and excess iodine was removed by washing the gel with distilled water. Effect of proteases Effect of proteases on AI was checked using 8% starch PAGE. The commercial bovine trypsin, chymotrypsin, pepsin and papaya papain were used for the experiments with the concentration of 1 mg/ml. Seed extract (5 lg of protein) was incubated with 20 ll of trypsin or chymotrypsin or pepsin or

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papain or T. confusum or H. armigera proteases for 1 h at 37C. The mixture was then resolved on 8% starch PAGE. Amylase inhibitor bands were detected by iodine staining as previously described. Thermal stability Thermal stability of AI was checked by subjecting the AI to different temperatures or at 100C for different time intervals. Seed extracts (5 lg of protein) were heated at 20, 40, 60, 80 and 100C for 30 min respectively or at 100C for 20, 30, 40, 50 and 60 min, respectively. After heating, the samples were immediately cooled and electrophoresed on 8% PAGE containing 0.5% soluble starch. AI bands were detected by using iodine staining as previously described. Acetone precipitation To the supernatant, three volumes of chilled acetone was added and kept overnight at -20C and centrifuged at 10,000 rpm for 20 min; acetone was removed by air drying and precipitate was collected. Precipitate was dissolved in minimum amount of 0.1 M phosphate buffer pH 7.0, centrifuged at 10,000 rpm for 20 min and supernatant was collected. Purication Three grams of Sephadex G-50 gel was dissolved in 0.1 M phosphate buffer, pH 7.0, and allowed to swell for overnight and column was packed. Acetone precipitate (5 ml) was loaded onto Sephadex G-50 column (1.2 9 50 cm) equilibrated with 0.1 M phosphate buffer, pH 7.0. Fractions of 2 ml were collected, and protein concentration was estimated by Lowrys method (Lowry et al.1951) using bovine serum albumin (BSA) as standard. All collected samples were screened for amylase inhibitor activity (AI). Molecular mass determination Molecular mass of puried AI was determined with sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) according to the procedure of Laemmli, (Laemmli 1970). Crude extract (20 lg), acetone precipitate (20 lg) and puried AI (20 lg) were loaded on 12% SDS-PAGE with standard molecular weight markers, and electrophoresis was carried out at constant current of 20 mA. After electrophoresis, the gel was stained with CBBR-250 and distained to visualize protein bands.

Dietary studies Bioassays were conducted by feeding T. confusum larvae on a control diet containing defatted wheat powder and on a test diet containing puried A. lebbeck AI (10 g/kg) incorporated into defatted wheat powder. Twenty (on each diet) early second-instar larvae were reared on these diets, and their rates of pupation and mortality were recorded at 24-h intervals. The assay was started at time zero and continued up to 5 days. The larvae surviving after the rst day continued to be fed on the same diet on the second day and so on. The experiment was repeated at least three times in triplicate. Extraction of amylases from insects Whole 200 larvae (34th instars) of T. confusum were weighed and homogenized in 6 volumes of 0.1 M phosphate buffer pH 7 (w/v). For H. armigera, midguts of fty 45th instars larvae were removed and homogenized in 6 volumes of 0.1 M Glycine NaOH buffer, pH 9.6 (w/v). The homogenates were centrifuged at 10,000 rpm at 4C for 10 min, and the supernatant was used as crude preparation of amylases. The supernatant was stored at -20C until needed. Amylase assay Amylase activity was assayed using modied Bernfelds method (Bernfelds 1955). Starch solution (1%) was prepared in 20 mM phosphate buffer, pH 6.9, containing 6.7 mM NaCl. Starch solution (0.5 ml) was taken in glass tubes and incubated at 37C. T. confusum and H. armigera amylase extracts (0.1 ml) were added to starch pre-incubated in 20 mM phosphate buffer (pH 6.9). The reaction mixtures were incubated at 37C for 15 min, and the reaction was terminated by adding DNS reagent (0.5 ml). All the tubes were kept in boiling water bath after the addition of DNS reagent for 5 min, cooled immediately under tap water and the reaction mixtures were diluted by adding 3 ml of distilled water. Amylase activity was determined from the absorbance of the color formed at 530 nm. Amylase activity was dened as one milligram of maltose liberated per milligram of protein per min at 37C. Amylase inhibitor assay Appropriate amounts (20 ll) of puried AI were mixed with amylase extracts of T. confusum and H. armigera larvae, and the volume of reaction mixture was made up by adding corresponding extracting medium. The reaction mixture was assayed for residual amylase activity as described above. Amylase inhibitor activity was dened as

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decrease in one milligram of maltose liberation per milligram of protein per min at 37C. Ingel inhibition of insect amylase isoenzymes Inhibition of T. confusum and H. armigera amylase isozymes was checked using 8% PAGE according to Davis buffer system (Davis 1964). Extracts (20 ll) of insect amylases were pre-incubated with 20 ll of puried AI for 30 min at 37C. The mixtures were electrophoresed; electrophoresis was carried out at constant current of 20 mA. After electrophoresis, the gel was washed with distilled water and equilibrated in 0.1 M phosphate buffer pH 6.9 for 10 min. Then the gel was placed in 1% starch solution (prepared in same buffer), incubated for 1 h at 37C and dipped in iodine solution (10 mM I2 and 14 mM KI) for 510 min. Finally, the gel was washed with distilled water, and amylase isozymes were visualized.

Results Detection of AI from A. lebbeck Ingel detection of AI was carried out using 8% starch PAGE. In the seed extract of A. lebbeck, two prominent AI activity bands were well separated on native gel and designated as AI1 and AI2. The mobility of AI1 was low as compared to AI2 (Fig. 1). Stability of AI to proteolysis T. confusum and H. armigera digestive proteases and commercial proteases such as trypsin, chymotrypsin, pepsin and papain were used to check the stability of AI to proteolysis. Study revealed that AI was not degraded by proteases. Any used protease was not able to cleave AI. We also found that the isolated inhibitor was resistant to T. confusum and H. armigera digestive proteases (Fig. 2). Stability of AI to heat Stability of AI was checked by subjecting it at different temperatures or at 100C for different time intervals. Study revealed that AI was able to retain its activity up to 60C and further the AI activity was declined at 80 and 100C (Fig. 3a). At 100C, AI activity was not detected at any time intervals (Fig. 3b). Purication and molecular weight determination AI from seed extract of A. lebbeck was puried by gel ltration chromatography, and we recorded 36.15-fold
Fig. 1 In-gel detection of amylase inhibitors. Seed extract of A. lebbeck was loaded on 8% native starchpolyacrylamide gel containing 0.5% soluble starch, and amylase inhibitor bands were visualized by iodine staining. Lane 1 and 2, 20 ll seed extract. Activity bands separated on native gel were designated as AI1 and AI2

Fig. 2 Stability of AI to proteolysis by commercial proteases, T. confusum and H. armigera digestive proteases. Amylase inhibitors (20 ll) incubated with 20 ll of each commercial protease and 20 ll of T. confusum and H. armigera enzymes for 37C for 60 min. Reaction mixture was resolved on 8% starch PAGE, and the gel was stained as described in Materials and methods. Lane 1 20 ll seed extract, lane 2 seed extract treated with trypsin, lane 3 seed extract treated with chymotrypsin, lane 4 seed extract treated with pepsin, lane 5 seed extract treated with papain, lane 6 seed extract treated with T. confusum proteases and lane 7 seed extract treated with H. armigera gut proteases

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Fig. 3 Detection of heat stability of amylase inhibitors of A. lebbeck on 8% starch PAGE. a Stability at different temperatures. Seed extracts (30 ll) were pre-incubated at 20, 40, 60, 80 and 100C for 30 min and loaded on 8% starch PAGE. AI bands were visualized by iodine staining. Lane 1 seed extract, lane 2 20C, lane 3 40C, lane 4 60C, lane 5 80C and lane 6 100C. b Stability at 100C for different Table 1 Purication of A. lebbeck amylase inhibitor (AI) Purication steps Crude seed extract Acetone precipitate Gel ltration Vol in ml 100 20 20 Protein in mg/ml 0.63 0.33 0.13

time intervals. Seed extracts (30 ll) were boiled at 100C for 20, 30, 40, 50 and 60 min, respectively. Inhibitors were separated and visualized by starch PAGE method. Lane 1 seed extract, lane 2 20 min, lane 3 30 min, lane 4 40 min, lane 5 50 min and lane 6 60 min, respectively

Total protein (mg) 63 6.6 2.6

Total AIU 3,176 765.4 236.95

Total AIU/mg of protein 50.41 115.96 1,822.6

Purication fold 1 2.30 36.15

Total protein, total AIU and purication fold of each step of purication are given in the table

increase in the AI activity after purication (Table 1). The molecular weight of the puried AI was obtained to be *14.4 kDa (Fig. 4). We could not purify all of the two AIs as we observed only one band on SDS-PAGE. Bioassay Tribolium confusum larvae were fed on control (wheat powder) and test (wheat powder plus puried AI) diets. The results of the feeding experiments in terms of mortality rate recorded for 5 days are summarized in Fig. 5. On the rst day, mortality rate of larvae was slightly higher on test diet as compared to control diet. On the second day of feeding, due to the toxic action of the inhibitor, it was observed that the mortality rate of larvae was signicantly increased for larvae fed on the test diet compared with those fed on the control diet. This pattern continued over the 5-day period. Therefore, throughout the feeding assay, a rate of survival was strongly affected by the toxic effect of puried AI from A. lebbeck (F = 0.015, df = 5, P \ 0.01) (Fig. 5). Interaction of AI with insect amylases Interaction of AI with T. confusum and H. armigera amylase was checked in solution assays as well as in

electrophoretic analysis. Electrophoretic study revealed that all the amylase isozymes of T. confusum and H. armigera were inhibited by isolated inhibitor (Fig. 6a). In solution assays also, we observed that the amylase activity of T. confusum and H. armigera amylases was strongly inhibited by isolated amylase inhibitor (Fig. 6b).

Discussion Since insect a-amylases play an important role in carbohydrate metabolism, an important pest control strategy focuses on the understanding of a-amylase inhibitor activity and specicity (Franco et al. 2000; Laskowski and Kato 1980). In spite of the presence of inhibitors, insects feed on seeds and overcome plant defense. Two factors seem to be responsible to this phenomenon. First, many plants suffer reductions in defense compounds during domestication (Hilder et al. 1987). Secondly, just as plants evolve defenses, their predators evolve means to evade those defense mechanisms (Franco et al. 2002). Several inhibitors of amylases have been characterized and proved their effectiveness against variety of storage pests (Franco et al. 2002). It is therefore necessary to study novel host and non-host plant amylase inhibitors as potential sources to overcome the adaptive strategies of insect pests.

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Fig. 4 SDS-PAGE analysis of purication steps of A. lebbeck inhibitor on 12% polyacrylamide gel followed by Coomassie Brilliant Blue R-250 staining. Lane 1 molecular weight marker proteins, lane 2 crude protein (20 lg), lane 3 acetone precipitate (20 lg) and lane 4 puried amylase inhibitor from Sephadex G-50 (20 lg)

Fig. 5 Effect of puried amylase inhibitor (AI) from A. lebbeck on the survival of T. confusum larvae. The bioassay was conducted by feeding T. confusum larvae on a diet containing puried AI. The assay was started at time zero and continued up to 5 days and mortality of larvae was recorded. The experiment was repeated at least three times in triplicate. The rate of mortality was signicantly increased in larvae fed on the AI-containing diet as compared with the control diet throughout the bioassay. Vertical bars represent standard deviations. Signicant differences were calculated by Students t tests: *P \ 0.05; **P \ 0.01

In the present work, we found two a-amylase inhibitor isoforms in the seed extracts of A. lebbeck. The AIs from A. lebbeck showed strong inhibitory activity toward human salivary, T. confusum and H. armigera amylases. Two AI bands were detected on starch PAGE, and they were

characterized with respect to their biochemical properties and inhibitor potential toward the insect amylases. A. lebbeck AI was partially puried with gel ltration chromatography. In this purication step, the amylase inhibitor activity was increased up to 36.15-fold. Molecular weight of puried AI was obtained to be *14.4 kDa. On the molecular weight estimate of *14.4 kDa, the identied AI may belong to cereal type amylase inhibitor family (Franco et al. 2002). Thermostability of AI from A. lebbeck was checked, and we found that the AI retained its activity up to 60C and further it was degraded by heating. This property of the AI is very important when it will be used for transgenic purpose. After transgenic expression, the AI should be degraded by heating while using as human food. If the AI from A. lebbeck transgenically expressed, it will be degraded by cooking and became safe for human consumption. Therefore, the AI from A. lebbeck might be a good candidate for transgenic expression. It is also important that the AI should be stable to proteolysis, because proteinacious plant AIs can be degraded by insect gut proteases when it is ingested in digestive tracts of insects. In our investigation, we observed that the AI from A. lebbeck was stable to proteolysis and could not be degraded by commercial proteases. This property of AI from A. lebbeck is also important to use it as biocontrolling agent. It is also important to report that both the AI isoforms were totally resistant to hydrolysis by gut enzymes of H. armigera and T. confusum. Similar study was carried out by Chrispeels et al. (1998) on Phaseolus acutifolius who showed that a-AI2 and a-AI Pa of Phaseolus acutifolius are resistant to proteases of Z. subfasciatus and supports the use of these candidates for genetic engineering approaches. Feeding analysis conrmed the efcacy of the AI of A. lebbeck against the storage pest T. confusum. Our observations indicate that incorporation of the puried AI in the diet affects the rate of survival of larvae as compared with the control. We also observed that, due to the insufciency of energy source, larvae eventually died. Similar results were observed when C. maculates, H. armigera and Periplaneta americana larvae were fed on non-host plant proteinase inhibitors (Harsulkar et al. 1999; Hivrale et al. 2011a; Hivrale et al. 2011b; Lomate and Hivrale 2011). Several insect pests synthesize at least two a-amylase isozymes at digestive tract (Franco et al. 2005; Silva et al. 1999). Study of Shivakumar et al. (2006) revealed that more than one isozymes of a-amylase were detected in midgut crude extracts of eight insect pests. Silva et al. (1999) reported the presence of large number of a-amylase isozymes is an efcient strategy of insects to escape from inhibitors for toxicity. As observed before, proteinacious inhibitors that inhibit digestive a-amylases could induce the expression of insensitive a-amylases as observed for H. armigera guts treated with pigeonpea inhibitors (Giri

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Fig. 6 Inhibition of insect amylases by A. lebbeck AI. a Ingel inhibition of T. confusum and H. armigera amylases. Insect amylases and inhibitors were pre-incubated at 37C for 30 min prior to electrophoresis and then stained with iodine for visualization as described in Materials and methods. Lane 1 20 ll T. confusum amylase extract, lane 2 20 ll T. confusum amylase extract incubated with 10 ll of A. lebbeck, lane 3 20 ll H. armigera amylase extract and lane 4 20 ll T. confusum amylase extract incubated with 10 ll of

A. lebbeck. b Inhibition of T. confusum and H. armigera amylases in solution assay. Insect amylases and inhibitors were pre-incubated at 37C for 30 min, and the assay was carried out as described in Materials and methods. CAT. confusum amylase extract, CA ? AIT. confusum amylase extract ? amylase inhibitor, HA H. armigera amylase extract, HA ? AIH. armigera amylase extract ? amylase inhibitor

and Kachole 1998). Taking all these facts in mind, it is important to nd out the amylase inhibitors that should be stable resistant to a variety of amylases present in insect digestive tracts. In this connection, our study revealed that the AIs from A. lebbeck were able to inhibit the amylase activity from representative coleopteran and lepidopteran insects. Electrophoretic analysis and solution assays revealed that AI from A. lebbeck inhibited the amylase activity from T. confusum and H. armigera. Due to the existence of more than one a-amylase isoform in a given insect species, the inhibitory specicity is an important initial step in the discovery of molecules that could be useful for generating insect resistant transgenic plants (Morton et al. 2000). The a-amylase diversity found in studied insects (Shivakumar et al. 2006) indicates that unless an a-amylase inhibitor with reasonably broad specicity capable of inhibiting all a-amylase isozymes produced by target insect, their expression in transgenic plants would probably have no impact on starch digestion by insect pests. So, the use of a-amylase inhibitors with different insect specicities could be combined to improve pest control (Franco et al. 2002). The characterization of AI from A. lebbeck and its promising properties such as degradation by heat, resistant to proteolysis and inhibitor specicity toward the amylases of different insect species reported in the present study might provide novel information that could contribute to pest management programs.

Acknowledgments VKH wish to express her gratitude to University Grant Commission, New Delhi, for providing the nancial support to carry out the present work.

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