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Digestive amylase from the larger grain borer, Prostephanus truncatus Horn
Comparative Biochemistry and Physiology Part B 126 (2000) 425 – 433 www.elsevier.com/locate/cbpb
Digestive amylase from the larger grain borer, Prostephanus truncatus Horn E. Mendiola-Olaya a, A. Valencia-Jime´nez b, S. Valde´s-Rodrı´guez a, J. De´lano-Frier a, A. Blanco-Labra a,* a Department of Biotechnology and Biochemistry, Centro de In6estigacio´n y de Estudios, A6anzados del IPN, Unidad de Biotecnologı´a e Ingenierı´a Gene´tica de Plantas, Apto. Postal 629, C.P. 36500 Irapuato, Gto, Mexico b Facultad de Ciencias Exactas y Naturales, Uni6ersidad de Caldas, Manizales, Colombia
Received 25 November 1999; received in revised form 9 March 2000; accepted 20 March 2000
Abstract A combination of ion-exchange chromatography, preparative electrophoresis and gel filtration chromatography allowed a 1209-fold purification of one of the two major digestive a-amylases from larvae of the larger grain borer, Prostephanus truncatus Horn. The purified enzyme showed a molecular mass of 60.2 kDa, an isoelectric point of 4.7 and an optimal pH for activity of 6.0. The enzyme was heat labile and it was recognized by proteinaceous inhibitors from amaranth seeds (Amaranthus hypochondriacus), whereas extracts from maize (Zea mays) and tepary bean (Phaseolus acutifolius) produced very low inhibition. When the enzyme was measured at different stages of development, maximal activity was found in the second instar larvae. Activity drastically decreased to a very low level during the pupae stage and increased again at the adult stage. A zymogram of the different developmental stages showed two main bands of a-amylase activity, which almost disappeared at the pupae stage to increase again during the adult stage, revealing a new, smaller band. This new band may be required for a better adaptation of the adult insect to its new environment. © 2000 Elsevier Science Inc. All rights reserved. Keywords: a-Amylase; Insect enzymes; Larger grain borer; Prostephanus truncatus; Enzyme purification; Amylase inhibitors; Stored products; Insect pests
1. Introduction Many organisms, including insects that constitute serious pests of stored grains, live on a polysaccharide-rich diet and depend to a large extent on the effectiveness of their a-amylases for survival. However, the role amylases play in starch digestion by insects feeding on grains is * Corresponding author. Tel.: +52-462-3-9600; fax: 52-4624-5996. E-mail address: [email protected] (A. Blanco-Labra)
sometimes compromised by the presence of aamylase inhibitors, which are considered to act as feeding deterrents. To further our knowledge on the interactions of this group of inhibitors with the corresponding insect a-amylases, it is necessary to have access to purified enzymes. Several insect a-amylases have already been described, some of which occur as mixtures of different isozymes. For instance, in eight amy strains of Drosophila melanogaster, at least two major a-amylase isozymes were found (Doane, 1969). Conversely, single molecular forms of aamylases have been reported in Callosobruchus
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chinensis L. (Podoler and Applebaum, 1971), Tenebrio molitor L. (Buonocore et al., 1976), Bombyx mori L. (Kanekatsu, 1978) and Zabrotes subfasciatus Boh. (Campos et al., 1989). Some other insect amylases have also been characterized. The properties of the a-amylases from Sitophilus zeamais Motch. and Sitophilus granarius L. were reported by Baker (1983), who found that these enzymes were activated by chloride and stabilized by calcium ions. He found two isoforms in S. zeamais and a single form in S. granarius. Chen et al., (1992) found two a-amylase isozymes in Sitophilus oryzae L. and Tribolium castaneum Herbst, whereas only a single form was detected in T. molitor. All these enzymes have shown a similar amino acid composition, with apparent molecular masses ranging from 53 to 56 kDa. A number of a-amylases of diverse origin have been purified. Some of them have been crystallized and their three-dimensional structures have been determined (MacGregor, 1993). The X-ray structure of the a-amylase from the bacteria Alteromonas haloplanctis showed that it was remarkably similar to that from mammalian origin (Aghajari et al., 1998). Also, the crystal structure of the porcine pancreatic a-amylase complexed with the microbial inhibitor tendamistat has been determined (Wiegand et al., 1995). Amylase inhibitors have been proposed to act as resistance factors against insect attack (Whitaker, 1983; Baker, 1991; Feng et al., 1991). However, weevils such as Sitophilus sp. have such high levels of a-amylase that they are able to overcome the inhibitors in their diet (Baker and Woo, 1985). Moreover, the bruchid insect species, Acanthoscelides obtectus Say and Z. subfasciatus, have a-amylases that are not inhibited by most of the known a-amylase inhibitors of plant origin. It is believed that this property allows these bruchids to infest the common bean Phaseolus 6ulgaris, known to be a rich source of a-amylase inhibitors. The large grain borer, Prostephanus truncatus, is now considered a serious pest of farm-stored maize and cassava (Hodges and Meik, 1994). The economic importance of this insect has increased progressively. For many years, it was widely recognized as a minor pest of field and farm-stored maize, limited in extension to parts of Mexico, Central and South America (Hoppe, 1986; Dobie, 1988) and, occasionally, Brazil (Hodges and
Meik, 1994). However, serious damage caused by this insect has been reported recently in Nicaragua and Africa, where weight losses up to 40% have been recorded for farm-stored unshelled maize over 6 months (Giles and Leon, 1975). Also, post-harvest losses of maize on small farms in Costa Rica, caused by both S. zeamais and P. truncatus, reached 13% after a similar period (Bo¨ye, 1988). This pest has also been reported to cause extensive damage to cassava roots (Manihot esculenta Crantz) in Africa (Hodges et al., 1985), where they reported losses of up to 73% on fermented and 52% on unfermented dried cassava chips. In the dry conditions prevalent in Tanzania (Hodges et al., 1983) and in some places of Nicaragua (Giles and Leon, 1975), P. truncatus was the predominant storage pest among at least seven other species. It is believed that the ability of this insect to develop in grain with low moisture content contributes to its recent success and expansion. Recently, cellulolytic bacteria were isolated from the intestinal track of these insects, a finding that may explain the reported damage in wood caused by these insects (Va´zquez-Arista et al., 1997). The negative impact of P. truncatus on economically important crops, especially in developing countries, makes this insect a very important subject of study. We undertook the study of the a-amylases of this insect to gain a better understanding of its digestive physiology, which we hope will lead to new strategies of control.
2. Materials and methods
2.1. Insects P. truncatus specimens were reared on whole maize seeds (cacahuazintle race) at 289 2°C, 60% relative humidity, under continuous darkness.
2.2. Extraction procedures Initially, three different extraction procedures were explored. First, whole extracts from carefully dissected guts were obtained. For this purpose, second instar larvae were collected and weighed. The guts were subsequently separated with the aid of a dissection microscope and floated on a 0.15 M NaCl solution. The dissected guts (including lumen content) were homogenized
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in a microcentrifuge tube, using a glass rod in a 0.01 M Tris–HCl buffer equilibrated at pH 7.2, containing 0.4% Triton X-100. Second, the guts in the 0.15 M NaCl solution were centrifuged at 17 500×g for 10 min. Only the soluble components present in the supernatant were used for a-amylase activity and protein determinations. Third, whole P. truncatus larvae were homogenized in 0.01 M Tris – HCl (pH 7.2) using a 1:5 w/v ratio. The homogenates were centrifuged at 31 000×g for 20 min. The crude supernatants were filtered through paper (Whatman No. 4). After comparison of the three extraction methods, subsequent enzyme purification and activity assays were conducted with clarified crude larval extracts. All operations described were carried out at 4°C.
2.3. Measurement of a-amylase le6els during different de6elopmental stages The a-amylase levels present in whole aqueous extracts of larvae and adults were assayed at five stages (life cycle, 34 days). Cultures were sampled for larvae (6, 14 and 26 days after eclosion), pupae and adults. Weevils and larvae were weighed and homogenized in 0.5 ml cold water. The homogenates were centrifuged in 1.5 ml microfuge tubes, and 20 ml aliquots of the supernatants were assayed for amylase activity and protein content.
2.4. Zymograms Zymograms for amylase activity were carried out on polyacrylamide gels. After separation, the polyacrylamide gels were placed on top of agarose–starch gels (Baker, 1983) and incubated for 20 min at 30°C. Amylase activity appeared on the agarose–starch matrix as clear bands on a purple-colored background after staining with a 0.01 N I2-KI solution.
2.5. Amylase acti6ity assays a-Amylase activity was assayed by the dinitrosalicylic acid (DNS) procedure (Bernfeld, 1955), using soluble starch as substrate. One unit of amylase activity was defined as the amount of enzyme required to produce 1 mg maltose in 3 min at 30°C.
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2.6. Protein determination Protein was determined by the bicinchoninic acid procedure (Smith et al., 1985). Bovine serum albumin was used as standard.
2.7. Purification procedure The clarified extracts (60 ml) were first applied to a Q-Sepharose column (1.7× 31.7 cm), equilibrated with 0.01 M Tris–HCl buffer (pH 7.2). Proteins were subsequently eluted using a linear salt gradient ranging from 0 to 1.3 M NaCl in the same buffer. The column was run at a flow rate of 21.5 ml/h, and 3 ml fractions were collected. Protein was monitored at 280 nm and amylase activity was assayed as already described. Fractions containing enzymatic activity were pooled and concentrated by ultrafiltration using a YM30 Amicon membrane. The concentrate (4 ml) was further purified on a Econo-Pac High Q cartridge, 1× 5 cm (Bio-Rad), equilibrated in 0.01 M Tris– HCl buffer (pH 7.2). This second ion-exchange chromatography step was performed using a shallower salt gradient, which increased from 0 to 0.6 M NaCl over 150 min at a flow rate of 1 ml/min. One milliliter fractions were collected. The active fractions eluting from this column were pooled and concentrated as already mentioned.
2.8. Preparati6e gel electrophoresis in nondenaturing conditions A 7.5% polyacrylamide resolving gel (8 cm) and a 4% polyacrylamide stacking gel (2.5 cm) were cast in the 28 mm i.d. gel tube of the Bio-Rad Prep-Cell apparatus, Model 491. Nondenaturing polyacrylamide gel electrophoresis (PAGE) was carried out using a discontinuous electrophoresis buffer mixture. The electrode running buffer present in both the upper and the lower chambers was 0.025 M Tris with 0.192 M glycine (pH 8.3). The elution was performed using a 0.01 M Tris– HCl (pH 7.2) buffer. The procedure was carried out at 4°C. Five milligrams of the partially purified enzyme preparation obtained from the Econo Pac High Q column were dissolved in 400 ml of 0.0625 M Tris–HCl (pH 6.8), with 10% glycerol and 0.025% bromophenol blue tracking dye, and loaded on top of the stacking gel. Electrophoresis was performed at 12 W, constant power. Elution (1 ml/
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min) and fraction collection (1 ml fractions) began just prior to the elution of the tracking dye. Fractions were subsequently assayed for a-amylase activity. In the final purification step, a Sephadex G-75 gel filtration column (167 ×2.25 cm) equilibrated with 0.01 M Tris–HCl buffer (pH 7.2) was used. Elution was carried out with the same buffer at a flow rate of 19 ml/h, and 3 ml fractions were collected. These were assayed for protein content and amylase activity as already described.
2.9. Optimum pH for acti6ity The optimum pH for activity of the purified P. truncatus a-amylase was determined using buffers with different molarity and a constant ionic strength (0.15). The enzyme activity was assayed using 3 min incubation periods at 30°C. The buffer solutions used were: succinate (pH 4.0 – 6.5) and Tris (pH 7.0–9.0). Each assay was performed in triplicate.
2.10. Thermal stability Thermal stability of the enzyme was determined in the presence of 0.06 M NaCl and 0.02 M CaCl2 in a 0.2 M succinate buffer solution at pH 6.0. Diluted enzyme solutions were heated to 30, 40, 45, 50, 55 and 60°C for 15 min. Aliquots (20 ml) were then immediately cooled in an ice bath before measuring the residual a-amylase activity as previously described. Each assay was performed in triplicate.
2.12. Screening for inhibitory acti6ity Partially purified a-amylase inhibitors from amaranth, tepary beans and maize seeds were tested. Inhibition of a-amylase was assayed looking for the amount of inhibitor to reach 50% inhibition. However, this was possible only with amaranth inhibitor, since the others presented very low inhibition even at high concentrations. Inhibition of a-amylase activity was assayed as follows: 15 ml aliquots of the purified enzyme solution with a protein content of approximately 1.23× 10 − 4 mg were pre-incubated for 15 min at 30°C with 5 ml (amaranth), 80 ml (tepary beans) and 100 ml (maize) aliquots of each of the inhibitor-containing solutions. The inhibitors were dissolved in a 0.2 M succinate buffer (pH 6.0) containing 0.06 M NaCl and 0.02 M CaCl2. The total protein content added was 0.0036, 0.0130 and 0.058 mg for amaranth, tepary and maize inhibitors, respectively. Residual a-amylase activity was determined after pre-incubation with the inhibitors by the DNS method.
3. Results Initially, an electrophoretic analysis of P. truncatus crude larval homogenates was performed using 7.5% polyacrylamide slab gels. The presence of two major amylase forms was observed (Fig. 1a). These amylases were strongly anionic and moved near the tracking dye in this gel system.
2.11. Electrophoresis
3.1. Amylase extraction
The molecular mass of the purified a-amylase was determined by sodium dodecyl sulfate (SDS)PAGE following Laemmli (1970)). Acrylamide concentrations were 4% (w/v) for the stacking gel and 10% (w/v) for the separating gel, and gels were silver-stained using the method of Blomm et al. (1987). Phosphorylase B (112 kDa), bovine serum albumin (84 kDa), ovalbumin (53.2 kDa), carbonic anhydrase (34.9 kDa), soybean trypsin inhibitor (28.7 kDa) and lysozyme (20.5 kDa) were use as molecular mass standards. Isoelectric focusing was carried out using a Rotofor Cell (Bio-Rad) with ampholytes in a pH range 3–9. Focusing was carried out at 15 W constant power for 4 h at 4°C. Twenty fractions were collected for pH measurement.
Three different extraction procedures were performed to verify the origin of the extracted amylases. First, guts were individually dissected from the specimen and extracted in a detergent-containing buffer solution. Second, to obtain the lumen content, the supernatants obtained after centrifuging whole guts in a 0.15 M NaCl solution were collected. The third procedure involved the maceration and extraction of whole larva in buffer. The zymograms clearly showed that the soluble larval gut content, the macerated gut extract and the whole larval extract had the same a-amylase pattern (Fig. 1a), indicating that the same enzymes were extracted in all cases. The difference found in the level of specific activity could be due to changes in the amount of protein extracted.
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However, a new band appeared in the extract from adult guts (data not shown). This information allowed us to simplify the purification procedure of the major P. truncatus a-amylase by using whole larval extracts as starting material.
3.2. a-Amylase le6els during different de6elopmental stages The amylolytic activity per milligram of tissue (fresh weight) was almost identical in the larval and adults stages (Fig. 1b). The mean activity measured in 6–7-day-old larvae was 0.087 U/mg
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tissue. The a-amylase activity reached its highest value (0.109 U/mg tissue) in the second-instar larvae, whereas only trace amounts of activity (B 0.024 U/mg tissue) were detected in the pupae. The activity present in the whole-body extracts of adults was also relatively high (0.089 U/mg). The general trend was a slight decrease in specific activity as the weevils became older. The results also indicated that a-amylase activity in P. truncatus increased concurrently with development of the larvae up to the third stage, where a small decrease in activity was observed. A more notable decrease in activity occurred in the pupae stage, followed by a final increase in the adult stage. For the most part, the values for specific activity (U/mg protein) followed the same pattern. Zymograms run with samples obtained from the different developmental stages consistently produced two bands. These bands, however, almost disappeared in the pupal stage. This result, coupled with the low activity detected in vitro, suggests that during the pupal stage, the insect survives in a dormant stage characterized by barely perceptible digestive processes. As expected, the two bands re-appeared again during the adult stage, accompanied by a new, intermediate activity band. The appearance of an additional band of activity suggests that, at the stage when the insect fully matures and changes its environmentally confined location, an additional amylase is probably required to meet its new digestive requirements.
3.3. Purification procedure
Fig. 1. (a) a-Amylase specific activity and the corresponding zymogram from different P. truncatus extracts. L, Whole larvae; HG1 and HG2, homogenized guts (10 and 20 ml, respectively; 1:5 w/v); CG1 and CG2, centrifuged guts (10 and 20 ml, respectively; 1:5 w/v). (b) a-Amylase levels during different developmental stages and the corresponding zymogram. L-1, 6-day-old larvae; L-2, 14-day-old larvae; L-3, 26-day-old larvae; P, pupae; A, adults. Mean values 9 SD (n= 3).
Two active peaks, represented as A1 and A2 according to their retention time, were obtained after subjecting a soluble larval homogenate to anion-exchange chromatography on Q-Sepharose. Peak A1 eluted with the void volume, whereas peak A2 eluted when the NaCl concentration gradient reached 0.6 M (Fig. 2a). The proteins eluting in the region corresponding to peak A2, which had most of the amylolytic activity, were subsequently fractionated by passage through an Econo-Pac High-Q anion-exchange chromatography cartridge. The a-amylase activity eluted as a single, symmetrical, peak when the salt gradient used reached a concentration of 0.4 M at pH 7.2 (Fig. 2b). The active fractions obtained from several runs were pooled and concentrated. This material was further fractionated by preparative
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3.5. Effect of pH and temperature on the enzyme acti6ity Similar to most insect a-amylases, which have optimal activities at neutral or slightly acid pH values, the purified P. truncatus a-amylase showed an optimal pH of 6 (Fig. 5a). Its thermal stability was monitored by measuring residual enzymatic activity after incubation for 15 min at temperatures from 30 to 70°C (Fig. 5b). The rapid decrease in amylolytic activity observed above 40°C, was indicative of its high thermal instability. The values shown are the average ( 9 S.D.s) of three independent measurements.
3.6. Inhibition of amylase acti6ity by natural inhibitors The major a-amylase inhibitors from amaranth (Chagolla-Lo´pez et al., 1994), tepary beans (Blanco-Labra et al., 1996) and maize seeds (Blanco-Labra et al., 1995) were tested against the 60 kDa P. truncatus a-amylase. The results (Table Fig. 2. Elution pattern of P. truncatus a-amylase through the different purification steps. (a) Anion-exchange chromatography on a Q-Sepharose column (1.7 × 31.7 cm) at pH 7.2. (b) Anion-exchange chromatography on an Econo-Pac High Q cartridge (1 cm× 5 cm) at pH 7.2. See text for separation conditions.
electrophoresis in nondenaturing conditions, and the protein fractions were assayed for a-amylase activity. Enzymatic activity was detected just after the bromophenol blue dye front eluted (Fig. 3a). This material was finally applied to a Sephadex G-75 gel filtration column (167×2.25 cm). All the a-amylase activity eluted from this column as a single peak (Fig. 3b). Table 1 summarizes the purification scheme that finally yielded a 1209fold purification.
3.4. Electrophoresis The a-amylase purified by the presented procedure appeared as a single homogeneous band after SDS-PAGE. This protein band had an estimated molecular mass of 60.2 kDa (Fig. 4). The isoelectric point of the 60 kDa P. truncatus a-amylase was 4.7 when determined by isoelectric focusing (data not shown).
Fig. 3. Elution profile of P. truncatus a-amylase after (a) preparative electrophoresis in native conditions, and (b) sizeexclusion chromatography on a Sephadex G-75 column (2.25 cm × 167 cm).
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Table 1 Purification of the major a-amylase present in P. truncatus Procedure
Protein (mg)
Total activity
Crude extract Anion-exchange chromatography (Q-Sepharose) Anion-exchange chromatography (Econo Pac High Q) Preparative electrophoresis (Prep-Cell) Gel-filtration chromatography (Sephadex G-75)
457.56 1.67
1184 466
0.91
361
446
0.07 0.03
180 103
2654 3131
2), indicate that the inhibitors purified from maize seeds and tepary beans had little effect on the amylolytic activity of this specific enzyme, whereas that isolated from amaranth seeds was active against it.
4. Discussion Comparison of the amylolytic activity patterns obtained after gel electrophoresis of whole larval, gut and lumen extracts allowed us to confirm the digestive origin of the P. truncatus a-amylase. The similarity observed in all patterns indicated that no other a-amylase forms were present in significant quantities in the whole larval extract. The presence of two well-defined bands of aamylase activity, detected in both adults and larvae of P. truncatus, has previously been reported (Va´zquez-Arista et al., 1999). Similarly, two main forms were detected at the L-1 and L-2 stages, whereas only one form was present at the L-3 stage. Conversely, the in-gel activity was reduced to a very minor band in the pupal stage. These observations correlated with the activity levels measured in vitro, which indicated that maximum levels were reached during the second larval stage (L-2). This peak of activity was followed by a sharp decline, especially evident at the pupal stage (Fig. 1b), possibly indicating that almost no digestive processes are taking place during this developmental stage. Interestingly, a novel band of activity appeared during the adult stage. This could indicate the need for a different type of a-amylase, required to cope with a changed habitat and modified feeding activities. The main a-amylase form present in larvae and adults was purified. The purification scheme al-
Specific activity (U/mg) 2.6 279
Yield (%)
Purification (fold)
100 40
1 108
31
172
15 8.7
1025 1209
lowed a 1209-fold purification of this enzyme. As shown in Table 1, the most effective step in the purification procedure was the preparative electrophoresis separation. The approximate molecular mass determined for the purified a-amylase was 60.2 kDa. This is within the range of most molecular masses reported for other insect a-amylases, even though some, such as the 33 kDa a-amylase isolated from Callosobruchus maculatus, are smaller (Campos et al., 1989). The isoelectric point determined in vitro was 4.7. Again, this was in general agreement with those reported for other insect a-amylases, most of which fall in the acidic region. For example, the two reported a-amylases of Rhizopertha dominica had isoelectric points of 3.4 and 3.5, respectively (Baker, 1991). Similarly, the isoelectric
Fig. 4. Molecular mass determination of the purified P. truncatus a-amylase by SDS-PAGE. Lane 1, Molecular mass markers (phosphorylase B, 112 kDa; bovine serum albumin, 84 kDa; ovalbumin, 53.2 kDa; carbonic anhydrase, 34.9 kDa; soybean trypsin inhibitor, 28.7 kDa; and lysozyme, 20.5 kDa). Lane 2, Purified a-amylase after size-exclusion chromatography on Sephadex G-75.
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Only the low molecular weight a-amylase inhibitor from amaranth seeds was active against this purified a-amylase. This is indicative of the high degree of selectivity that plant-derived inhibitors have developed to recognize specific enzymes from select groups of insects. It also implies the need for more detailed studies concerning the characterization of new inhibitors, especially when great variations in specificity might occur between different isozymes. In our opinion, the purification and characterization of more insect a-amylases will greatly facilitate the understanding of the mechanisms responsible for this selectivity and will help to design new and more specific strategies for insect control.
Acknowledgements The authors are grateful to the personal of the insectary at Cinvestav. They also thank Ana Lilia Ruı´z and Armando Guerrero for their skillful technical assistance.
References Fig. 5. (a) pH activity curve of the purified a-amylase. The enzymatic activity was assayed at 30°C using buffers having the same molarity and a constant ionic strength. (b) Effect of temperature on the stability of the P. Truncatus a-amylase. Thermal stability was determined by measuring the residual a-amylase activity remaining in the enzyme preparations after 15 min at the temperatures shown. Mean values 9 SD (n= 3).
point reported for the a-amylase isolated from T. molitor was 4.0 (Buonocore et al., 1976). Maximum activity was detected at pH 6 and at 30°C. The enzyme proved, however, to be heat labile since a rapid decrease in activity was already observed at 40°C and activity was almost undetectable after incubation at 60°C. Table 2 Effect of protein a-amylase inhibitors isolated from different sources on the a-amylase activity of P. truncatus (n= 3) Source of inhibitor
Relative inhibition (%)
IU/mg powder (mean 9 S.D.)
Amaranth Tepary bean Maize
100.0 7.0 2.1
29.5 9 1.44 1.82 9 0.20 0.549 0.12
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Hodges, R.J., Dunstan, W.R., Magazini, I., Golob, P., 1983. An outbreak of Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae) in East Africa. Protein Ecol. 5, 183 – 194. Hodges, R.J., Meik, J., Denton, H., 1985. Infestation of dried cassava (Manihot esculenta Crantz) by Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae). J. Stored Prod. Res. 21, 73 – 77. Hoppe, T., 1986. Storage insects of basic grains in Honduras. Trop. Sci. 26, 25 – 38. Kanekatsu, R., 1978. Studies on further properties for an alkaline amylase in the digestive juice of the silkworm, Bombyx mori. J. Fac. Text. Sci. Technol. (Series E) 76 (9), 1 – 21. Laemmli, U.K., 1970. Cleavage of the structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680 – 685. MacGregor, E.A., 1993. Relationships between structure and activity in the a-amylase family of starchmetabolizing enzymes. Starch 45, 232 – 237. Podoler, H., Applebaum, S.W., 1971. The a-amylase of the beetle Callosobruchus chinensis: properties. Biochem. J. 121, 321 – 325. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76 – 85. Va´zquez-Arista, M., Smith, R.H., Olalde-Portugal, V., Hinojosa, R.E., Herna´ndez-Delgadillo, R., BlancoLabra, A., 1997. Cellulolytic bacteria in the digestive system of Prostephanus truncatus (Coleoptera: Bostrichidae). J. Econ. Entomol. 90 (5), 1371 – 1376. Va´zquez-Arista, M., Smith, R.H., Martı´nez-Gallardo, N., Blanco-Labra, A., 1999. Enzymatic differences in the digestive system of the adult and larvae of Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae). J. Stored Prod. Res. 35 (2), 167 – 174. Whitaker, J.R., 1983. Protease and amylase inhibitors in biological materials. In: Finley, J.W., Schwass, D.E. (Eds.), Xenobiotics in Foods and Feeds. Reprinted from ACS Symposium Series, No. 234. American Chemical Society, pp. 15 – 46. Wiegand, G., Epp, O., Huber, R., 1995. The crystal structure of porcine pancreatic a-amylase in complex with the microbial inhibitor tendamistat. J. Mol. Biol. 247, 99 – 110.
Digestive amylase from the larger grain borer, Prostephanus truncatus Horn E. Mendiola-Olaya a, A. Valencia-Jime´nez b, S. Valde´s-Rodrı´guez a, J. De´lano-Frier a, A. Blanco-Labra a,* a Department of Biotechnology and Biochemistry, Centro de In6estigacio´n y de Estudios, A6anzados del IPN, Unidad de Biotecnologı´a e Ingenierı´a Gene´tica de Plantas, Apto. Postal 629, C.P. 36500 Irapuato, Gto, Mexico b Facultad de Ciencias Exactas y Naturales, Uni6ersidad de Caldas, Manizales, Colombia
Received 25 November 1999; received in revised form 9 March 2000; accepted 20 March 2000
Abstract A combination of ion-exchange chromatography, preparative electrophoresis and gel filtration chromatography allowed a 1209-fold purification of one of the two major digestive a-amylases from larvae of the larger grain borer, Prostephanus truncatus Horn. The purified enzyme showed a molecular mass of 60.2 kDa, an isoelectric point of 4.7 and an optimal pH for activity of 6.0. The enzyme was heat labile and it was recognized by proteinaceous inhibitors from amaranth seeds (Amaranthus hypochondriacus), whereas extracts from maize (Zea mays) and tepary bean (Phaseolus acutifolius) produced very low inhibition. When the enzyme was measured at different stages of development, maximal activity was found in the second instar larvae. Activity drastically decreased to a very low level during the pupae stage and increased again at the adult stage. A zymogram of the different developmental stages showed two main bands of a-amylase activity, which almost disappeared at the pupae stage to increase again during the adult stage, revealing a new, smaller band. This new band may be required for a better adaptation of the adult insect to its new environment. © 2000 Elsevier Science Inc. All rights reserved. Keywords: a-Amylase; Insect enzymes; Larger grain borer; Prostephanus truncatus; Enzyme purification; Amylase inhibitors; Stored products; Insect pests
1. Introduction Many organisms, including insects that constitute serious pests of stored grains, live on a polysaccharide-rich diet and depend to a large extent on the effectiveness of their a-amylases for survival. However, the role amylases play in starch digestion by insects feeding on grains is * Corresponding author. Tel.: +52-462-3-9600; fax: 52-4624-5996. E-mail address: [email protected] (A. Blanco-Labra)
sometimes compromised by the presence of aamylase inhibitors, which are considered to act as feeding deterrents. To further our knowledge on the interactions of this group of inhibitors with the corresponding insect a-amylases, it is necessary to have access to purified enzymes. Several insect a-amylases have already been described, some of which occur as mixtures of different isozymes. For instance, in eight amy strains of Drosophila melanogaster, at least two major a-amylase isozymes were found (Doane, 1969). Conversely, single molecular forms of aamylases have been reported in Callosobruchus
0305-0491/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 3 0 5 - 0 4 9 1 ( 0 0 ) 0 0 2 1 6 - 9
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chinensis L. (Podoler and Applebaum, 1971), Tenebrio molitor L. (Buonocore et al., 1976), Bombyx mori L. (Kanekatsu, 1978) and Zabrotes subfasciatus Boh. (Campos et al., 1989). Some other insect amylases have also been characterized. The properties of the a-amylases from Sitophilus zeamais Motch. and Sitophilus granarius L. were reported by Baker (1983), who found that these enzymes were activated by chloride and stabilized by calcium ions. He found two isoforms in S. zeamais and a single form in S. granarius. Chen et al., (1992) found two a-amylase isozymes in Sitophilus oryzae L. and Tribolium castaneum Herbst, whereas only a single form was detected in T. molitor. All these enzymes have shown a similar amino acid composition, with apparent molecular masses ranging from 53 to 56 kDa. A number of a-amylases of diverse origin have been purified. Some of them have been crystallized and their three-dimensional structures have been determined (MacGregor, 1993). The X-ray structure of the a-amylase from the bacteria Alteromonas haloplanctis showed that it was remarkably similar to that from mammalian origin (Aghajari et al., 1998). Also, the crystal structure of the porcine pancreatic a-amylase complexed with the microbial inhibitor tendamistat has been determined (Wiegand et al., 1995). Amylase inhibitors have been proposed to act as resistance factors against insect attack (Whitaker, 1983; Baker, 1991; Feng et al., 1991). However, weevils such as Sitophilus sp. have such high levels of a-amylase that they are able to overcome the inhibitors in their diet (Baker and Woo, 1985). Moreover, the bruchid insect species, Acanthoscelides obtectus Say and Z. subfasciatus, have a-amylases that are not inhibited by most of the known a-amylase inhibitors of plant origin. It is believed that this property allows these bruchids to infest the common bean Phaseolus 6ulgaris, known to be a rich source of a-amylase inhibitors. The large grain borer, Prostephanus truncatus, is now considered a serious pest of farm-stored maize and cassava (Hodges and Meik, 1994). The economic importance of this insect has increased progressively. For many years, it was widely recognized as a minor pest of field and farm-stored maize, limited in extension to parts of Mexico, Central and South America (Hoppe, 1986; Dobie, 1988) and, occasionally, Brazil (Hodges and
Meik, 1994). However, serious damage caused by this insect has been reported recently in Nicaragua and Africa, where weight losses up to 40% have been recorded for farm-stored unshelled maize over 6 months (Giles and Leon, 1975). Also, post-harvest losses of maize on small farms in Costa Rica, caused by both S. zeamais and P. truncatus, reached 13% after a similar period (Bo¨ye, 1988). This pest has also been reported to cause extensive damage to cassava roots (Manihot esculenta Crantz) in Africa (Hodges et al., 1985), where they reported losses of up to 73% on fermented and 52% on unfermented dried cassava chips. In the dry conditions prevalent in Tanzania (Hodges et al., 1983) and in some places of Nicaragua (Giles and Leon, 1975), P. truncatus was the predominant storage pest among at least seven other species. It is believed that the ability of this insect to develop in grain with low moisture content contributes to its recent success and expansion. Recently, cellulolytic bacteria were isolated from the intestinal track of these insects, a finding that may explain the reported damage in wood caused by these insects (Va´zquez-Arista et al., 1997). The negative impact of P. truncatus on economically important crops, especially in developing countries, makes this insect a very important subject of study. We undertook the study of the a-amylases of this insect to gain a better understanding of its digestive physiology, which we hope will lead to new strategies of control.
2. Materials and methods
2.1. Insects P. truncatus specimens were reared on whole maize seeds (cacahuazintle race) at 289 2°C, 60% relative humidity, under continuous darkness.
2.2. Extraction procedures Initially, three different extraction procedures were explored. First, whole extracts from carefully dissected guts were obtained. For this purpose, second instar larvae were collected and weighed. The guts were subsequently separated with the aid of a dissection microscope and floated on a 0.15 M NaCl solution. The dissected guts (including lumen content) were homogenized
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in a microcentrifuge tube, using a glass rod in a 0.01 M Tris–HCl buffer equilibrated at pH 7.2, containing 0.4% Triton X-100. Second, the guts in the 0.15 M NaCl solution were centrifuged at 17 500×g for 10 min. Only the soluble components present in the supernatant were used for a-amylase activity and protein determinations. Third, whole P. truncatus larvae were homogenized in 0.01 M Tris – HCl (pH 7.2) using a 1:5 w/v ratio. The homogenates were centrifuged at 31 000×g for 20 min. The crude supernatants were filtered through paper (Whatman No. 4). After comparison of the three extraction methods, subsequent enzyme purification and activity assays were conducted with clarified crude larval extracts. All operations described were carried out at 4°C.
2.3. Measurement of a-amylase le6els during different de6elopmental stages The a-amylase levels present in whole aqueous extracts of larvae and adults were assayed at five stages (life cycle, 34 days). Cultures were sampled for larvae (6, 14 and 26 days after eclosion), pupae and adults. Weevils and larvae were weighed and homogenized in 0.5 ml cold water. The homogenates were centrifuged in 1.5 ml microfuge tubes, and 20 ml aliquots of the supernatants were assayed for amylase activity and protein content.
2.4. Zymograms Zymograms for amylase activity were carried out on polyacrylamide gels. After separation, the polyacrylamide gels were placed on top of agarose–starch gels (Baker, 1983) and incubated for 20 min at 30°C. Amylase activity appeared on the agarose–starch matrix as clear bands on a purple-colored background after staining with a 0.01 N I2-KI solution.
2.5. Amylase acti6ity assays a-Amylase activity was assayed by the dinitrosalicylic acid (DNS) procedure (Bernfeld, 1955), using soluble starch as substrate. One unit of amylase activity was defined as the amount of enzyme required to produce 1 mg maltose in 3 min at 30°C.
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2.6. Protein determination Protein was determined by the bicinchoninic acid procedure (Smith et al., 1985). Bovine serum albumin was used as standard.
2.7. Purification procedure The clarified extracts (60 ml) were first applied to a Q-Sepharose column (1.7× 31.7 cm), equilibrated with 0.01 M Tris–HCl buffer (pH 7.2). Proteins were subsequently eluted using a linear salt gradient ranging from 0 to 1.3 M NaCl in the same buffer. The column was run at a flow rate of 21.5 ml/h, and 3 ml fractions were collected. Protein was monitored at 280 nm and amylase activity was assayed as already described. Fractions containing enzymatic activity were pooled and concentrated by ultrafiltration using a YM30 Amicon membrane. The concentrate (4 ml) was further purified on a Econo-Pac High Q cartridge, 1× 5 cm (Bio-Rad), equilibrated in 0.01 M Tris– HCl buffer (pH 7.2). This second ion-exchange chromatography step was performed using a shallower salt gradient, which increased from 0 to 0.6 M NaCl over 150 min at a flow rate of 1 ml/min. One milliliter fractions were collected. The active fractions eluting from this column were pooled and concentrated as already mentioned.
2.8. Preparati6e gel electrophoresis in nondenaturing conditions A 7.5% polyacrylamide resolving gel (8 cm) and a 4% polyacrylamide stacking gel (2.5 cm) were cast in the 28 mm i.d. gel tube of the Bio-Rad Prep-Cell apparatus, Model 491. Nondenaturing polyacrylamide gel electrophoresis (PAGE) was carried out using a discontinuous electrophoresis buffer mixture. The electrode running buffer present in both the upper and the lower chambers was 0.025 M Tris with 0.192 M glycine (pH 8.3). The elution was performed using a 0.01 M Tris– HCl (pH 7.2) buffer. The procedure was carried out at 4°C. Five milligrams of the partially purified enzyme preparation obtained from the Econo Pac High Q column were dissolved in 400 ml of 0.0625 M Tris–HCl (pH 6.8), with 10% glycerol and 0.025% bromophenol blue tracking dye, and loaded on top of the stacking gel. Electrophoresis was performed at 12 W, constant power. Elution (1 ml/
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min) and fraction collection (1 ml fractions) began just prior to the elution of the tracking dye. Fractions were subsequently assayed for a-amylase activity. In the final purification step, a Sephadex G-75 gel filtration column (167 ×2.25 cm) equilibrated with 0.01 M Tris–HCl buffer (pH 7.2) was used. Elution was carried out with the same buffer at a flow rate of 19 ml/h, and 3 ml fractions were collected. These were assayed for protein content and amylase activity as already described.
2.9. Optimum pH for acti6ity The optimum pH for activity of the purified P. truncatus a-amylase was determined using buffers with different molarity and a constant ionic strength (0.15). The enzyme activity was assayed using 3 min incubation periods at 30°C. The buffer solutions used were: succinate (pH 4.0 – 6.5) and Tris (pH 7.0–9.0). Each assay was performed in triplicate.
2.10. Thermal stability Thermal stability of the enzyme was determined in the presence of 0.06 M NaCl and 0.02 M CaCl2 in a 0.2 M succinate buffer solution at pH 6.0. Diluted enzyme solutions were heated to 30, 40, 45, 50, 55 and 60°C for 15 min. Aliquots (20 ml) were then immediately cooled in an ice bath before measuring the residual a-amylase activity as previously described. Each assay was performed in triplicate.
2.12. Screening for inhibitory acti6ity Partially purified a-amylase inhibitors from amaranth, tepary beans and maize seeds were tested. Inhibition of a-amylase was assayed looking for the amount of inhibitor to reach 50% inhibition. However, this was possible only with amaranth inhibitor, since the others presented very low inhibition even at high concentrations. Inhibition of a-amylase activity was assayed as follows: 15 ml aliquots of the purified enzyme solution with a protein content of approximately 1.23× 10 − 4 mg were pre-incubated for 15 min at 30°C with 5 ml (amaranth), 80 ml (tepary beans) and 100 ml (maize) aliquots of each of the inhibitor-containing solutions. The inhibitors were dissolved in a 0.2 M succinate buffer (pH 6.0) containing 0.06 M NaCl and 0.02 M CaCl2. The total protein content added was 0.0036, 0.0130 and 0.058 mg for amaranth, tepary and maize inhibitors, respectively. Residual a-amylase activity was determined after pre-incubation with the inhibitors by the DNS method.
3. Results Initially, an electrophoretic analysis of P. truncatus crude larval homogenates was performed using 7.5% polyacrylamide slab gels. The presence of two major amylase forms was observed (Fig. 1a). These amylases were strongly anionic and moved near the tracking dye in this gel system.
2.11. Electrophoresis
3.1. Amylase extraction
The molecular mass of the purified a-amylase was determined by sodium dodecyl sulfate (SDS)PAGE following Laemmli (1970)). Acrylamide concentrations were 4% (w/v) for the stacking gel and 10% (w/v) for the separating gel, and gels were silver-stained using the method of Blomm et al. (1987). Phosphorylase B (112 kDa), bovine serum albumin (84 kDa), ovalbumin (53.2 kDa), carbonic anhydrase (34.9 kDa), soybean trypsin inhibitor (28.7 kDa) and lysozyme (20.5 kDa) were use as molecular mass standards. Isoelectric focusing was carried out using a Rotofor Cell (Bio-Rad) with ampholytes in a pH range 3–9. Focusing was carried out at 15 W constant power for 4 h at 4°C. Twenty fractions were collected for pH measurement.
Three different extraction procedures were performed to verify the origin of the extracted amylases. First, guts were individually dissected from the specimen and extracted in a detergent-containing buffer solution. Second, to obtain the lumen content, the supernatants obtained after centrifuging whole guts in a 0.15 M NaCl solution were collected. The third procedure involved the maceration and extraction of whole larva in buffer. The zymograms clearly showed that the soluble larval gut content, the macerated gut extract and the whole larval extract had the same a-amylase pattern (Fig. 1a), indicating that the same enzymes were extracted in all cases. The difference found in the level of specific activity could be due to changes in the amount of protein extracted.
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However, a new band appeared in the extract from adult guts (data not shown). This information allowed us to simplify the purification procedure of the major P. truncatus a-amylase by using whole larval extracts as starting material.
3.2. a-Amylase le6els during different de6elopmental stages The amylolytic activity per milligram of tissue (fresh weight) was almost identical in the larval and adults stages (Fig. 1b). The mean activity measured in 6–7-day-old larvae was 0.087 U/mg
429
tissue. The a-amylase activity reached its highest value (0.109 U/mg tissue) in the second-instar larvae, whereas only trace amounts of activity (B 0.024 U/mg tissue) were detected in the pupae. The activity present in the whole-body extracts of adults was also relatively high (0.089 U/mg). The general trend was a slight decrease in specific activity as the weevils became older. The results also indicated that a-amylase activity in P. truncatus increased concurrently with development of the larvae up to the third stage, where a small decrease in activity was observed. A more notable decrease in activity occurred in the pupae stage, followed by a final increase in the adult stage. For the most part, the values for specific activity (U/mg protein) followed the same pattern. Zymograms run with samples obtained from the different developmental stages consistently produced two bands. These bands, however, almost disappeared in the pupal stage. This result, coupled with the low activity detected in vitro, suggests that during the pupal stage, the insect survives in a dormant stage characterized by barely perceptible digestive processes. As expected, the two bands re-appeared again during the adult stage, accompanied by a new, intermediate activity band. The appearance of an additional band of activity suggests that, at the stage when the insect fully matures and changes its environmentally confined location, an additional amylase is probably required to meet its new digestive requirements.
3.3. Purification procedure
Fig. 1. (a) a-Amylase specific activity and the corresponding zymogram from different P. truncatus extracts. L, Whole larvae; HG1 and HG2, homogenized guts (10 and 20 ml, respectively; 1:5 w/v); CG1 and CG2, centrifuged guts (10 and 20 ml, respectively; 1:5 w/v). (b) a-Amylase levels during different developmental stages and the corresponding zymogram. L-1, 6-day-old larvae; L-2, 14-day-old larvae; L-3, 26-day-old larvae; P, pupae; A, adults. Mean values 9 SD (n= 3).
Two active peaks, represented as A1 and A2 according to their retention time, were obtained after subjecting a soluble larval homogenate to anion-exchange chromatography on Q-Sepharose. Peak A1 eluted with the void volume, whereas peak A2 eluted when the NaCl concentration gradient reached 0.6 M (Fig. 2a). The proteins eluting in the region corresponding to peak A2, which had most of the amylolytic activity, were subsequently fractionated by passage through an Econo-Pac High-Q anion-exchange chromatography cartridge. The a-amylase activity eluted as a single, symmetrical, peak when the salt gradient used reached a concentration of 0.4 M at pH 7.2 (Fig. 2b). The active fractions obtained from several runs were pooled and concentrated. This material was further fractionated by preparative
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3.5. Effect of pH and temperature on the enzyme acti6ity Similar to most insect a-amylases, which have optimal activities at neutral or slightly acid pH values, the purified P. truncatus a-amylase showed an optimal pH of 6 (Fig. 5a). Its thermal stability was monitored by measuring residual enzymatic activity after incubation for 15 min at temperatures from 30 to 70°C (Fig. 5b). The rapid decrease in amylolytic activity observed above 40°C, was indicative of its high thermal instability. The values shown are the average ( 9 S.D.s) of three independent measurements.
3.6. Inhibition of amylase acti6ity by natural inhibitors The major a-amylase inhibitors from amaranth (Chagolla-Lo´pez et al., 1994), tepary beans (Blanco-Labra et al., 1996) and maize seeds (Blanco-Labra et al., 1995) were tested against the 60 kDa P. truncatus a-amylase. The results (Table Fig. 2. Elution pattern of P. truncatus a-amylase through the different purification steps. (a) Anion-exchange chromatography on a Q-Sepharose column (1.7 × 31.7 cm) at pH 7.2. (b) Anion-exchange chromatography on an Econo-Pac High Q cartridge (1 cm× 5 cm) at pH 7.2. See text for separation conditions.
electrophoresis in nondenaturing conditions, and the protein fractions were assayed for a-amylase activity. Enzymatic activity was detected just after the bromophenol blue dye front eluted (Fig. 3a). This material was finally applied to a Sephadex G-75 gel filtration column (167×2.25 cm). All the a-amylase activity eluted from this column as a single peak (Fig. 3b). Table 1 summarizes the purification scheme that finally yielded a 1209fold purification.
3.4. Electrophoresis The a-amylase purified by the presented procedure appeared as a single homogeneous band after SDS-PAGE. This protein band had an estimated molecular mass of 60.2 kDa (Fig. 4). The isoelectric point of the 60 kDa P. truncatus a-amylase was 4.7 when determined by isoelectric focusing (data not shown).
Fig. 3. Elution profile of P. truncatus a-amylase after (a) preparative electrophoresis in native conditions, and (b) sizeexclusion chromatography on a Sephadex G-75 column (2.25 cm × 167 cm).
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Table 1 Purification of the major a-amylase present in P. truncatus Procedure
Protein (mg)
Total activity
Crude extract Anion-exchange chromatography (Q-Sepharose) Anion-exchange chromatography (Econo Pac High Q) Preparative electrophoresis (Prep-Cell) Gel-filtration chromatography (Sephadex G-75)
457.56 1.67
1184 466
0.91
361
446
0.07 0.03
180 103
2654 3131
2), indicate that the inhibitors purified from maize seeds and tepary beans had little effect on the amylolytic activity of this specific enzyme, whereas that isolated from amaranth seeds was active against it.
4. Discussion Comparison of the amylolytic activity patterns obtained after gel electrophoresis of whole larval, gut and lumen extracts allowed us to confirm the digestive origin of the P. truncatus a-amylase. The similarity observed in all patterns indicated that no other a-amylase forms were present in significant quantities in the whole larval extract. The presence of two well-defined bands of aamylase activity, detected in both adults and larvae of P. truncatus, has previously been reported (Va´zquez-Arista et al., 1999). Similarly, two main forms were detected at the L-1 and L-2 stages, whereas only one form was present at the L-3 stage. Conversely, the in-gel activity was reduced to a very minor band in the pupal stage. These observations correlated with the activity levels measured in vitro, which indicated that maximum levels were reached during the second larval stage (L-2). This peak of activity was followed by a sharp decline, especially evident at the pupal stage (Fig. 1b), possibly indicating that almost no digestive processes are taking place during this developmental stage. Interestingly, a novel band of activity appeared during the adult stage. This could indicate the need for a different type of a-amylase, required to cope with a changed habitat and modified feeding activities. The main a-amylase form present in larvae and adults was purified. The purification scheme al-
Specific activity (U/mg) 2.6 279
Yield (%)
Purification (fold)
100 40
1 108
31
172
15 8.7
1025 1209
lowed a 1209-fold purification of this enzyme. As shown in Table 1, the most effective step in the purification procedure was the preparative electrophoresis separation. The approximate molecular mass determined for the purified a-amylase was 60.2 kDa. This is within the range of most molecular masses reported for other insect a-amylases, even though some, such as the 33 kDa a-amylase isolated from Callosobruchus maculatus, are smaller (Campos et al., 1989). The isoelectric point determined in vitro was 4.7. Again, this was in general agreement with those reported for other insect a-amylases, most of which fall in the acidic region. For example, the two reported a-amylases of Rhizopertha dominica had isoelectric points of 3.4 and 3.5, respectively (Baker, 1991). Similarly, the isoelectric
Fig. 4. Molecular mass determination of the purified P. truncatus a-amylase by SDS-PAGE. Lane 1, Molecular mass markers (phosphorylase B, 112 kDa; bovine serum albumin, 84 kDa; ovalbumin, 53.2 kDa; carbonic anhydrase, 34.9 kDa; soybean trypsin inhibitor, 28.7 kDa; and lysozyme, 20.5 kDa). Lane 2, Purified a-amylase after size-exclusion chromatography on Sephadex G-75.
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Only the low molecular weight a-amylase inhibitor from amaranth seeds was active against this purified a-amylase. This is indicative of the high degree of selectivity that plant-derived inhibitors have developed to recognize specific enzymes from select groups of insects. It also implies the need for more detailed studies concerning the characterization of new inhibitors, especially when great variations in specificity might occur between different isozymes. In our opinion, the purification and characterization of more insect a-amylases will greatly facilitate the understanding of the mechanisms responsible for this selectivity and will help to design new and more specific strategies for insect control.
Acknowledgements The authors are grateful to the personal of the insectary at Cinvestav. They also thank Ana Lilia Ruı´z and Armando Guerrero for their skillful technical assistance.
References Fig. 5. (a) pH activity curve of the purified a-amylase. The enzymatic activity was assayed at 30°C using buffers having the same molarity and a constant ionic strength. (b) Effect of temperature on the stability of the P. Truncatus a-amylase. Thermal stability was determined by measuring the residual a-amylase activity remaining in the enzyme preparations after 15 min at the temperatures shown. Mean values 9 SD (n= 3).
point reported for the a-amylase isolated from T. molitor was 4.0 (Buonocore et al., 1976). Maximum activity was detected at pH 6 and at 30°C. The enzyme proved, however, to be heat labile since a rapid decrease in activity was already observed at 40°C and activity was almost undetectable after incubation at 60°C. Table 2 Effect of protein a-amylase inhibitors isolated from different sources on the a-amylase activity of P. truncatus (n= 3) Source of inhibitor
Relative inhibition (%)
IU/mg powder (mean 9 S.D.)
Amaranth Tepary bean Maize
100.0 7.0 2.1
29.5 9 1.44 1.82 9 0.20 0.549 0.12
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