Biochemical and kinetic characterization of the digestive trypsin-like activity of the lesser grain borer Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae)

Journal of Stored Products Research 51 (2012) 41e48

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Biochemical and kinetic characterization of the digestive trypsin-like activity of the lesser grain borer Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) Pablo S. Osuna-Amarillas a, Francisco J. Cinco-Moroyoqui a, *, José L. Cárdenas-López a, Josafat M. Ezquerra-Brauer a, Rogerio Sotelo-Mundo b, Mario O. Cortez-Rocha a, Jesús M. Barrón-Hoyos a, Ofelia Rouzaud-Sández a, Jesús Borboa-Flores a a

Departamento de Investigación y Posgrado en Alimentos, Grupo de Investigación en Química Agrícola y Manejo Postcosecha (QAMPO), Universidad de Sonora, 83000 Hermosillo, Sonora, Mexico Centro de Investigación en Alimentación y Desarrollo, A.C. Carretera a la Victoria Km. 0.6, Hermosillo, Sonora, Mexico

b

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 19 June 2012

The digestive trypsin-like activity of the lesser grain borer Rhyzopertha dominica was characterized in some of its biochemical and kinetic properties. The enzyme activity from insect midguts was isolated using hydrophobic interaction chromatography with phenyl-sepharose CL-4B. Eight bands (identified from A through H) with caseinolytic activity and molecular weights in the range of 22e51.3 kDa were detected by zymography in casein-polyacrylamide gels. The strongest bands were D, G, and H, and showed estimated molecular weights of 33.6, 25.4, and 22 kDa, respectively. In-gel inhibition of caseinolytic activity showed that the serine protease inhibitors TLCK and SBTI inhibited all the proteases, except E. In-vitro inhibitory assays showed that SBTI and TLCK suppressed the BApNAase activity by 92.3% and 79.2%, respectively, indicating the presence of serine proteases. Wheat hexaploid albumin extracts were highly effective in inhibiting all the proteolytic activity. The chymotrypsin inhibitor TPCK did not affect the BApNAase activity, indicating that the proteolytic activity in R. dominica belongs to the trypsin-like type. With BApNA as the substrate, the proteolytic activity was high across a broad pH range of 6e11 with two peaks of maximum activity at pH 8 and 10 with an optimum temperature of 50
C. SBTI inhibited the BApNAase activity with IC50 and Ki values of 0.02 mM and 1.17  108 M, respectively. The kinetic constants Km and Vmax were 0.07 mM and 2.8 mM/min, respectively. The activation energy (Ea) for BApNA hydrolysis was 33.5 kJ/mol. The results of this study confirm that R. dominica rely on serine protease activity for food digestion. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Insect serine proteases Trypsin-like proteases Rhyzopertha dominica Biochemical characterization Kinetic properties

1. Introduction Insect proteases have attracted attention due to their role in planteinsect interactions (Reeck et al., 1999). Significant advances have been made in characterizing the different properties of the proteases from different stored-product pests, which have contributed to a better understanding of the way they work on hydrolyzing proteins from different sources (Houseman and Thie, 1993; Elpidina et al., 2005; Hosseininaveh et al., 2007; Oppert et al., 2010; Vinokurov et al., 2006). The coleopteran Rhyzoperta dominica is a primary pest that feeds in intact wheat kernels under storage causing significant economical losses (Zhu and Baker, 1999; Cinco-Moroyoqui et al., 2008). The developing larva feeds inside

* Corresponding author. Tel.: þ52 662 259 2208; fax: þ52 662 259 2207. E-mail address: [email protected] (F.J. Cinco-Moroyoqui). 0022-474X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jspr.2012.06.005

grain kernels and cause weight loss and damage to the germ and endosperm in wheat (Janarthanan et al., 2008). The female R. dominica lays eggs on the exterior of the kernels, and the larvae bores through the hull and actively feeds inside the kernel where they reach the adult stage creating a large exit hole in the kernel causing significant damages to the kernels (Chanbang et al., 2007). Several studies have related certain physical characteristics of the wheat kernel with its susceptibility to the feeding activity of R. dominica (McGaughey et al., 1990; Toews et al., 2000). Proteinaceous amylase inhibitors found in the wheat kernel are effective in suppressing the amylase activity of R. dominica (Baker, 1991) and variations in amylase inhibitory activity of infested wheat cultivars were found to negatively correlate with F-1 progeny sizes (CincoMoroyoqui et al., 2006). Proteases from stored-product pests play an important role in the digestion process of dietary proteins and some have been studied in detail (Houseman and Thie, 1993; Reeck et al., 1999;

42

P.S. Osuna-Amarillas et al. / Journal of Stored Products Research 51 (2012) 41e48

Elpidina et al., 2005; Oppert et al., 2005; Vinokurov et al., 2006; Hosseininaveh et al., 2007). Although some coleopterans such as Tenebrio molitor (Vinokurov et al., 2006), Tribolium castaneum (Murdock et al., 1987; Oppert et al., 2003) and Callosobruchus maculatus (Kitch and Murdock, 1986; Silva et al., 2001) rely mainly on cysteine proteases for protein digestion, others like Trogoderma granariun (Hosseininaveh et al., 2007) and R. dominica (Zhu and Baker, 1999) does it on serine proteases. Other stored-product pests such as Sitophilus zeamais (Silva et al., 2010a, 2010b), Prostephanus truncatus (Houseman and Thie, 1993), and Zabrotes subfasciatus (Silva et al., 2001) use a combination of cysteine and serine proteases for food digestion. Studies have demonstrated that the presence of protease inhibitors in the diet of stored-product pests lead them to produce insensitive proteases as a mechanism of adaptation to overcome the effect of those inhibitors (Oppert et al., 2005; Amorim et al., 2008). Serine proteases are common in many insects and are involved in the digestion of dietary protein (Reeck et al., 1999; Terra and Ferreira, 1994), protein activation in the melanization cascade (Terra and Ferreira, 1994; Lee et al., 2002; Kan et al., 2008) and antibacterial activity and insect immune response (Ma and Kanost, 2000). Among the digestive enzymes of R. dominica, only the amylases of this coleopteran has been studied in detail (CincoMoroyoqui et al., 2006, 2008), whereas those belonging to the proteolytic activity has been relatively little studied (Zhu and Baker, 1999). The objectives of the present study were to characterize certain biochemical and kinetic properties of the R. dominica digestive trypsin-like proteolytic activity. 2. Materials and methods

ultrafiltration cell (Fisher Co.) under a stream of nitrogen to a final volume of approximately 50 mL. The flow rate was 1.0 mL/ min at 4.0 psi through a YM-10 Millipore ultrafiltration membrane 44.5 mm in diameter and a nominal MW limit of 10 kDa. 2.3. Protease assay Total proteolytic activity in crude extracts and in chromatographic fractions was determined using 2% (w/v) azocasein as substrate (Araújo et al., 2008). The reaction mixture contained 100 mL enzyme extract, 100 mL of phosphate buffer (0.01 M phosphate buffer, pH 8, containing 1 mM dithiothreitol or 1 mM bmercaptoethanol), and 300 mL substrate. The mixture was incubated at 37
C for 60 min for azocasein hydrolysis. The reaction was stopped by adding 500 mL of 25% (w/v) trichloroacetic acid. The samples were rested on ice for 15 min for protein precipitation and centrifuged for 5 min at 10,000  g at 4
C for protein removal. The supernatants were combined with 300 mL of 1 N NaOH and their absorbance was read at 428 nm. One unit of azocaseinolytic activity was defined as the change in 0.01 absorbance units under the conditions of the assay. Trypsin-like activity in crude extracts and in chromatographic fractions was determined using N-a-benzoyl-DL-arginine p-nitroanilide (BApNA) as substrate (Hosseininaveh et al., 2007). Usually, 75 mL of the enzyme preparation was combined with 325 mL of 1 mM phosphate buffer, pH 8, at 37
C. The reaction was started by adding 200 mL of 1 mM BApNA and stopped after 10 min by the addition of 150 mL of 30% acetic acid. The released p-nitroaniline was estimated at 410 nm. One unit of proteolytic activity was defined as the amount of enzyme causing an increase in absorbance of 0.01 at 410 nm under the conditions of the assay.

2.1. Insect rearing 2.4. Protein determination A population of 100 unsexed adults of R. dominica was used to infest 1 kg of hard winter wheat and held at 27
C and 70% RH (12:12 photophase period) in a rearing chamber for a 15-day oviposition period. The adults were sieved and the wheat samples were incubated under the same conditions for 45 days. The progeny emerged was collected and used as the source of proteolytic activity. 2.2. Preparation of proteolytic extracts Intestinal tracts were removed from R. dominica adults by pressing their abdomen and removing the head with the help of forceps. The tracts were placed in a mortar seated in an ice-water bath containing chilled assay buffer (100 mM phosphate buffer, pH 8) and homogenized. The mixture was centrifuged at 10,000  g for 30 min at 4
C. The supernatant was removed and further filtered through 0.45 mm Cameo 17N nylon syringe filters (Osmonics Laboratory Products, Minnetonka, MN). The protease homogenate was saturated with 25% ammonium sulfate and left for 1 h. The precipitate formed was pelleted by centrifugation at 10,000  g for 60 min, re-dissolved in 40 mL of assay buffer, and filtered through 0.45 mm nylon filters. Ten milliliters (equivalent to approximately 45 mg protein) of the resulting fraction was loaded onto a 1.2  40 cm phenyl sepharose CL-4B column (Sigma Aldrich Chemical Co., St. Louis, MO, USA) equilibrated with assay buffer containing 25% (w/v) saturation of ammonium sulfate. After all the unbound protein was removed from the column with equilibration buffer, the proteolytic activity was eluted with a linear gradient of ethylene glycol (0e50%; 400 mL) in assay buffer without ammonium sulfate. The flow rate was 24 mL/h and fractions of 3 mL were collected. Fractions showing azocaseinolytic and BApNAase activity were pooled and concentrated in an Amicon

Protein content of the insect proteolytic crude extract and wheat albumin extracts were determined using the dye binding assay of Bradford (1976) with bovine serum albumin as standard. 2.5. Polyacrylamide gel electrophoresis (PAGE) Crude proteolytic homogenates and pooled fractions from hydrophobic interaction chromatography were analyzed under non-denaturing conditions (Laemmli, 1970). Bands of protein were visualized by a silver nitrate method (Blum et al., 1987). 2.6. Zymography analysis For zymography, protease aliquots (approximately 2 units of BApNAase activity) were combined with equal volumes of sample buffer containing no b-mercaptoethanol and were not heat-treated. Thirty microliters of the mixture was loaded onto a polyacrylamide gel system consisting of a 12% acrylamide separation gel and a 3.83% acrylamide stacking gel, both containing 1.35% bisacrylamide (Laemmli, 1970) without sodium dodecyl sulfate. The protein separation was done under non-denaturing conditions at a constant voltage of 200 V for 45 min. The running buffer was 25 mM TriseHCl and 192 mM glycine, pH 8.3. After electrophoresis, the gel containing the proteases was recovered and placed onto a polyacrylamide gel containing 0.33% (w/v) casein co-polymerized with the acrylamide matrix following the procedure of Heussen and Dowdle (1980). Both gels were transferred into a plastic container that was floated for 2 h in a water bath set at 37
C to allow casein digestion. After that, the gel containing the copolymerized casein was recovered and stained for 2 h with 0.1% (w/v) Amido black in 7% acetic acid (v/v). Destaining was

P.S. Osuna-Amarillas et al. / Journal of Stored Products Research 51 (2012) 41e48

performed washing the gel several times with 7% acetic acid until clear bands against dark background were visible indicating the presence of proteases. 2.7. In vitro inhibition assays The proteolytic activity was assayed against several inhibitors: 1-chloro-3-tosylamido-7-amino-2-heptanone-HCl (TLCK), phenylmethyl sulfonyl fluoride (PMSF), 1-chloro-3-tosylamido-4-phenyl2-butanone (TPCK), epoxisuccinyl-L-leucylamido-(4-guanidino) butane (E-64), pepstatin A, bestatin hydrochloride (bestatin), and soybean Kunitz trypsin inhibitor type IIS (Sigma Chemical Co.). Stock solutions of bestatin and TPCK were prepared in ethanol, whereas the rest of the inhibitors were dissolved in deionized water. All inhibition assays were performed by combining 75 mL of each inhibitor preparation with 75 mL of enzyme solution (containing approximately 2 units of BApNAase activity). The final inhibitor concentration in the assay mixture was 5 mg/mL for SBTI and TPCK, 2.5 mg/mL for PMSF and bestatin, 5 mg/mL for pepstatin, and 18 mg/mL for E-64. The mixtures were pre-incubated for 1 h at 37
C before determining the residual protease activity as described above. Appropriate controls prepared with R. dominica proteolytic extract without inhibitor were included. One inhibition unit was defined as the change in 0.01 absorbance unit at 410 nm under the conditions of the assay. 2.8. In-gel inhibition of proteases The inhibitors 1-chloro-3-tosylamido-7-amino-2-heptanoneHCl (TLCK), phenylmethyl sulfonyl fluoride (PMSF), 1-chloro-3tosylamido-4-phenyl-2-butanone (TPCK), trans-epoxisuccinyl-Lleucylamido-(4-guanidino) butane (E-64), pepstatin A, bestatin hydrochloride and soybean Kunitz trypsin inhibitor (SBTI) type IIS (all obtained from Sigma Aldrich; St. Louis, MO, USA) were tested on the proteolytic activity of R. dominica. The final inhibitor concentration in the assay mixture, once combined with approximately 2 units of BApNAase activity, was 10 mg/mL for SBTI and TPCK, 5 mg/mL for PMSF and bestatin, 10 mg/mL for pepstatin, and 36 mg/mL for E-64. For inhibition using a wheat albumin extract, kernels from the hexaploid wheat Kronstandt (which was selected from a set of wheat cultivars due to its high inhibitory protease activity) were finely ground to pass a 60 mesh sieve to optimize protein extraction. One gram of the powder was extracted mechanically for 1 h at ambient temperature with 50 mL of deionized water. After centrifugation at 10,000  g, the supernatant was recovered and used as inhibitor extract. For inhibition of the proteolytic activity, an aliquot of the proteolytic extract containing approximately 2 units of BApNAase activity was combined with an equal volume of the wheat albumin preparation. The mixtures were incubated for 1 h at 37
C before the addition of sample buffer (0.125 M TriseHCl, pH 6.8, containing 20% glycerol and 0.004% bromophenol blue) and were not heat-treated. Twenty five microliters of the mixtures were loaded onto a 12% acrylamide gel and analyzed by non-denaturing electrophoresis. The residual activity of the proteolytic bands was visualized by zymography as previously described. 2.9. Determination of pH optimum The proteolytic activity was determined at several pH values using acetate buffer (pH 4.0e5.5), phosphate buffer (pH 6.0e7.0), TriseHCl (pH 7.5e8.0), and glycine-NaOH (pH 8.5e11.0). Aliquots containing approximately 2 units of BApNAase activity were tested. Each buffer was 100 mM and a blank for each pH value was used.

43

2.10. Determination of optimum temperature For optimum temperature determination, aliquots of the enzyme preparation containing approximately 2 units of BApNAase activity were combined with 375 mL of assay buffer and incubated for 10 min in a temperature range from 30
C to 70
C at intervals of 10
C. BApNAase activity was determined according to the standard assay described before. 2.11. Molecular weight determination The electrophoretic procedure of Laemmli (Laemmli, 1970) was used to determine the molecular weight of R. dominica proteases. Aliquots of the proteolytic preparation (containing approximately 2 units of BApNAase activity) and of a molecular weight markers preparation consisting of myosin (200 kDa), b-galactosidase (116.2 kDa), phosphorylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa) and aprotinin (6.5 kDa) were separated at 200 V for 45 min in a Mini-Protean II Cell (Bio-Rad, Hercules, CA) using a 12% acrylamide separation gel and a 3.83% acrylamide stacking gel, both containing 1.35% bisacrylamide. After electrophoresis, the gel was cut into two halves, one containing the molecular weight markers and the other the insect proteases. Detection of molecular weight markers was performed by staining the gel for 1 h using a Coomassie Blue G250 commercial preparation (GelCode blue stain reagent; Pierce, Rockford, IL). Destaining of the gel was carried out by several washes with deionized water until dark blue bands of the molecular weight markers were visualized against a clear background. The half of the gel containing the insect proteases was subjected to zymography to detect the protease bands. The molecular weight of each insect protease was calculated using a graph of log of the molecular weight vs Rm of the molecular weight markers. 2.12. Determination of kinetic parameters To determine Km and Vmax, five BApNA concentrations in the range 0.1e1.0 M were combined with 2 units of protease activity in 100 mM phosphate buffer, pH 8.0. The reactions were carried out as described previously. Estimation of the parameters was obtained by fitting the observed values of V0 versus substrate concentration to the MichaeliseMenten equation using non-linear correlation curve-fitting software (Graph-Pad Prism 5.00; San Diego, CA, USA). IC50 determination was performed using increasing concentrations of SBTI in the range from 1.25  102 to 6.25  102 mM with 1.0 mM BApNA as substrate. The IC50 value was obtained visually by curve fitting the percent inhibition data versus inhibitor concentration i, whereas the inhibition constant Ki was determined by means of Dixon plots (Burlingham and Widlanski, 2003) using two fixed BApNA concentrations (0.015 and 0.05 mM), which were combined with final SBTI concentrations of 2.5  103, 5.0  103, 7.5  103, 1.0  103, and 1.25  102 mM. Dixon plots were prepared by plotting the reciprocal velocity 1/V0 against the inhibitor concentrations. The inhibitor constant Ki was calculated from the intersection of the lines obtained. The activation energy of the enzymatic reaction was determined by measuring the slope of the Arrhenius plot prepared in the temperature range from 30
C to 50
C by plotting ln V0 versus T1, and employing the equation:

ln V0 ¼ lnðAÞ  Ea =RT where Ea is the activation energy, T (
K) is the corresponding absolute temperature, and R is the gas constant (8.314 J mol1 K1). V0 values used in the Arrhenius plot were determined at intervals of 5
C in the given temperature range above indicated.

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These results are in agreement with others in that serine proteases from different sources are stable at temperatures of 50
C (Tsybina et al., 2005; Xavier et al., 2005).

3. Results 3.1. Protease purification One broad peak with proteolytic activity was eluted from the phenyl sepharose column with a linear gradient of ethylene glycol from 0 to 50% (Fig. 1). This broad peak showed the presence of both azocaseinolytic as well as trypsin-like activities. Addition of reducing agents such as dithiothreitol or b-mercaptoethanol to the assay buffer did not increase the hydrolysis of the substrates indicating the absence of cysteine proteases. The chromatographic procedure allowed removal of proteinaceous material lacking proteolytic activity, leaving behind the proteases bound to the phenyl sepharose column. Several attempts were carried out trying to separate and purify the proteases. However, we experienced difficulties in their separation mainly due to their very similar hydrophobic character.

3.4. Detection of proteases by zymography Digestion of the casein co-polymerized with the acrylamide matrix allowed the detection of eight proteolytic bands, which were designated as A, B, C, D, E, F, G, and H according to their position in the gel (Fig. 4). The most active bands were D, G, and H, and their estimated molecular weights were 33.6, 25.4, and 22 kDa, respectively. Other faint proteolytic bands with higher molecular weights of 52.3, 48.9 and 38.1 kDa (bands A, B, and C, respectively) and lower intermediate molecular weights of 29.7 and 27.9 kDa (bands E and F, respectively) were also observed. Molecular weights of a serine protease of 22.7 kDa have been reported for Trogoderma granarium (Hosseininaveh et al., 2007), whereas a 25.5 kDa trypsinlike proteinase was observed in T. molitor (Tsybina et al., 2005). Vinokurov et al. (2006) detected trypsin-like proteases in T. molitor with molecular weights of 17 and 59 kDa. Similar molecular weight values were also observed for serine proteases from T. castaneum (Oppert et al., 2005). Other coleopterans such as Sitophilus seamais, T. granarium, T. molitor, and T. castaneum (Reeck et al., 1999; Hosseininaveh et al., 2007; Vinokurov et al., 2006; Oppert et al., 2003) use multiple proteases to digest dietary proteins. Some stored-product pests use a combination of serine and cysteine proteases such as S. zeamais and P. truncatus (Houseman and Thie, 1993), T. molitor (Vinokurov et al., 2006) and Z. subfasciatus (Silva et al., 2001).

3.2. Determination of pH optimum The trypsin-like activity of R. dominica was detected over a broad range of pH (pH range 6e11) with two peaks observed at pH 8 and 10 using BApNA as the substrate (Fig. 2). At slightly acid (pH 6) and neutral pH (pH 7) high trypsin-like activity was observed accounting approximately 71.2% and 91.0% and 68% and 87% of that observed at pH 8 and 10, respectively. 3.3. Determination of optimum temperature The trypsin-like activity of R. dominica extracts held at different temperatures is shown in Fig. 3. The highest trypsin-like proteolytic activity on BApNA as the substrate was 50
C. A “break point” in activity was observed at 40
C, which might be due to a conformational change in the enzymes(s) structure allowing the substrate to interact easily with the catalytic site (Rao et al., 2009). The BApNAase activity increased as the temperature rose from 30
C to 50
C. At 30
C and 40
C the activity was approximately 43% and 91%, respectively, of the maximum observed at 50
C. When heated at 60
C, the BApNAase activity retained approximately 70% of the maximum observed at 50
C and virtually disappeared at 70
C.

25

3.5. In-gel inhibition of proteases Susceptibility of R. dominica proteases to in-gel inhibition by SBTI, TLCK, PMSF, wheat albumin inhibitory extract, TPCK, E-64, pepstatin A, and bestatin is shown in Fig. 5. It was observed that the serine protease inhibitors TLCK (lane 2) and SBTI (lane 3) inhibited all the proteases, except E. The inhibitor PMSF, an inhibitor for serine proteases, strongly inhibited band E, whereas bands A, B, and C were slightly inhibited, suggesting that the serine proteases of R. dominica are not only of the trypsin-like type. All protease bands

4

20

10

50

50

8

40

40

6

30

30

20

1

5

0

20

0 0

50

100

150

200

250

300

2

10

10

0

0

0

% Ethylene Glycol

4

UA (Azocasein)

10

2

UA (BApNA)

15

Absorbance 280 nm

% Ammonium Sulfate

3

350

Fraction Fig. 1. Phenyl sepharose chromatography of the proteolytic activity from Rhyzopertha dominica. The active fractions were eluted with a linear gradient of ethylene glycol from 0 to 50% and tested against azocasein and BApNA.

P.S. Osuna-Amarillas et al. / Journal of Stored Products Research 51 (2012) 41e48

45

40 35

Activity Units

30 25 20 15 10 5 0 3

4

5

6

7

8

9

10

11

12

13

pH Fig. 2. Determination of pH optimum of the proteolytic activity of Rhyzopertha dominica. Each point represents the average of three replicates  SE.

were inhibited by wheat inhibitory extracts (Lane 5). The serine protease inhibitor TPCK (lane 6), a specific inhibitor of chymotrypsin, was unable to cause inhibition of the proteolytic activity suggesting that chymotrypsin-like activity is not present in the proteolytic extract. The inhibitors E-64, pepstatin, and bestatin, which are specific inhibitors for cysteine proteases, aspartic proteases, and metalloproteases, respectively, showed no noticeable inhibitory activity on the crude proteolytic extract (lanes 6, 7, and 8). Fig. 4. Molecular weight determination of the proteases from Rhyzopertha dominica. (1) Molecular weight markers, and (2) zymogram of Rhyzopertha dominica proteases.

3.6. In-vitro inhibition of proteolytic activity The trypsin-like activity of R. dominica was characterized using synthetic inhibitors as well as wheat kernel inhibitors. Table 1 shows that the serine protease inhibitors SBTI and TLCK inhibited the proteolytic activity by 92.3% and 79.2%, respectively. The rest of the inhibitors did not affect the proteolytic activity of the extract used as control. It was also observed that the wheat inhibitory extract completely inhibited the trypsin-like activity of the insect, which is in agreement with previous reports (McGaughey et al.,

1990). However, the high inhibitory effect of the wheat protein preparation used in the present study deserves attention as that, although it was highly effective in inhibiting the trypsin-like activity of R. dominica in vitro, it was observed that the beetle fed and reproduced efficiently in the same wheat variety from which the inhibitor extract was obtained. These results suggest that R. dominica have adapted to overcome the inhibitory effect of wheat proteins found in the kernel from which it feeds.

Specific Activity (Units/mg protein)

60

50

40

30

20

10

0 20

30

40

50

60

70

80

Temperature (°C) Fig. 3. Determination of optimum temperature for proteolytic activity of Rhyzopertha dominica. Each point is the mean of three repetitions  SE.

Fig. 5. Effect of synthetic and natural inhibitors on the proteolytic activity of Rhyzopertha dominica. (1) Control, no inhibitor; (2) TLCK; (3) Soybean trypsin inhibitor; (4) PMSF; (5) Wheat albumin extract; (6) TPCK; (7) E-64; (8) Pepstatin; (9) Bestatin.

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Table 1 Effect of inhibitors on Rhyzopertha dominica proteolytic activity. Concentration (mg/mL)

Residual activity (%)a

Control TLCK SBTI PMSF TPCK E-64 PEPS BEST Wheat albumin extract

e 0.560 0.560 0.280 0.560 0.020 0.056 0.280 80.0

100.0  0 20.8  1.6 6.7  2.0 95.0  2.4 100.0  3.2 94.7  1.1 101.1  2.1 89.7  2.0 0

a

0.05

1/V0

Inhibitor

0.06

Ki

0.04

0.03

Values are expressed as means  SE.

0.02

3.7. Kinetic parameters 0.01

3.8. Inhibition constants The proteolytic digestive activity of R. dominica was very sensitive to the inhibition effect of SBTI with an IC50 value of 0.02 mM/mL (95% confidence interval, 0.0165e0.0226). This value is significantly lower than that reported by Oppert et al. (2003), who observed an average IC50 value of 58 mM/mL for the digestive proteinases from T. castaneum. Silva et al. (2010a) determined average IC50 values of 36.90 and 140.17 mM/mL of SBTI for the trypsin-like proteinase activity of S. zeamais strains showing resistance and susceptibility to pyrethroid insecticides, respectively. A similar low IC50 value (0.1 mg/mL) to that observed for R. dominica trypsin-like activity was determined for the M. brassicae serine proteolytic activity (Chougule et al., 2008). The Dixon plot analysis showed that SBTI is a competitive inhibitor of the trypsinlike activity of R. dominica with a Ki value of 1.17  108 M (Fig. 6). This value is similar to that reported for a serine protease inhibitor highly active against digestive proteases from Z. subfasciatus and C. maculatus (Ki value of 1.65  108 M) (Oliveira et al., 2007). Other studies have reported similar low Ki values such as that of a proteinaceous inhibitor isolated from seeds of the tamarind tree (Tamarindus indica) that showed a Ki value of 1.7  109 M and was highly active against trypsin-like serine proteinases from insect pests (Araújo et al., 2005). The serine protease inhibitor from amaranth (Amaranthus hypochondriacus) seeds was similarly highly active against the trypsin-like activity of P. truncatus with a Ki value of 1.187  109 M (Valdés-Rodríguez et al., 1993).

-0.05

-0.04

-0.03

-0.02

-0.01

0.00 0.00

0.01

0.02

[SBTI] M Fig. 6. Determination of the dissociation constant Ki for soy bean trypsin inhibitor (SBTI) binding to trypsin-like proteases from Rhyzopertha dominica. Different concentrations of the substrate BApNA were used: (C) 0.05 mM (F ¼ 124; df ¼ 14; R2 ¼ 0.94; P < 0.0001), and (B) 0.015 mM (F ¼ 37.8; df ¼ 14; R2 ¼ 0.94; P < 0.0001). Each point represents the average of three replicates  SE.

4. Discussion Results from pH proteolytic activity using BApNA as substrate showed the presence of proteases that were active over a broad range of pH (pH range 6e11). These results differ from those described by Zhu and Baker (1999), who found that the proteolytic activity of R. dominica was most active in a narrower pH range from 7 to 9.5. Serine proteases from different sources are also active in slightly acid to slightly alkaline pH conditions (Oberoi et al., 2001; Xavier et al., 2005). In agreement with our findings, maximum serine proteolytic activity over a broad alkaline pH range (pH 8e11) was observed in T. castaneum (Oppert et al., 2003, 2005) and T. granarium (Hosseininaveh et al., 2007). Chougule et al. (2008) found that the serine proteolytic activity from the cabbage moth M. brassicae was active in the pH range 7e11.5. Similarly, Zeng et al.

4.6

4.4

4.2

4.0

ln V0

The parameters Km and Vmax were 0.07 mM and 2.8 mM/min, respectively, using BApNA as substrate. Ahsan and Watabe (2000) found that the trypsin-like activity from the Japanese anchovy Engraulis japonicas showed an average Km value of 0.035 mM, whereas Xavier et al. (2005) observed Km and Vmax values of 0.23 mM and 1.38 mM, respectively, for the trypsin-like activity of Anticarsia gemmatalis. An isolated trypsin from sardinelle Sardinella aurita showed a Km value of 0.083 mM using BApNA as substrate (Khaled et al., 2011). The Km for the trypsin activity of Mamestra brassicae was 0.069 mM (Chougule et al., 2008) and for the serine proteinase amidolytic activity of several strains of the maize weevil S. zeamais varied in the range 0.22e0.34 mM (Araújo et al., 2008; Silva et al., 2010a).

3.8

3.6

3.9. Activation energy

3.4

The activation energy (Ea) for hydrolysis of BApNA in the temperature range from 30
C to 50
C was 33.5 kJ/mol (Fig. 7), which indicates the high efficiency of the proteolytic digestive activity of R. dominica compared to that of an alkaline protease from Bacillus circulans that showed an Ea value of 59.5 kJ/mol (Rao et al., 2009).

3.2 0.00305

0.00310

0.00315

0.00320

0.00325

0.00330

0.00335

0.00340

1/T °K Fig. 7. Arrhenius plot for the BApNA hydrolysis reaction by proteases from Rhyzopertha dominica. The estimated activation energy Ea was 33.5 kJ mol1 (F ¼ 368.2; df ¼ 14; R2 ¼ 0.993; P < 0.0001). Each point represents the average of three replicates  SE.

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(2002) reported that serine protease activity of Lygus lineolaris was active in the alkaline pH range with maximum at 10. The differences observed in the number of digestive proteases in the present work with respect to those found by Zhu and Baker (1999) could be indicative of the different origins of R. dominica populations used in both studies. A total of eight bands with proteolytic activity were observed with the casein zymogram following electrophoresis. Two unusual high molecular weight trypsins were found in our study (52 and 48 kDa). However, a protease with an apparent molecular weight of 60 kDa found in the digestive tract of T. castaneum activity was reported by Oppert et al. (2005). Vinokurov et al. (2006) found a heavy trypsin-like protease with a molecular weight of 59 kDa, which was assumed that might be conformed by aggregated proteases considering the tendency of these molecules to aggregate in aqueous buffers. The ability of the proteolytic extract to hydrolyze the specific substrate BApNA, the elucidation of the pH range at which maximal hydrolysis occurs, and the effect on trypsins inhibitors (SBTI and TLCK) suggest the presence of trypsin-like proteases. The activity of band E was partially suppressed by PMSF suggesting that it belongs to the serine-protease type, but not the trypsin or quimotrypsin type according to the inhibitory effects observed with SBTI and TLCK, respectively. No activity of other proteases was detected in the digestive tract of the insect according to the null results obtained with the protease inhibitors E-64, pepstatin A, bestatin and TPCK. The kinetic parameter Km calculated for the midgut protease activity of R. dominica was similar to that reported for the trypsinlike activity of M. brassicae (Chougule et al., 2008) and S. aurita (Khaled et al., 2011), however it was lower than those reported for different strains of S. zeamais (Araújo et al., 2008; Silva et al., 2010a). Results of this study indicate that main endoproteinases present in the digestive tract of R. dominica are serine-proteases, specifically trypsin-like proteases. The proteolytic activity of those digestive endoproteinases was inhibited in vitro by inhibitors found in the albumin fraction present in the wheat grain. It would useful to fractionate and evaluate the different protease inhibitors from the wheat kernel to investigate whether all they have the same ability to inhibit R. dominica proteases, something that can be useful to devise a strategy for preventing grain infestation by this insect. Acknowledgment The authors thank to Pedro Figueroa-López (INIFAP, Dr. Norman E. Borlaug Experiment Station, Cd. Obregón, Son., Mexico) for kindly donating the wheat samples used in this study. Author P.S. Osuna-Amarillas acknowledges financial support from the Consejo Nacional de Ciencia y Tecnología (CONACyT, México). References Ahsan, M.N., Watabe, S., 2000. Kinetic and structural properties of two isoforms of trypsin isolated from the viscera of Japanese anchovy, Engraulis japonicas. Journal of Protein Chemistry 1, 49e58. Amorim, T.M.L., Leonardo, L.P., Macedo, L.L.P., Uchoa, A.F., Oliveira, A.S., Joelma, C.M., Pitanga, J.C.M., Macedo, F.P., Santos, E.A., de Sales, M.P., 2008. Proteolytic digestive enzymes and peritrophic membranes during the development of Plodia interpunctella (Lepidoptera: Piralidae): targets for the action of soybean trypsin inhibitor (SBTI) and chitin-binding vicilin (EvV). Journal of Agricultural and Food Chemistry 56, 7738e7745. Araújo, C.L., Bezerra, I.W.L., Oliveira, A.S., Moura, F.T., Macedo, L.L.P., Gomes, C.E.M., Barbosa, A.E.A.D., Macedo, F.P., Souza, T.N.M.S., Franco, O.L., Bloch-, J.C., Sales, M.P., 2005. In vivo bioinsecticidal activity toward ceratitis capitata (fruit fly) and Callosobruchus maculatus (cowpea weevil) and in vitro bioinsecticidal activity toward different orders of insect pests of a trypsin inhibitor purified from tamarind tree (Tamarindus indica) seeds. Journal of Agricultural Food Chemistry 53, 4381e4387.

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Araújo, R.A., Guedes, R.N.C., Oliveira, M.G.A., Ferreira, G.H., 2008. Enhanced proteolytic and cellulolytic activity in insecticide-resistant strains of the maize weevil, Sitophilus zeamais. Journal of Stored Products Research 44, 354e359. Baker, J.E., 1991. Purification and partial characterization of a-amylase allozymes from the lesser grain borer, Rhyzopertha dominica. Insect Biochemistry 21, 303e311. Blum, H., Beier, H., Gross, H.J., 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 78, 93e99. Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248e254. Burlingham, B.T., Widlanski, T.S., 2003. An intuitive look at the relationship of Ki and IC50: a more general use for the Dixon plot. Journal of Chemical Education 2, 214e218. Chanbang, Y., Arthur, F.H., Wilde, G.E., Throne, J.E., 2007. Efficacy of diatomaceous earth to control Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) in rough rice: Impacts of temperature and relative humidity. Crop Protection 26, 923e929. Chougule, N.P., Doyle, E., Fitches, E., Gatehouse, J.A., 2008. Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays. Journal of Insect Physiology 54, 563e572. Cinco-Moroyoqui, F.J., Díaz-Malváez, F.I., Alanís-Villa, A., Barrón-Hoyos, J.M., Cárdenas-López, J.L., Cortez-Rocha, M.O., Wong-Corral, F.J., 2008. Isolation and partial characterization of three isoamylases of Rhyzopertha dominica F. (Coleoptera: Bostrichidae). Comparative Biochemistry and Physiology 150B, 153e160. Cinco-Moroyoqui, F.J., Rosas-Burgos, E.C., Borboa-Flores, J., Cortez-Rocha, M.O., 2006. a-Amylase activity of Rhyzopertha dominica (Coleoptera: Bostrichidae) reared on several wheat varieties and its inhibition with kernel extracts. Journal of Economic Entomology 6, 2146e2150. Elpidina, E.N., Tsybina, T.A., Dunaevsky, Y.E., Belozersky, M.A., Zhuzhikov, P., Oppert, B., 2005. A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae. Biochimie 87, 771e779. Heussen, C., Dowdle, E.B., 1980. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Analytical Biochemistry 102, 196e202. Hosseininaveh, V., Bandani, A., Azmayeshfard, P., Hosseinkhani, S., Kazzazi, M., 2007. Digestive proteolytic and amylolytic activities in Trogoderma granarium Everts (Dermestidae: Coleoptera). Journal of Stored Products Research 43, 515e522. Houseman, J.G., Thie, N.M.R., 1993. Difference in digestive proteolysis in the stored maize beetles: Sitophilus zeamais (Coleoptera: Curculionidae) and Prostephanus truncatus (Coleoptera: Bostrichidae). Journal of Economic Entomology 4, 1049e1054. Janarthanan, S., Suresh, P., Radke, G., Morgan, T.D., Oppert, B., 2008. Arcelins from an Indian wild pulse, Lablab purpureus, and insecticidal activity in storage pests. Journal of Agricultural and Food Chemistry 56, 1676e1682. Kan, H., Kim, C.H., Kwon, H.M., Park, J.W., Roh, K.B., Lee, H., Park, B.J., Zhang, R., Zhang, J., Söderhäll, K., Ha, N.C., Lee, B.L., 2008. Molecular control of phenoloxidase-induced melanin synthesis in an insect. Journal of Biological Chemistry 37, 25316e25323. Khaled, H.B., Jellouli, K., Souissi, N., Ghorbel, S., Barkia, A., Nasri, M., 2011. Purification and characterization of three trypsin isoforms from viscera of sardinelle (Sardinella aurita). Fish Physiology and Biochemistry 37, 123e133. Kitch, L.W., Murdock, L.L., 1986. Partial characterization of a major gut thiol proteinase from larvae of Callosobruchus maculatus (F.). Archives of Insect Biochemistry and Physiology 3, 561e575. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 22, 77e80. Lee, K.Y., Zhang, R., Kim, M.S., Park, J.W., Park, H.Y., Kawabata, S., Lee, B.L., 2002. A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca2þ in coleopteran and Tenebrio molitor larvae. European Journal of Biochemistry 17, 4375e4383. Ma, C.C., Kanost, M.R., 2000. A beta 1,3-glucan recognition protein from an insect, Manduca sexta agglutinates microorganisms and activates the phenoloxidase cascade. Journal of Biological Chemistry 275, 7505e7514. McGaughey, W.R., Speirs, R.D., Martin, C.R., 1990. Susceptibility of classes of wheat grown in the United States to stored-grain insects. Journal of Economic Entomology 83, 1122e1127. Murdock, L.L., Brookhart, G., Dunn, P.E., Foard, D.E., Kelley, S., 1987. Cysteine digestive proteinases in Coleoptera. Comparative Biochemistry and Physiology 87B, 783e787. Oberoi, R., Beg, Q.K., Puri, S., Saxena, R.K., Gupta, R., 2001. Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp. World Journal of Microbiology and Biotechnology 17, 493e497. Oliveira, A.S., Migliolo, L., Aquino, R.O., Ribeiro, J.K.C., Macedo, L.L.P., Andrade, L.B.S., Bemquerer, M.P., Santos, E.A., Kiyota, S., Sales, M.P., 2007. Purification and characterization of a trypsinepapain inhibitor from Pithecelobium dumosum seeds and its in vitro effects towards digestive enzymes from insect pests. Plant Physiology and Biochemistry 45, 858e865. Oppert, B., Elpidina, E.N., Toutges, M., Mazumdar-Leighton, S., 2010. Microarray analysis reveals strategies of Tribolium castaneum larvae to compensate for cysteine and serine protease inhibitors. Comparative Biochemistry and Physiology 5D, 280e287.

48

P.S. Osuna-Amarillas et al. / Journal of Stored Products Research 51 (2012) 41e48

Oppert, B., Morgan, T.D., Hartzer, K., Kramer, K.J., 2005. Compensatory proteolytic responses to dietary proteinase inhibitors in the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae). Comparative Biochemistry and Physiology 140C, 53e58. Oppert, B., Morgan, T.D., Hartzer, K., Lenarcic, B., Galesa, K., Brzin, J., Turk, V., Yozad, K., Ohtsubod, K., Kramer, K.J., 2003. Effects of proteinase inhibitors on digestive proteinases and growth of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Comparative Biochemistry and Physiology 134C, 481e490. Rao, Ch. S., Sathish, T., Ravichandra, P., Prakasham, R.S., 2009. Characterization of thermo and detergent stable serine protease from isolated Bacillus circulans and evaluation of eco-friendly applications. Process Biochemistry 44, 262e268. Reeck, G.R., Oppert, B., Denton, M., Kanost, M., Baker, J.E., Kramer, K.J., 1999. Insect proteinases. In: Turk, V. (Ed.), Proteases: New Perspectives. Birkhauser, Boston, pp. 125e148. Silva, C.P., Terra, W.R., Lima, R.M., 2001. Differences in midgut serine proteinases from larvae of the bruchid beetles Callosobruchus maculatus and Zabrotes subfasciatus. Archives of Insect Biochemistry and Physiology 1, 18e28. Silva, L.B., Reis, A.P., Pereira, E.J.G., Oliveira, M.G.A., Guedes, R.N.C., 2010a. Partial purification and characterization of trypsin-like proteinases from insecticideresistant and -susceptible strains of the maize weevil, Sitophilus zeamais. Comparative Biochemistry and Physiology 155B, 12e19. Silva, L.B., Reis, A.P., Pereira, E.J.G., Oliveira, M.G.A., Guedes, R.N.C., 2010b. Altered cysteine proteinase activity in insecticide-resistant strains of the maize weevil: purification and characterization. Comparative Biochemistry and Physiology 157B, 80e87. Terra, W.R., Ferreira, C., 1994. Insect digestive enzymes: properties, compartmentalization and function. Comparative Biochemistry and Physiology 109B, 1e62.

Toews, M.D., Cuperus, G.W., Phillips, T.W., 2000. Susceptibility of eight U.S. wheat cultivars to infestation by Rhyzopertha dominica (Coleoptera: Bostrichidae). Environmental Entomology 29, 250e255. Tsybina, T.A., Dunaevsky, Y.E., Belozersky, M.A., Zhuzhikov, D.P., Oppert, B., Elpidina, E.N., 2005. Digestive proteinases of yellow mealworm (Tenebrio molitor) larvae: purification and characterization of a trypsin-like proteinase. Biochemistry (Moscow) 3, 300e305. Valdés-Rodríguez, S., Segura-Nieto, M., Chagolla-López, A., Verver y VargasCortina, A., Martínez-Callardo, N., Blanco-Labra, A., 1993. Purification, characterization, and complete amino acid sequence of a trypsin inhibitor from amaranth (Amaranthus hypochondriacus) seeds. Plant Physiology 103, 1407e1412. Vinokurov, K.S., Elpidina, E.N., Oppert, B., Prabhakar, S., Zhuzhikov, D.P., Dunaevsky, Y.E., Belozersky, M.A., 2006. Fractionation of digestive proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein digestion. Comparative Biochemistry and Physiology 145B, 138e146. Xavier, L.P., Oliveira, M.G.A., Guedes, R.N.C., Santos, A.V., de-Simone, S.G., 2005. Trypsin-like activity of membrane-bound midgut proteases from Anticarsia gemmatalis (Lepidoptera: Noctuidae). European Journal of Entomology 102, 147e153. Zeng, F., Zhu, Y., Cohen, A., 2002. Partial characterization of trypsin-like protease and molecular cloning of a trypsin-like precursor cDNA in salivary glands of Lygus lineolaris. Comparative Biochemistry and Physiology 131B, 453e463. Zhu, Y.C., Baker, J.E., 1999. Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae). Insect Biochemical Molecular Biology 29, 1053e1063.