Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 13, Num. 4, 1996

Determination And in vitro Inhibition Of Alpha- amylase And Protease Activity In Spodoptera frugiperda

Julio Alfonso,^1 Rosa Sanchez-Monge,^2 Gloria Garcia-Casado,^2 Yamilet Coll,^1 Raul Armas,^1 Merardo Pujol^1 and Gabriel Salcedo^2

^1 Center for Genetic Engineering and Biotechnology, P.O. Box. 83, C.P. 60200, Sancti-Spiritus, Cuba. E-mail: pujol@cigbss.ingen.edu.cu ^2 Biotechn. Dept., E.T.S.I.A., Polytechn. Univ., Madrid 28040, Spain.

Code Number: BA96114
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Introduction

Proteinaceous inhibitors of digestive enzymes from insects might play an important role in plant protection (1). Different inhibitor preparations from wheat and barley endosperms inhibited alpha-amylases from 23 agricultural insect pests (2), or trypsin activities (3). We determined the main digestive activity of the armyworm Spodeptera frugiperda, and evaluated its inhibition by dimeric, monomeric and tetrameric alpha-amylases inhibitors from wheat and a trypsin inhibitor (CMe) from barley cv. Bomi.

Materials and Methods

Insects

Larvae of S. frugiperda were grown on diets as (4).

Insect extracts

Extracts from various instars S. frugiperda larvae were made as (1) with Tris and Glycine buffer to elucidate alpha- amylase activity during larval cycle. Extracts for trypsin inhibition was prepared in HCl.

Inhibition tests

Alpha-amylase activity and in vitro inhibition was made essentially as (5). L-BAPA was used for trypsin activity of extracts and in vitro tests were made according to (6).

Inhibitors

Monomeric, dimeric, and tetrameric inhibitor fractions from wheat were purified as (7), and CMe proteins were prepared following reported protocols (8).

Results and Discussion

S. frugiperda alpha-amylase showed maximal activity at pH 8.5-9.5 in Tris buffer. Figure 1 shows maximal alpha- amylase activity during 11 and 12 days of larvae life, coinciding with the highest feeding activity of the insect for life stage change. S. frugiperda alpha-amylase activity in non-denaturing PAGE was observed during larval life, and no difference among the 5 different isoenzymes was detected. The tetrameric inhibitor inhibits 25 and 60 % respectively of the total alpha-amylase activity at 7 and 11 days of larval life cycle, whereas inhibition with dimeric and monomeric inhibitors was not significant.

Figure 1. Alpha-amylase activity and inhibition.

Figure 1 shows also alpha-amylase inhibition with 5ug of wheat tetrameric inhibitor. This was minimal between 7-10 days of larvae life cycle, probably because of the high quantity of insect pigment compounds interfering the assay. Figure 2 shows in vitro alpha-amylase inhibition.

Protease activity of the trypsin type in the larvae midgut was detected and it was inhibited at 64 % by 5ug of CMe proteins from barley cv. Bomi. We concluded that the tetrameric and CMe trypsin inhibitors could be good candidates for protecting plants against S. frugiperda attack by genetic engineering.

Figure 2. In vitro alpha-amylase inhibition; a- S. frugiperda alpha-amylase activity; b-g, S. frugiperda alpha-amylase activity inhibition by: b-d, 1, 3, 5 ug wheat tetrameric inhibitor; e-g, 1, 3, 5 ug wheat dimeric inhibitor; h-j, 1, 3, 5 ug wheat monomeric inhibitor.

References

1. Garcia F, et al. Oxf Surv P Mol Cell Biol 1987;4:275-280.

2. Gutierrez C, et al. Plant Sci 1990; 72: 37-44.

3. Moralejo M, et al. Plant Sci 1993;89: 23-29.

4. Ayala J, Armas J. C Agricola UCLV 1987;14(4):88-90.

5. Benfeld P. beta, alpha-amylases. Meth Enzymol 1995;1:49- 158.

6. Boisen S, Djurtoft R. Cereal Chem 1981;58:194-198.

7. Sanchez R, et al. Theor Appl Gen 1986;74:811-816.

8. Salcedo, et al. J Exper Bot 1982;33: 1325-1331.

Copyright 1996 Elfos Scientiae

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