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Characterisation of Bacillus thuringiensis variety israelensis delta-endotoxin

Published online by Cambridge University Press:  19 September 2011

Fred W. Wamunyokoli  and
Ellie O. Osir
Fred W. Wamunyokoli
Affiliation:
Department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology, P. O. Box 62000, Nairobi, Kenya
Ellie O. Osir
Affiliation:
International Centre of Insect Physiology and Ecology, P. O. Box 30772, Nairobi, Kenya
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Abstract

Crystals of a local isolate of Bacillus thuringiensis variety israelensis were isolated by centrifugation on a continuous sucrose gradient (40–70%). Analysis of the crystals by SDS-PAGE revealed three major protein subunits of Mr˜25,000, ˜66,000 and Mr˜140,000. The crystals were solubilised using high pH and reducing conditions. Analysis of the soluble (protoxin) and insoluble fractions by SDS-PAGE showed the presence of proteins of Mr˜21,000 and ˜61,000, respectively. Proteolytic treatment of these fractions resulted in no apparent change in the molecular weights of the proteins. The crystals contained carbohydrate moieties as determined by periodic acid Schiff (PAS) and fluorescein isothiocyanate (FITC) Concanavalin A staining. In double radial immunodiffusion experiments, antisera raised against the Mr˜21,000 and ˜66,000 subunits did not react with other B. thuringiensis protoxins known to be active against the tsetse fly, Glossina morsitans morsitans, and the stemborer, Chile partellus.

Résumé

Les cristaux d'un isolat local de Bacillus thuringiensis var. israelensis ont été isolés sur un gradient de saccharose continu (40–70%). L'analyse des crystaux par SDS-PAGE a mis en évidence trois sous-unités protéiques majeures de Mr ˜25.000, ˜66.000 et Mr ˜140.000. Les crystaux ont été solubilisés en utilisant un pH élevé en conditions réductrices. L'analyse des fractions solubles (protoxines) et insolubles par SDS-PAGE a montré la présence des protéines de Mr ˜21.000 et ˜61.000. respectivement. Le traitement protéolytique de ces fractions a eu comme résultat un changement non apparent dans les poids moléculaires des protéines. Les cristaux contenaient des moïetés d'hydrates de carbone telles que determinées par l'acide périodique de Schiff (PAS) et l'isothiocianate de flourecéine (FTTC) qui colore la canavaline A. Dans les expériences de double immunodiffusion radiale, les antisérums produits contre les sous-unités Mr ˜21.000 et ˜66.000 n'ont pas réagi avec les autres protoxines de B. thuringiensis qui sont reconnues comme actives contre la mouche tsé-tsé, Glossina morsitans morsitans, et la foreur de tiges, Chilo partellus.

Keywords

Bacillus thuringiensiscrystalsendotoxinsphysico-chemical propertiesBacillus thuringiensiscrystauxendotoxinespropriétés physico-chimiques
Type
Research Articles
Information
International Journal of Tropical Insect Science , Volume 16 , Issue 3-4 , December 1995 , pp. 343 - 349
Copyright
Copyright © ICIPE 1995

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