Isolation, purification and characterization of Bacillus thuringiensis o-endotoxin active against Mosquito larvae

Abstract

During the past decade, growing public awareness of the impact of pesticides on food and enviromental safety has significantly affected the pest control industry. Several organizations (scientific, consumer, enviromental and governmental) have called for more severe restrictions on the use of toxic chemicals and increased funding for the development of alternative pest control methods (Gelernter, 1990). This has been a major stimulus for renewed interest in the use of microbial control agents, which have an excellent safety record and maybe produced using renewable raw-materials. Many of these agents can be integrated with chemical and other pest management techniques. Among the microbial agents that offer great potential in this respect is Bacillus thuringiensis (B.t.) (Davidson and Sweeney, 1983). B.t. based products account for 90-95% of the total bio-pesticide markeb_(Feitelson et al., 1992). Worldwide sales of B.t. products have. grown from $24 million in 1980 to $107 million -in 1989. Annual sale~ a~e forecast to expand at a rate of 11% teaching $300 millio.n by the year 1999 (Feitelson et al., 1992). In this study, a newly isolated B.t. strain was cultured by shake flask fe-rmentation involving two inoculum stages and a production stage. The a-endotoxin crystals so produced were isolated by centrifugation on a continuous sucrose gradient (40-70%). Analysis of the amorphous protein crystals by SDS polyacrylamide gel electrophoresis, revealed three major sub-units of molecular weights -25 KD, -66 KD, and -140 KD. Upon solubilization of the crystal under high pH and reducing conditions, the resulting supernatant solution had one major protein sub-unit band (Mr -21 KD), while the pellet also had one major sub-unit band (Mr -66 KD). Upon protease treatment of the solubilized protoxin and insoluble protoxin (pellet) fractions using bovine pancreatic trypsin, a-chymotrypsin, insect larval gut homogenates (Aedes aegypti, Chilo partellus, Musca domestica) and partially purified trypsin like protease from Glossina spp midgut, no apparent change in the molecular weight of the major proteins was observed. The crystal was shown to be a glycoprotein with high mannose sugar residues as demonstrated by staining with periodic acid Schiff reagent and fluorescein~isothiocyanate conjugated concanavalin-A~ respectively. The carbohydrate content of , the crystal estimated by phenol sulphuric acid method was 0.023 ± 0.0016%. Double radial immunodiffusion experiments showed that the antisera raised against the Mr -21 KD and Mr -66 KD protein sub-units did not cro~~-react with the solubilized protoxins a9tive against Glossina spp (TIKI) and Chilo partellus/~podoptera exempta (MFB4/2), respectively.