The insect pathogen Bacillus thuringiensis (Bt) holds great promise as an effective and friendly way for management of the pests with safety for nontarget animals and humans. However, high capital investment due to high production and formulation cost of commercial Bt preparations has caused prohibitive effect on companies. The present study mainly aimed at developing a low cost medium that supports the growth of different Bt strains and their specific bioinsecticidal &amp / #948 / -endotoxins (crystal proteins). A comparison was made between the representative members of three different subspecies of Bt to observe toxin yields in response to certain nutritional conditions. Three different Bt subspecies were Bt kurstaki (strain 81), Bt israelensis (strain HD500) and Bt tenebrionis (strain 3203), producing lepidoptera- and diptera-specific Cry1 and Cry2, diptera-specific Cry4Ba and Cry11Aa and coleoptera-specific Cry3Aa toxins, respectively. Studies were conducted to optimize glucose and inorganic phosphate concentrations in standard DSM medium for the production of these Bt-based biopesticides. General suppression of toxin yields in high glucose medium (10 g/L) thought the generality of carbon catabolite regulation for biosynthesis of different types of toxins. Inorganic phosphate (Pi) level was important for Cry4Ba, Cry11Aa and Cry3Aa biosynthesis while Cry1 and Cry2 production was not responsive to high Pi. Wastewater sludge, fruit residues and broiler litter were next tested as cheap raw materials for Bt-based biopesticide production in batch cultures. Broiler litter seemed to be a much better substrate among all since some degree of production of each toxin was observed at almost every stage of fermentation. The processing of broiler litter was found to significantly improve toxin yields. The medium prepared from processed broiler litter was successfully used to cultivate all Bt stains and obtain bioinsecticidal proteins in high yields which were comparable or higher than those that can be obtained on standard semi-synthetic media.

The control of pest populations by using insect pathogens has been an attractive alternative to the application of chemical pesticides employed for the same purpose. As these chemicals not only damage the environment, but also trigger development of resistance by the pests and can harm other organisms together with the target pest, biological control is preferable and Bacillus thuringiensis (Bt) subspecies have been the most widely used bioinsecticides in forestry, agriculture and mosquito/ black fly control. The most important property of Bt subspecies is the synthesis of protoxins named as delta-endotoxins (crystal proteins). In this study, response surface optimization of Bt subsp. israelensis HD500 batch fermentation for high level production of its toxin proteins Cry4Ba and Cry11Aa was performed. As the interaction of the medium components as well as cultivation conditions are expected to influence the production of the toxin proteins, an experimental chart was prepared by accepting the previously reported optimal values for the most important parameters as zero points: [Mn], 10-6 M / [K2HPO4], 50 mM / C:N ratio, 20:1 and incubation temperature / 30&deg / C. When the combinations of these variables at different levels were studied at 30 batch cultures and analysed for the optimum toxin protein concentrations, temperature: 28.3&amp / #61616 / C, [Mn]: 3.3x10-7M, C:N ratio: 22.2 and [K2HPO4]: 66.1mM yielded the highest concentrations of both Cry4Ba and Cry11Aa toxin proteins.

Cry3Aa is a 73 kDa protoxin toxic to insect larvae of Coleoptera order. It is processed to form a stable 65 kDa &amp / #61540 / -endotoxin by endogenous proteases. The first part of this study involved the determination of the patterns of biosynthesis of Coleoptera-specific &amp / #61540 / -endotoxin by a local isolate of Bacillus thuringiensis subsp. tenebrionis (Btt) in relation to its growth and sporulation. Among four different media compared (DSM, GYS, HCT and C2) Cry3Aa production was the highest in DSM, especially at 72nd h and 120th h of incubation. For improvement of Cry3Aa production, the effects of different carbon and nitrogen sources, inorganic phosphate and other mineral elements were tested. Increasing concentrations (5-10 g.L-1) of glucose or sucrose decreased the toxin yield probably by suppressing sporulation. Inorganic phosphate was found to have the most striking effect on toxin biosynthesis. 200 mM inorganic phosphate concentration resulted in 5 fold increase in Cry3Aa yield. Cry3Aa production was greatly reduced when various combinations of organic and inorganic nitrogen sources, especially ammonium sulphate and Casamino acids were replaced with Nutrient broth in DSM. The highest Cry3Aa production was obtained in the media containing 10-5-10-7 M MnCl2, 10-5 M FeSO4 and 5.10-4 M MgSO4, corresponding to their original concentrations in DSM. Decrease of iron concentration or its omission from the medium decreased the toxin yield. Toxin production capacity of our local isolate was compared with those of 30 different anti-Coleopteran Bt strains. Most of the strains producing this protein gave general protein banding patterns quite similar to that of our local isolate. Lastly, the cry3Aa gene of the Btt local isolate was PCR-amplified and cloned into the E. coli/Bacillus shuttle vector pNW33N. The recombinant plasmid was amplified in E. coli and the sequence of the cry3Aa was determined. Amino acid sequence deduced was found to be 97.4 %-99.2 % identical to the cry3Aa sequences (GenBank) of 10 different quaternary ranks. In this respect, the gene has to represent the 11th quaternary rank of the cry3Aa ones. The recombinant plasmid carrying cry3Aa gene was next used to transform Bs 168 as an intermediate host and low level of expression was seen.

Made available in DSpace on 2014-08-20T14:31:29Z (GMT). No. of bitstreams: 1 dissertacao_ana_paula_de_lara.pdf: 4627911 bytes, checksum: 887ee69dd56b73509c1a603e3b25aebd (MD5) Previous issue date: 2013-03-08 / Bacillus thuringiensis (Bt) is a Gram-positive bacteria, ubiquitous, facultative anaerobic, and form spores. During sporulation produce a parasporal crystals inclusion. Within these inclusions there are δ-endotoxin proteins well known for its insecticides proprieties. Among them, the Cry (crystal) is wide employed for biological control of plagues. The δ-endotoxin has an advantage of been more specific than chemical insecticides, thus been consider more favorable for the environment. The aim of this study was to obtain the Cry 11Aa recombinant protein of Bacillus thuringiensis var. israelensis in Escherichia coli, active for use in biocontrol. Two expression E. coli strains were tested: BL 21 (DE3) C41 and BL 21 (DE3) Ril. The protein Cry 11Aa was expressed and secreted in a soluble form by the two strains. The expression was demonstrated by Western Blot using anti-histidin monoclonal antibody. The strain BL 21 (DE3) C41 express the protein Cry 11Aa ~3.6 times more than the strain Rill, and showed a biologic efficiency of 95% of mortality for Culex quinquefaciatus larvae. The data obtained in this study suggest that the protein recombinant Cry 11Aa expressed in E. coli has a potential to be used in biological control. / Bacillus thuringiensis (Bt) é uma bactéria Gram-positiva, de ocorrência ubíqua, anaeróbica facultativa, formadora de esporos. Produz cristais, como inclusões parasporal durante a esporulação. Estas inclusões contêm proteínas chamadas de δ-endotoxinas, que são bem conhecidas pelas suas propriedades inseticidas. Dentre elas as toxinas Cry (crystal) são largamente empregadas no controle biológico de pragas. As δ-endotoxinas têm a vantagem de serem mais específicos do que os inseticidas químicos sintéticos, portanto, são considerados como agentes de controles favoráveis ao meio ambiente. O objetivo deste estudo foi a obtenção da proteína Cry 11Aa recombinante de Bacillus thuringiensis var. israelensis em Escherichia coli, ativa, para utilização no controle biológico.Duas cepas de E. coli de expressão foram testadas: BL 21 (DE3) C41 e BL 21 (DE3) Ril. A proteína Cry 11Aa foi expressa e secretada na forma solúvel pelas duas cepas. A expressão foi demonstrada por Western blot utilizando-se anticorpo monoclonal anti-histidina. A cepa BL 21 (DE3) C41 expressou a proteína Cry 11Aa ~3.6 vezes mais que a cepa BL 21 (DE3) Ril, e apresentou, em teste biológico, uma eficácia de 95% de mortalidade sobre larvas de Culex quinquefaciatus. Com os dados obtidos neste trabalho podemos sugerir que a proteína recombinante Cry 11Aa expressa em E. coli é um potencial candidato para ser utilizado no controle biológico.