O recente aumento da procura por implantes osseointegráveis consequentemente levou ao aumento dos procedimentos de enxertos ósseos. Com isso, novos materiais têm surgido para estes procedimentos. Um destes materiais é o osso bovino integral (OBIN), cujo diferencial é o seu método de processamento, que visa manter suas características o mais próximas possível ao osso in natura. Por se tratar de um material novo, ainda existem poucos estudos avaliando seu desempenho, sendo a maioria deles estudos pré-clínicos testando sua biocompatibilidade. Portanto, o objetivo deste trabalho foi avaliar o desempenho do OBIN na região maxilofacial através de um modelo experimental em animais. Foram criados defeitos ósseos na base da mandíbula de coelhos, onde foram enxertados blocos de OBIN nos grupos experimentais e deixados vazios nos grupos controle. Os animais foram sacrificados nos tempos 0 (imediatamente após a cirurgia), 3 e 6 meses após o procedimento cirúrgico. As mandíbulas foram analisadas através de tomografias computadorizadas de feixe cônico, onde foi analisada a regeneração óssea qualitativamente e quantitativamente. Na análise qualitativa observou-se que aos três e seis meses, nos animais do grupo controle, não houve regeneração total, restando um defeito côncavo na base da mandíbula, confirmando que o defeito criado possuía tamanho crítico. No grupo experimental, no tempo 0, observou-se grande variação de densidade entre os enxertos e esta variação pareceu influenciar seu desempenho, havendo mais regeneração óssea nos menos densos. No tempo 3, todos os enxertos haviam integrados, com perda de densidade, sugerindo uma reabsorção parcial. Aos seis meses, a reabsorção e regeneração pareciam mais avançadas do que aos três meses. Na análise quantitativa houve diferença estatisticamente significante nos grupos controles entre os tempos 0 e os tempos 3 e 6. Nos grupos experimentais não houve diferença entre os tempos e na comparação entre os grupos controles e experimentais houve diferença estatisticamente significante entre todos os tempos. Os resultados observados sugerem que o OBIN é um material indicado para a realização de procedimentos de enxerto na região maxilofacial, com aparente reabsorção e substituição por osso regenerado, ideal para a reabilitação com implantes ósseo integráveis. / The recent increase in the demand for dental implants consequently led to increased bone grafting procedures. Therefore, new materials have emerged for these procedures. One of these materials is integral bovine bone (IBB) whose differential is its processing, which aims to maintain its characteristics as close as possible to the raw bone. Because it is a new material, there are only a few studies evaluating its performance, with the majority of them constituted of preclinical studies testing its biocompatibility. For this reason, the aim of this study was to evaluate the performance of the IBB at the maxillofacial region through an experimental model in animals. Bone defects were created on the basis of the mandible of rabbits, which were grafted with blocks of IBB in the experimental groups, while control groups were left empty. The animals were sacrificed at time 0 (immediately after surgery), 3 and 6 months after surgery. The jaws were analyzed using cone beam computed tomography, where bone regeneration was analyzed qualitatively and quantitatively. Qualitative analysis showed that at three and six months, in the control group, there was not full regeneration, leaving a concave defect in the base of the mandible, confirming that the defect created had critical size. In the experimental group, at time 0, there was wide variation in density between the grafts and this variation appeared to influence their performance, seeming to be more bone regeneration in less dense ones. At time 3, all grafts were integrated, and a density loss, suggesting a partial resorption of the graft, was also observed. At six months, resorption and regeneration seemed more advanced than at three months. The quantitative analysis did not showed statistically significant difference in controls between time 0 and times 3 e 6. In experimental groups there was no difference between times and comparison between control and experimental groups were statistically significant different between all time. The data suggest that the IBB is a material suitable for the realization of grafting procedures in the maxillofacial region, with apparent resorption and replacement by regenerated bone, ideal for rehabilitation with dental implants.

The wide diversity of plant secondary products results from different modifications undergone during biosynthesis, including glucosylation. These modification reactions result in production of the compounds actually found in plants and to unique chemical and biochemical properties, including some bitter compounds in grapefruit. While the presence of a PSPG box motif allows for identification of a clone as a putative glucosyltransferase (PGT), diversity of GT primary structures makes it difficult to accurately assign specific function. Our approach is to identify and isolate putative GT clones, express them heterologously, and biochemically characterize the proteins. Eleven putative GT clones have been isolated from Citrus paradise and some have been biochemically characterized. The current hypothesis being tested is that PGT3 and PGT9 clones are plant secondary product GTs. Due to issues with inclusion bodies when using E. coli, proteins were expressed in Pichia pastoris using the pPICZA vector. Recombinant protein expression was confirmed by Western blot and proteins were enriched by IMAC. Over 30 flavonoid and simple phenolic substrates, representing many compounds found in grapefruit, were screened for activity with PGT3 and PGT9 proteins. No significant activity was found and the biochemical function of the proteins encoded by these clones will be further investigated.

A produção de biocombustíveis e a obtenção de alguns compostos químicos a partir de materiais renováveis, como a biomassa lignocelulósica, ainda não são processos triviais, principalmente devido a recalcitrância destes materiais. Estudos recentes reconheceram as enzimas acessórias, como xilanases e enzimas com Atividade Auxiliar, como potencializadores da atividade de celulases no processo de despolimerização da lignocelulose. A prospecção de enzimas com características termoestáveis é vantajosa para este tipo de aplicação e além disso, estudos sobre o secretoma de diversos fungos cultivados em biomassa como fonte de carbono, tem encontrado enzimas com mecanismo oxidativo, dentre eles, o fungo termofílico Myceliophthora thermophila M77. Porém, estas enzimas tem sido pouco estudadas quanto a sua aplicação na sacarificação da biomassa. Sendo assim, este trabalho visou a expressão heteróloga, a caracterização bioquímica e a ação da enzima oxidativa celobiose desidrogenase do fungo M. thermophila (M77CDH) em conjunto com outras celulases no processo de sacarificação da biomassa. Pela análise filogenética a M77CDH prospectada foi classificada como pertencente a Classe IIB das CDHs. O gene que codifica esta enzima foi clonado no vetor pEXPYR e heterólogamente expresso em A. nidulans. A proteína recombinante M77CDH foi purificada e teve sua identidade confirmada por espectrometria de massas. Nas análises bioquímicas, apresentou atividade ótima a 65 °C e reteve mais de 80% da sua atividade a 50°C por 2 horas e pela análise de dicroísmo circular apresentou um desenovelamento da sua estrutura na temperatura de transição de 62,8 °C. Apresentou mais de 80% de atividade em uma faixa ampla de pH (4,5 - 9), em que o domínio citocromo mostrou maior afinidade em pHs alcalinos, característica incomum entre as CDHs descritas na literatura. A atividade da M77CDH foi ligeiramente aumentada pela adição de MgCl2 e Na2MoO4 e altamente afetada por CuSO4 e FeCl3. A eficiência catalítica (kcat/km=266 mM-1s-1) utilizando celobiose foi bastante similar aos valores indicados por CDHs da Classe IIA. O envelope da M77CDH gerado por SAXS foi satisfatório e conveniente com a literatura. Na sacarificação de bagaço de cana pré-tratado hidrotermicamente, utilizando coquetel de A. niveus suplementado com M77CDH, foi possível observar que a adição de M77CDH modificou o perfil de produtos liberados na desconstrução da biomassa. Por fim, na sacarificação do PASC observou-se a sacarificação e produção de ácido celobiônico. / The production of biofuels and chemicals from renewable materials such as lignocellulosic biomass are non-trivial processes mainly due to the recalcitrance of the material. Recent studies have recognized accessory enzymes such as xylanases and Auxiliary Activity enzymes as potentiators in cellulase activity during the depolymerization of lignocellulose. The prospection of thermostable enzymes can be an advantage the improve the depolymerization of these materials. In addition, several enzymes showing oxidative mode of action were found in the secretoma of the thermophilic fungus Myceliophthora thermophila strain M77. However, these enzymes are poor studied regarding their application in biomass saccharification. Therefore, this project aimed the heterologous expression and biochemical characterization of the oxidative enzyme cellobiose dehydrogenase of the fungus M. thermophila (M77CDH). By phylogenetic analysis the M77CDH was classified as belonging to Class IIB of CDHs. The gene encoding this enzyme was cloned and heterologously expressed in A. nidulans, the M77CDH was purified and had its identity confirmed by mass spectrometry. In the biochemical analyzes the M77CDH showed an optimum activity at 65 °C and retained more than 80% of its activity at 50 °C for 2 hours. The circular dichroism analysis showed a denaturation of its structure at the transition temperature of 62.8 ° C. M77CDH also kept more than 80% of its activity in a wide pH range (4.5 - 9), in which the cytochrome domain showed higher affinity at alkaline pH, an unusual behavior compared with other CDHs described in the literature. The activity of M77CDH was increased slightly in the presence of MgCl2 and Na2MoO4 and was highly affected by CuSO4 and FeCl3. The catalytic efficiency (kcat/km = 266 mM-1s-1) in cellobiose was quite similar to the values indicated by CDHs from Class IIA. The envelope of M77CDH generated by SAXS was satisfactory and convenient with the literature. In saccharification of sugarcane bagasse hydrothermally pretreated using A. niveus cocktail supplemented with M77CDH was possible to observe the addition of M77CDH modified the profile of released products in the deconstruction of the biomass. Finally, in the action on PASC was observed the saccharification and production of cellobionic acid.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of carbohydrate metabolism in all living organisms. The main aim of this thesis was to investigate the function and regulation of plant UGP genes as well as the UGPase proteins. Both in vivo and in vitro approaches were used, including the use of transgenic plants deficient in UGPase activity, and using purified proteins and their mutants to elucidate the structure/ function properties of UGPase. In both Arabidopsis and aspen, there are two highly similar UGP genes being actively transcribed, but not to the same extent. For both species, the UGP genes could be classified into two categories: a “house-keeping” gene and a subsidiary gene, with the former functioning universally in all the tissues to support the normal growth, whereas the latter usually expressed at a lower level in most of the organs/tissues tested. Besides, the two UGP genes were also found being differentially regulated under abiotic stress conditions, e.g. low temperature. By investigating the Arabidopsis T-DNA insertion mutants, which respectively have one or both of the UGP genes knocked out, we noticed that as little as 10% of the remaining UGPase activity could still support normal growth and development under controlled conditions, with little or no changes in carbohydrate contents, including soluble sugars (e.g. sucrose), starch and cell wall polysaccharides. Those plants, however, had a significantly decreased fitness under field conditions, i.e. the plants most deficient in UGPase activity produced up to 50% less seeds than in wt. Therefore, we concluded that UGPase is not a rate-limiting enzyme in carbohydrate synthesis pathways, but still is essential in viability of Arabidopsis plants. In order to characterize two Arabidopsis UGPase isozymes, both proteins were heterologously overexpressed in prokaryotic cells and purified by affinity chromatography. The two isozymes showed little differences in physical and biochemical properties, including substrate specificity, Km values with substrates in both directions of the reaction, molecular masses, isoelectric point (pI), and equilibrium constant. On the other hand, possibilities of distinct post-translational regulatory mechanisms were indicated, based on amino acid (aa) motif analyses, and on 3D analyses of derived crystal structures of the two proteins. We used the heterologous bacterial system also to overexpress barley UGPase and several of its mutants, both single mutants and those with whole domains/ exons deleted. As a result, we have identified several aa residues/ protein domains that may be essential for structural integrity and catalytic/ substrate-binding properties of the protein. For instance, we found that the last exon of UGPase (8 aa at the end of C-terminus) was important for the protein ability to oligomerize and that Lys-260 and the second-to-last exon were essential for pyrophosphate (but not UDP-glucose) binding. The data emphasized the critical role of central part of the active site (so called NB-loop) in catalysis, but also pointed out to the role of N-terminus in catalysis and oligomerization, but not substrate binding, and that of C-terminus in both catalysis/substrate binding and oligomerization.

The primary objectives of this research were to demonstrate that: 1.) antibiotic resistant bacteria can promote the survival of antibiotic sensitive organisms when grown simultaneously as biofilms in antibiotics, 2.) community-level multiple antibiotic resistance of polymicrobial consortia can lead to biofilm formation despite the presence of multiple antibiotics, and 3.) biofilms may benefit plasmid retention and heterologous protein production in the absence of selective pressure. Quantitative analyses of confocal data showed that ampicillin resistant organisms supported populations of ampicillin sensitive organisms in steady state ampicillin concentrations 13 times greater than that which would inhibit sensitive cells inoculated alone. The rate of reaction of the resistance mechanism influenced the degree of protection. Spectinomycin resistant organisms did not support their sensitive counterparts, although flow cytometry indicated that GFP production by the sensitive strain was improved. When both organisms were grown in both antibiotics, larger numbers of substratum-attached pairs at 2 hours resulted in greater biofilm formation at 48 hours. For biofilms grown in both antibiotics, a benefit to spectinomycin resistant organism’s population size was detectable, but the only benefit to ampicillin resistant organisms was in terms of GFP production. Additionally, an initial attachment ratio of 5 spectinomycin resistant organisms to 1 ampicillin resistant organism resulted in optimal biofilm formation at 48 hours. Biofilms also enhanced the stability of high-copy number plasmids and heterologous protein production. In the absence of antibiotic selective pressure, plasmid DNA was not detected after 48 hours in chemostats, where the faster growth rate of plasmid-free cells contributed to the washout of plasmid retaining cells. The plasmid copy number per cell in biofilms grown without antibiotic selective pressure steadily increased over a six day period. Flow cytometric monitoring of bacteria grown in biofilms indicated that 95 percent of the population was producing GFP at 48 hours. This research supports the idea that ecological interactions between bacteria contribute to biofilm development in the presence of antibiotics, and demonstrates that community-level multiple antibiotic resistance is a factor in biofilm recalcitrance against antibiotics. Additionally, biofilms may provide an additional tool for stabilizing high copy number plasmids used for heterologous protein production.

No description available.

Microbial chlorate respiration plays an important role in the turnover of oxochlorates in nature and industrial waste management. This thesis deals with the characterization of the molecular components of chlorate respiration in Ideonella dechloratans. Chlorate respiration utilizes two soluble periplasmic enzymes, chlorate reductase and chlorite dismutase, to convert chlorate to chloride and oxygen. The genes encoding the enzymes participating in the chlorate degradation have been sequenced, and are found in close proximity, forming a gene cluster for chlorate metabolism. This work also includes the successful recombinant expression of three genes from Ideonella dechloratans. Two of the gene products, chlorite dismutase and the C subunit of chlorate reductase, participate in the chlorate respiration. The third gene, which is found close to the gene cluster for chlorate metabolism, encodes a soluble c-type cytochrome. The localization of the gene suggests the corresponding protein as a candidate for a role as electron donor to chlorate reductase. Also, the role of soluble periplasmic c cytochromes of Ideonella dechloratans in chlorate respiration was studied. At least one of the soluble c cytochromes was found capable of serving as electron donor for chlorate reduction. This c cytochrome, and several others, can also donate electrons to a terminal oxidase for subsequent reduction of oxygen, as required for the branched electron flow during chlorate respiration.

Orientador: João Pessoa Araujo Junior / Resumo: A suinocultura vem alcançando grande desenvolvimento de técnicas eficientes associadas ao melhoramento genético, nutrição, manejo e sanidade. Entretanto, devido aos métodos intensivos de criação, os suínos se tornaram mais susceptíveis a um grande número de doenças infecciosas. Entre os mais importantes patógenos que afetam a indústria suinícola mundial está o circovírus suíno 2 (PCV2), um pequeno vírus icosaédrico, não-envelopado, de DNA circular, de fita simples (ssDNA), ambisenso, composta por 1767-1768 nucleotídeos. Este vírus é altamente resistente a variações ambientais e agentes desinfetantes, é endêmico no mundo todo e está associado a várias manifestações clínicas distintas, que acarretam importantes perdas econômicas aos produtores. Um dos fatores possivelmente implicados na patogenicidade do PCV2 é a proteína Cap, a unidade fundamental constituinte do capsídeo deste vírus. Estudos realizados pela equipe do Prof. Dr. João Pessoa Araújo Jr., do Instituto de Biotecnologia da Unesp em Botucatu/SP, comprovaram que vírus com mutações em suas proteínas Cap isolados a partir de cultivo celular aumentavam a morte celular em culturas celulares infectadas. Estes resultados evidenciaram a importância do capsídeo nos mecanismos de infecção e patogenicidade do PCV2, tornando interessante a realização de estudos estruturais com as proteínas Cap mutantes. A execução de estudos estruturais in silico mostrou a baixa frequência das mutações identificadas na proteína Cap dos vírus pro... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Swine breeding has achieved a high development based on genetic improvement, nutrition, management and sanity. However, due to the intensive breeding methods, swine have become more susceptible to a higher number of infectious diseases. Among the most important pathogens that affect the swine world industry is the porcine circovirus 2 (PCV2), a small, icosahedral, non-enveloped virus, ambisense single-stranded circular DNA, composed by 1,767-1,768 nucleotides. This virus is highly resistant to environmental variations and disinfecting agents, endemic worldwide and has been associated to several distinct clinical manifestations that entail important economic losses to the producers. One of the factors possibly implicated on the PCV2 pathogenicity is the Cap protein, the fundamental unity that constitutes this virus capsid. Studies performed by the group of Dr. João Pessoa Araújo Jr. rom the Instituto de Biotecnologia da Unesp em Botucatu/SP, confirmed that viruses with mutated Cap proteins from cell culture increased cell death in infected cultures. Such results highlight the importance of capsid in the infection mechanisms and pathogenicity of PCV2 and the importance of structural and comparative studies with Cap protein structures. In silico structural studies showed the low frequency of the mutations identified in the mutant Cap proteins and also indicated a clear difference between the physico-chemical properties of the new amino acid residues in comparison to those found ... (Complete abstract click electronic access below) / Mestre

Submitted by Pâmela Merlo Marson (pam.marson@aluno.ibb.unesp.br) on 2018-06-05T17:11:43Z No. of bitstreams: 1 Dissertação_Mestrado_Pamela-Merlo-Marson_2018_vf.pdf: 1757229 bytes, checksum: ed7b8faed4d956e7a1ba89178199507d (MD5) / Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-06-07T14:10:28Z (GMT) No. of bitstreams: 1 marson_pm_me_bot.pdf: 1757229 bytes, checksum: ed7b8faed4d956e7a1ba89178199507d (MD5) / Made available in DSpace on 2018-06-07T14:10:28Z (GMT). No. of bitstreams: 1 marson_pm_me_bot.pdf: 1757229 bytes, checksum: ed7b8faed4d956e7a1ba89178199507d (MD5) Previous issue date: 2018-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A suinocultura vem alcançando grande desenvolvimento de técnicas eficientes associadas ao melhoramento genético, nutrição, manejo e sanidade. Entretanto, devido aos métodos intensivos de criação, os suínos se tornaram mais susceptíveis a um grande número de doenças infecciosas. Entre os mais importantes patógenos que afetam a indústria suinícola mundial está o circovírus suíno 2 (PCV2), um pequeno vírus icosaédrico, não-envelopado, de DNA circular, de fita simples (ssDNA), ambisenso, composta por 1767-1768 nucleotídeos. Este vírus é altamente resistente a variações ambientais e agentes desinfetantes, é endêmico no mundo todo e está associado a várias manifestações clínicas distintas, que acarretam importantes perdas econômicas aos produtores. Um dos fatores possivelmente implicados na patogenicidade do PCV2 é a proteína Cap, a unidade fundamental constituinte do capsídeo deste vírus. Estudos realizados pela equipe do Prof. Dr. João Pessoa Araújo Jr., do Instituto de Biotecnologia da Unesp em Botucatu/SP, comprovaram que vírus com mutações em suas proteínas Cap isolados a partir de cultivo celular aumentavam a morte celular em culturas celulares infectadas. Estes resultados evidenciaram a importância do capsídeo nos mecanismos de infecção e patogenicidade do PCV2, tornando interessante a realização de estudos estruturais com as proteínas Cap mutantes. A execução de estudos estruturais in silico mostrou a baixa frequência das mutações identificadas na proteína Cap dos vírus provenientes do cultivo in vitro e também indicou uma clara diferença entre as propriedades físico-químicas dos novos resíduos de aminoácidos em relação àqueles substituídos. Estas alterações, associadas à localização dos resíduos na superfície viral e a menor flexibilidade das proteínas Cap dos vírus mutantes, indicaram a possibilidade de alterações estruturais/funcionais relevantes, incluindo a alteração da afinidade por receptores celulares e diminuição da efetividade de anticorpos produzidos contra vírus vacinais. Foram também realizados trabalhos experimentais para a produção heteróloga da proteína Cap de um vírus selvagem, os quais envolveram ensaios de subclonagem da sequência de interesse em um vetor de expressão, testes de transcrição e experimentos de expressão protéica. Os resultados destes procedimentos foram compatíveis com a produção da proteína Cap, porém novos estudos são necessários para confirmar a produção da molécula alvo e melhorar o rendimento dos ensaios de expressão. / Swine breeding has achieved a high development based on genetic improvement, nutrition, management and sanity. However, due to the intensive breeding methods, swine have become more susceptible to a higher number of infectious diseases. Among the most important pathogens that affect the swine world industry is the porcine circovirus 2 (PCV2), a small, icosahedral, non-enveloped virus, ambisense single-stranded circular DNA, composed by 1,767-1,768 nucleotides. This virus is highly resistant to environmental variations and disinfecting agents, endemic worldwide and has been associated to several distinct clinical manifestations that entail important economic losses to the producers. One of the factors possibly implicated on the PCV2 pathogenicity is the Cap protein, the fundamental unity that constitutes this virus capsid. Studies performed by the group of Dr. João Pessoa Araújo Jr. rom the Instituto de Biotecnologia da Unesp em Botucatu/SP, confirmed that viruses with mutated Cap proteins from cell culture increased cell death in infected cultures. Such results highlight the importance of capsid in the infection mechanisms and pathogenicity of PCV2 and the importance of structural and comparative studies with Cap protein structures. In silico structural studies showed the low frequency of the mutations identified in the mutant Cap proteins and also indicated a clear difference between the physico-chemical properties of the new amino acid residues in comparison to those found in the wild-type virus. These mutations, associated with the location of the mutated residues on the viral surface and the lower mutated Cap protein flexibility, could lead to relevant structural/functional changes, including alteration of affinity for cellular receptors and decreased effectiveness of antibodies produced against vaccine viruses. Experimental works aiming the heterologous production of a wild-type Cap protein were also carried out, which involved expression vector subcloning, transcription tests and protein expression experiments. The results of these procedures were compatible with the production of the Cap protein, but further studies are needed to confirm the production of the target molecule and improve the yield of the expression assays.

BEZERRA, Walderly Melgaço. Expressão de uma proteína YD de Chromobacterium violaceum em sistemas heterólogos: potencial no controle de pragas e fungos. 2008. 53 f. Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2008. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-19T11:44:51Z No. of bitstreams: 1 2008_dis_wmbezerra.pdf: 488580 bytes, checksum: 8867bb56552a329eb81d971c78c66730 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:26:50Z (GMT) No. of bitstreams: 1 2008_dis_wmbezerra.pdf: 488580 bytes, checksum: 8867bb56552a329eb81d971c78c66730 (MD5) / Made available in DSpace on 2016-08-02T20:26:50Z (GMT). No. of bitstreams: 1 2008_dis_wmbezerra.pdf: 488580 bytes, checksum: 8867bb56552a329eb81d971c78c66730 (MD5) Previous issue date: 2008 / Chromobacterium violaceum is a free-living betaproteobacterium, which is found in the water and soil environments of tropical and subtropical regions. C. subtsugae, another species of the same genus, is toxic towards insects belonging to several orders. Among the genes that may be involved in the insecticidal activity displayed by these bacteria are those that encode chitinases. In addition to these chitinases, a protein containing YD repeats with potential insecticidal activity, encoded by the gene cv2776, was also identified in the genome of C. violaceum ATCC 12472. Similar proteins have also been found in the genomes of Xenorhabdus bovienii and Photorhabdus luminescens. The YD motif comprises 20 amino acids, with the consensus sequence Gx3-9YxYDx2GR(L, I or V)x3-10G, where x represents any amino acid. The protein TccC, from P. luminescens, has nine YD repeats in its sequence, as well as the protein SepC, from Serratia entomophila. SepC, along with two other toxins, SepA and SepB, are able to cause the "amber" disease in Costelytra zealandica (Coleoptera: Scarabaeidae). In the present work, the DNA coding seuquence of the gene cv2776 of C. violaceum ATCC 12472 was cloned in two different heterologous systems. In Escherichia coli, the coding sequence was expressed under control of the promoter araBAD, induced by L-arabinose. The protein rCV2776 was detected by imunoblotting in the insoluble fraction of induced E. coli cells. The rCV2776 present in the total insoluble cell fraction caused the death of Callosobruchus maculatus larvae, reducing the number of emerged adults in 78% and also decreasing the average weight of the insects. The E. coli cell fraction containing the recombinant protein also affected the vegetative growth of the phytopatogenic fungi Macrophomina phaseolina, Rhizoctonia solani and Sclerotium rolfsii. When the fraction containing rCV2776 was incorporated at 1.% (w/v) in the medium, the inhibition percentages of the mycelium growth were 38.5% (R. solani), 60.7% (M. phaseolina) and 71.1% (S. rolfsii). In the yeast Pichia pastoris, the coding sequence was expressed under the control of the promoter PAOX, which is induced by methanol as the sole carbon source. When expressed in P. pastoris, the recombinant protein was apparently degraded by endogenous proteases and could not be detected by SDS-PAGE or Western Blotting, even when 8 mg of protein was loaded into the gel. In conclusion, the recombinant protein CV2776 present in the insoluble fraction of induced E. coli cells was effective in controlling the emergence of the cowpea weevil, as well as slowing the growth of M. phaseolina and S. rolfsii / Chromobacterium violaceum é uma betaproteobactéria de vida livre encontrada em regiões tropicais e subtropicais. C. subtsugae, bactéria do mesmo gênero, possui atividade tóxica contra insetos de diversos gêneros. Entre os genes que podem estar envolvidos no potencial inseticida dessa bactéria, destacam-se aqueles que codificam quitinases. Em adição, identificou-se também no genoma de C. violaceum ATCC 12472 uma proteína contendo repetições YD com potencial atividade inseticida, codificada pelo gene cv2776, similar àquelas produzidas por Xenorhabdus bovienii e Photorhabdus luminescens. O motivo YD compreende 20 aminoácidos, com sequência consenso Gx3-9YxYDx2GR(L, I ou V)x3-10G (x representa qualquer aminoácido). A proteína TccC, de P. luminescens, possui 9 repetições YD, assim como a proteína SepC, de Serratia entomophila. SepC, juntamente com outras duas toxinas, SepA e SepB, são suficientes para causar a doença “âmbar” em Costelytra zealandica (Coleoptera: Scarabaeidae). No presente trabalho, a região codificadora do gene cv2776 de C. violaceum ATCC 12472 foi clonada em dois diferentes sistemas de expressão heteróloga. Em Escherichia coli, o gene foi expresso sob controle do promotor araBAD, induzido por L-arabinose. A proteína rCV2776 foi detectada por ensaio de imunoblotting na fração insolúvel das células de E. coli induzidas. A rCV2776 presente na fração insolúvel das células dessa bactéria teve efeito letal sobre larvas de Callosobruchus maculatus, diminuindo em até 78% o número de insetos adultos emergidos, além de diminuir o peso médio desses insetos. Além disso, a fração celular de E. coli contendo rCV2776 mostrou-se capaz de inibir o crescimento vegetativo de três fungos fitopatogênicos, Macrophomina phaseolina, Rhizoctonia solani e Sclerotium rolfsii. Quando incorporada no meio de cultura em uma concentração de 1% (m/v), essa fração causou percentuais de inibição de crescimento do micélio de 38.5% (R. solani), 60.7% (M. phaseolina) e 71.1% (S. rolfsii), respectivamente. Na levedura Pichia pastoris, a região codificadora foi expressa sob controle do promotor PAOX, que é induzido na presença de metanol como única fonte de carbono. Quando expressa em P. pastoris, a proteína recombinante foi aparentemente degradada por proteases endógenas, e não pôde ser detectada em análises de SDS-PAGE ou Western Bloting, mesmo quando foram aplicados 8,0 mg do sobrenadante concentrado e liofilizado no gel de eletroforese. Em conclusão, a proteína rCV2776 presente na fração insolúvel das células de E. coli induzidas foi efetiva em controlar a emergência do caruncho do feijão-de-corda, bem como em retardar o crescimento de M. phaseolina e S. rolfsii