<p>Pyrosequencing is a DNA sequencing method based on thedetection of released pyrophosphate (PPi) during DNA synthesis.In a cascade of enzymatic reactions, visible light isgenerated, which is proportional to the number of nucleotidesincorporated into the DNA template. When dNTP(s) areincorporated into the DNA template, inorganic PPi is released.The released PPi is converted to ATP by ATP sulfurylase, whichprovides the energy to luciferase to oxidize luciferin andgenerate light. The excess of dNTP(s) and the ATP produced areremoved by the nucleotide degrading enzyme apyrase.</p><p>The commercially available enzymes, isolated from nativesources, show batch-tobatch variations in activity and quality,which decrease the efficiency of the Pyrosequencing reaction.Therefore, the aim of the research presented in this thesis wasto develop methods to recombinantly produce the enzymes used inthe Pyrosequencing method. Production of the nucleotidedegrading enzyme apyrase by Pichia pastoris expression system,both in small-scale and in an optimized large-scale bioreactor,is described. ATP sulfurylase, the second enzyme in thePyrosequencing reaction, was produced in<i>Escherichia coli</i>. The protein was purified and utilizedin the Pyrosequencing method. Problems associated with enzymecontamination (NDP kinase) and batch-to-batch variations wereeliminated by the use of the recombinant ATP sulfurylase.</p><p>As a first step towards sequencing on chip-format,SSB-(single-strand DNA binding protein)-luciferase and KlenowDNA polymerase-luciferase fusion proteins were generated inorder to immobilize the luciferase onto the DNA template.</p><p>The application field for the Pyrosequencing technology wasexpanded by introduction of a new method for clone checking anda new method for template preparation prior the Pyrosequencingreaction.</p><p><b>Keywords:</b>apyrase, Pyrosequencing technology, Z_basictag fusion, luciferase, ATP sulfurylase, dsDNAsequencing, clone checking, Klenow-luciferase, SSB-luciferase,<i>Pichia pastoris, Echerichia coli</i>.</p>

Pseudozyma (Candida) antarctica lipase B (PalB) was expressed in Escherichia coli facilitating protein engineering. The lack of glycosylation was evaluated for a deeper understanding of the difficulties in expressing PalB in E. coli. Different systems were tested: periplasmic expression in Rosetta (DE3), cytosolic expression in Rosetta-gami 2(DE3), Origami 2(DE3), and coexpression of groES and groEL. Periplasmic expression resulted 5.2 mg/L active PalB at 16 °C in shake flasks. This expression level was improved by using the EnBase technology, enabling fed-batch cultivation in 24-deep well scale. The feed rate was titrated with the addition of α-amylase, which slowly releases glucose as energy source. Different media were evaluated where the EnBase mineral salt medium resulted in 7.0 mg/L of active PalB. Protein secreted directly into the media was obtained using the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter for screening and production of PalB in P. pastoris. A protease sensitive fusion protein CBM-PalB (cellulose-binding module) was used as a model system. When optimised, the expression system resulted in 46 mg/L lipase in 72 hours in shake flask, 37 mg/L lipase in 28 hours in 96-deep-well plate format, and 2.9 g PalB per 10 L bioreactor cultivation. The E. coli expression system was used to express a small focused library of PalB variants, designed to prevent water from entering the active site through a hypothesised tunnel. Screening of the library was performed with a developed assay, allowing for simultaneous detection of both transacylation and hydrolytic activity. From the library a mutant S47L, in which the inner part of the tunnel was blocked, was found to catalyse transacylation of vinyl butyrate in 20 mM butanol 14 times faster than hydrolysis. Water tunnels, assisting water in reaching the active sites, were furthermore found by molecular modelling in many hydrolases. Molecular modelling showed a specific water tunnel in PalB. This was supported by experimental data, where the double mutant Q46A S47L catalysed transacylation faster than hydrolysis compared to the wild type PalB. / <p>QC 20100818</p>

Techn. Hochsch., Diss., 2000--Aachen.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-06-23T11:41:54Z No. of bitstreams: 1 texto completo.pdf: 388479 bytes, checksum: 3f6213e3987bc7cbece94deab1128175 (MD5) / Made available in DSpace on 2017-06-23T11:41:54Z (GMT). No. of bitstreams: 1 texto completo.pdf: 388479 bytes, checksum: 3f6213e3987bc7cbece94deab1128175 (MD5) Previous issue date: 2001-03-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A região codificadora do gene LAC4, que codifica a β-galactosidase de Kluyveromyces marxianus var. lactis, foi isolada do plasmídeo pLAC4-12, por meio da técnica de reação de polimerização em cadeia (PCR) com oligonucleotídeos iniciadores, que amplificam um fragmento de aproximadamente 3.000 pb. Com os objetivos de expressar e secretar a β- galactosidase, este fragmento foi clonado em um vetor de expressão e secreção pPIC9 do sistema de expressão de Pichia pastoris. A região codificadora LAC4 intacta e uma versão truncada foram fundidas em fase com a seqüência sinal de secreção do fator-α, sob controle do promotor AOX1. A hidrólise dos DNAs recombinantes obtidos com as enzimas de restrição apropriadas confirmou a clonagem da região codificadora LAC4 intacta nas orientações anti-senso e senso e da versão truncada no vetor pPIC9. Os clones foram linearizados e usados para transformar células eletrocompetentes de Pichia pastoris GS115 (His - e Mut + ). Colônias recombinantes foram isoladas e as inserções da região codificadora LAC4 intacta e truncada no genoma da levedura foram confirmadas por “PCR” com iniciadores específicos. Colônias recombinantes positivas foram cultivadas em glicerol e a expressão dos genes recombinantes foi induzida com metanol. Amostras foram coletadas periodicamente e o meio extracelular e a massa de células analisados quanto à atividade de β -galactosidase. Nenhum aumento na atividade de β - galactosidase foi observado perante o controle. Espera-se que a transformação de linhagens da levedura Mut - com os clones obtidos possa resultar na produção de uma proteína funcional. / The codins Kluyveromyces region marxianus of var. the lactis β-galactosidase was isolated LAC4 from gene, pLAC4-12, from by polimerase chain reaction (PCR) technique with primers that amplify a fragment of approximately 3.000 bp. Expression and secretion vector to expresse and secrete β-galactosidase, the intact and truncated LAC4 coding regions were fused in phase with the factor-α secretion signal sequence in the Pichia pastoris pPIC9. Appropriate restriction enzyme hydrolates confirmed the insertion of the intact LAC4 coding region in sense and antisense orientations, as well as its truncated version in to the pPIC9 vector. The clones were linearized and used to transform electrocompetent Pichia pastoris GS115 (His - e Mut + ) cells. Recombinant cells were isolated and the insertions of intact and truncated LAC4 coding regions in the yeast genome were confirmed by PCR with specific primers. Positive recombinant colonies were grown in glycerol and the expression of the heterologous genes was induced with methanol. Samples were periodically collected and the extracellular medium and cell mass were analyzed for β-galactosidase activity. No increase in β-galactosidase activity was observed over the control. The transformation of Mut - yeast cell lines with the clones is expectial to result in the production of a functional protein. / Dissertação importada do Alexandria

MEDEIROS, Suelen Carneiro de. Expressão heteróloga de uma glicosil hidrolase da Família 18 (cv2736) de Chromobacterium violaceum em Pichia pastoris com potencial antibacteriano. 2012. 122 f. Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza-CE, 2012. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-11T13:31:54Z No. of bitstreams: 1 2012_dis_scmedeiros.pdf: 2847279 bytes, checksum: c0e6c68e1b479acad3f4108642933a87 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:21:01Z (GMT) No. of bitstreams: 1 2012_dis_scmedeiros.pdf: 2847279 bytes, checksum: c0e6c68e1b479acad3f4108642933a87 (MD5) / Made available in DSpace on 2016-08-02T20:21:01Z (GMT). No. of bitstreams: 1 2012_dis_scmedeiros.pdf: 2847279 bytes, checksum: c0e6c68e1b479acad3f4108642933a87 (MD5) Previous issue date: 2012 / Chitinases are enzymes capable of hydrolyzing chitin, a polysaccharide composed of units of N-acetyl-D- glucosamine, which is abundant in nature. The genome sequence of C. violaceum ATCC 12472 has revealed some genes encoding proteins with potential applications in agriculture and medicine, such as those encoding chitinases. This study aimed to express the protein CV2736 in Pichia pastoris KM71H and to evaluate its antimicrobial potential against microorganism of medical importance. To this purpose, the full ORF (encoding rCV2736 PS+) and a partial sequence of it, encoding a truncated protein without its putative signal peptide (rCV2736PS‒), were cloned into the vector pPICZ α A for expression in P. pastoris. The protein rCV2736 PS+ was detected in the cell-free culture medium of P. pastoris cells harboring the corre sponding expression cassete. The recombinant CV2736PS+ was produced in a heterogeneous manner, and when analyzed by SDS-PAGE, three protein bands with apparent molecular masses of 41. 3, 38.1 and 36. 2 kDa were detected. The protein band with the highest molecular mass (41.3 kDa) was stained with the Periodic acid - Schiff’s reagent, thus showing that this band corresponds to N-glycosylated rCV2736PS+. Furthermore, tryptic peptides identified by mass spectrometry (ESI-MS) confirmed the identity of the expressed protein. The recombinant protein produced without the first 22 amino acids(rCV2736PS‒) showed higher chitinase activity than that found for th e fraction containing rCV2736PS+, despite not being detected by SDS-PAGE. The recombinant protein CV2736PS+, present in the fraction F0/95 from the culture medium of induced cells, was active after heat treatment at 50 °C and its maximum chitinolytic activity was recorded at pH 3.0. This fraction was also able to degrade the synthetic substrates 4-nitrophenyl N,N'-diacetyl -β-D-chitobioside and 4-nitrophenyl N,N',N''-triacet yl chitotrioside, which characterizes an endochitinase activity. Abinitio molecular modeling demonstrated that the polypeptide of CV2736 likely folds as a(β/α)8 barrel (also known as TIM barrel), which is typical of the GH18 family proteins. The fraction F0/95 showed antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and growth inhibition against Salmonella cholera-suis. The same fraction were evaluated against the yeast Candida albicans, but was not detectable inhibition of this microorganism in the assay. These results suggest that rCV2736 should be further studied as a new anti-bacterial agent. / Quitinases são enzimas capazes de hidrolisar quitina, um polissacarídeo formado por unidades de N-acetil-β-D-glucosamina, que é extremamente abundante na natureza. Após o sequenciamento do genoma de C. violaceum ATCC 12472, alguns genes referentes a proteínas com potencial biotecnológico foram encontrados, dentre eles os que codificam para quitinases. Este trabalho teve como objetivos expressar a proteína CV2736 de C. violaceum ATCC 12472 de forma recombinante em células de Pichia pastoris KM71H e avaliar seu potencial antimicrobiano frente a microrganismos de importância médica. Para isto, a sequencia completa da ORF (CV2736PS+), bem como uma sequencia parcial, codificando uma proteína truncada, sem um possível peptídeo sinal N-terminal (CV2736PS‒), foram clonadas no vetor de expressão pPICZαA, para expressão em P. pastoris KM71H. A proteína completa (rCV2736PS+) foi detectada em uma fração proteica, contendo as proteínas secretadas para o meio de cultura, por células de P. pastoris transformadas com o respectivo cassete de expressão recombinante. A rCV2736PS+ apresentou-se de forma heterogênea, quando analisada por SDS-PAGE, exibindo três bandas com massas moleculares aparentes de 41,3, 38,1 e 36,2 kDa, respectivamente, sendo uma delas (41,3 kDa) maior do que se poderia deduzir a partir da sequencia de aminoácidos. Após coloração com reagente de Schiff para revelação de glicoproteínas, constatou-se que essa banda (41,3 kDa) correspondia a rCV2736PS+ na sua forma N-glicosilada. Além disso, rCV2736PS+ teve seus peptídeos identificados por espectrometria de massas, na fração F0/95 do meio de cultura livre de células. A proteína recombinante produzida sem os 22 aminoácidos iniciais (rCV2736PS‒) apresentou atividade quitinásica maior que a encontrada para a fração contendo rCV2736PS+, apesar de não ter sido detectada por SDS-PAGE. A fração F0/95 do meio de cultura contendo rCV2736PS+ mostrou-se termoestável até 50°C e com pico de atividade quitinolítica em pH 3,0. A mesma fração apresentou maior atividade endoquitinásica e ativa contra os substratos sintéticos 4-nitrofenil N,N’–diacetil-β-D-quitobiosídeo e 4-nitrofenil N,N’,N’’–triacetilquitotriose. A modelagem molecular evidenciou o dobramento na forma de barril (β/α)8 (barril TIM), típico das proteínas da família GH18. Do mesmo modo, a fração F0/95 foi testada contra seis espécies de bactérias, apresentando atividade microbicida contra Pseudomona aeruginosa, Escherichia coli e Staphylococcus aureus, e foi capaz de inibir o crescimento de Salmonella choleraesuis. A mesma fração foi testada contra a levedura Candida albicans, mas não foi detectada inibição do crescimento contra este microrganismo no ensaio. Portanto, a proteína recombinante rCV2736 pode, assim, ser considerada uma molécula com potencial antibacteriano. Palavras-chave: Quitinase; Pichia pastoris; antimicrobiano; Expressão heteróloga.

TEIXEIRA, C. S. Expressão de uma quitinase de Chromobacterium Violaceum em Pichia Pastoris: purificação e caracterização parcial da proteína recombinante. 2011. 110 f. Dissertação (Mestrado em Bioquímica) - Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2011. / Submitted by Francisco Lacerda (lacerda@ufc.br) on 2014-11-10T18:58:20Z No. of bitstreams: 1 2011_dis_csteixeira.pdf: 2266830 bytes, checksum: cf710a23bf6fe474186662c855e584ee (MD5) / Approved for entry into archive by José Jairo Viana de Sousa(jairo@ufc.br) on 2015-01-20T16:54:04Z (GMT) No. of bitstreams: 1 2011_dis_csteixeira.pdf: 2266830 bytes, checksum: cf710a23bf6fe474186662c855e584ee (MD5) / Made available in DSpace on 2015-01-20T16:54:04Z (GMT). No. of bitstreams: 1 2011_dis_csteixeira.pdf: 2266830 bytes, checksum: cf710a23bf6fe474186662c855e584ee (MD5) Previous issue date: 2011 / Microorganisms are a valuable tool for the expression of proteins from a variety of sources, including plants, animals and other microorganisms. Thus, chitinases, a group of glycosil hydrolases capable to hydrolize chitin, have already been expressed and purified from different systems including bacteria and yeast. Chitin, a linear polymer of N-acetyl-β-D-glucosamine (GlcNAc), is an important structural component found in the crustacean shells, in the peritrophic membrane of insect guts as well as in the fungi cell walls. The aim of this work was to express a chitinase (encoded by the ORF CV3316) from Chromobacterium violaceum ATCC 12472, using the methylotrophic yeast Pichia pastoris strains GS115 and KM71H. Furthermore, purification and partial characterization of the recombinant protein were also achieved. The GS115 strain carrying the expression cassette pPICZαA-CV3316 was selected due to its higher expression level as compared to KM71H strain. Immobilized metal ion affinity chromatography was employed to purify the recombinant chitinase which was eluted as a single peak at 0.04 M imidazol. The homogeneity of the purified protein was confirmed as judged by polyacrylamide gel electrophoresis (SDS-PAGE). In these conditions, the recombinant chitinase migrated as a single protein band with an apparent molecular mass of about 87 kDa. Thus, a chitinase from C. violaceum ATCC 12472 was successfully expressed in P. pastoris and the soluble recombinant protein purified. The content of secondary structure was investigated by circular dichroism (CD) spectroscopy. At 24 oC the CD spectrum revealed secondary structure contents of 37% (alpha helix), 26% (beta sheet) and 38% (random coil). The CD spectra obtained in the temperature range 10-50 oC were characteristic of beta sheet. In contrast, the CD spectra generated in the range 60-90 oC were characteristic of alpha helix. The midpoint temperature of this conformational transition was 59.6 1.2 oC as calculated from the CD experimental data. Fluorescence spectroscopy was carried out with excitation at 280 and 290 nm, producing emission spectra in which the wavelengths of maximum emission were 339 and 342 nm, respectively. This behavior is characteristic of tryptophan residues in limited contact with water. Chitinolytic activity against several substrates and the pH dependency of the enzymatic activity of the pure protein were all accessed. The purified enzyme showed hydrolytic activity on the following substrates: colloidal chitin (1,189.4 U.mgP-1), 4-nitrophenyl N-N’-diacetyl--D-chitobioside (30,411.0 U.mgP-1) and 4-nitrophenyl β-D-N-N’-N”-triacetylchitotriose (13,150.0 U.mgP-1); and, respectively. In contrast, no activity was detected using 4-nitrophenyl N-acetyl-β-D-glucosaminide as substrate. The enzyme presented an optimal chitinolytic activity at pH 5.0 using colloidal chitin as a substrate. Additionally, the antifungal activity against the phytopathogenic fungi, Fusarium solani, Fusarium oxysporum, Rhizoctonia solani and Penicillium herquei was investigated. The recombinant chitinase did not inhibit the spore germination and the mycelium growth of the tested fungi, at the 0.63 mgP.ml-1 concentration. Further studies should be carried out in order to discover potential applications of this protein as a biotechnological tool in the control of other phytopathogenic fungi as well as economically important pests. / A utilização de microrganismos como sistemas heterólogos de expressão de proteínas tem se mostrado uma estratégia alternativa e/ou complementar aos passos tradicionais utilizados no processo de purificação de proteínas. Diante deste contexto, quitinases (EC 3.2.1.14), enzimas hidrolíticas capazes de degradar quitina, têm sido expressas em diferentes sistemas heterólogos, incluindo bactérias e leveduras. Quitina é um polímero linear composto de resíduos de N-acetil-β-D-glucosamina (GlcNAc), sendo um importante constituinte estrutural da carapaça de crustáceos e membrana peritrófica de insetos e, ainda, da parede celular de fungos. Este trabalho teve por objetivo expressar uma quitinase, codificada pela ORF cv3316, de Chromobacterium violaceum ATCC 12472 na levedura metilotrófica Pichia pastoris, estirpes GS115 e KM71H, além de purificar e caracterizar a proteína recombinante (rCHI3316). A estirpe GS115, portando a construção (pPICZαA-CV3316), foi selecionada para os experimentos posteriores, pois apresentou um maior nível de expressão quando comparada à cepa KM71H. Cromatografia de afinidade em coluna de níquel imobilizado foi utilizada para purificar a quitinase recombinante (rCHI3316), que foi eluída como um único pico com imidazol 0,04 M. A proteína purificada se mostrou homogênea quando submetida à eletroforese em gel de poliacrilamida 15% em presença de SDS e -mercaptoetanol (SDS-PAGE). Nessas condições, uma única banda com massa molecular aparente de aproximadamente 87 kDa foi observada. A quitinase rCHI3316 de C. violaceum foi expressa de forma solúvel utilizando o sistema de expressão P. pastoris e, além disso, sua purificação foi realizada de forma satisfatória. O conteúdo de estrutura secundária foi estimado por espectroscopia de dicroísmo circular (CD), com a proteína submetida a diferentes temperaturas (10-90 °C). Na temperatura de 24 °C, o espectro de CD revelou a predominância do conteúdo de hélice alfa (38%), folha beta (26 %) e estrutura randômica (37%). Entre as temperaturas de 10-50 °C, rCHI3316 exibiu um espectro característico de folha beta. A partir de 60 °C até 90 °C, rCHI3316 adquiriu uma conformação característica de hélice alfa. A temperatura média para essa transição conformacional foi calculada como sendo 59,6  1,21 °C. Experimentos de espectroscopia de fluorescência, com excitação a 280 e 290 nm, produziram espectros de emissão com comprimentos de onda máximos iguais a 339 e 342 nm, respectivamente. Esses valores são característicos de resíduos de triptofano parcialmente expostos ao solvente. Atividade quitinolítica contra vários substratos e dependência de pH da atividade enzimática da proteína pura foram avaliados. A rCHI3316 exibiu atividade hidrolítica sobre os substratos quitina coloidal (1.189,40 U/mgP), 4-nitrofenil N-N’-diacetil-β-D-quitobiosídeo (30.411,0 U/mgP) e 4-nitrofenil N-N’-N’’-triacetilquitotriose (13.150,0 U/mgP); entretanto, nenhuma atividade enzimática da rCHI3316 foi detectada frente a 4-nitofenil N-acetil-β-D-glucosaminídeo. A enzima exibiu atividade quitinolítica ótima (100%) em pH 5,0; quando quitina coloidal foi utilizada como substrato. Em adição, a atividade antifúngica contra fungos fitopatogênicos, Fusarium solani, Fusarium oxysporum, Rhizoctonia solani e Penicillium herquei foi investigada. rCHI3316 não inibiu a germinação e o crescimento micelial dos esporos dos fungos testados, na concentração de 0,63 mgP.ml-1. Estudos subseqüentes deverão ser realizados na intenção de descobrir potenciais aplicações desta proteína como uma ferramenta biológica no controle de outros fungos fitopatogênicos bem como insetos considerados pragas.

Resumo: O gene GPD2 de Saccharomyces cerevisiae, que codifica a enzima glicerol-3- fosfato desidrogenase (G3PDH; EC 1.1.1.8; NAD+: oxidoredutase) foi clonado na levedura Pichia pastoris para expressar extracelularmente a enzima em meio de cultura. Essa enzima apresenta aplicação prática em diversos sistemas acoplados para determinação quantitativa de triacilglicerol, glicerol, ácido fosfatídico e outros fosfolipidios também podendo ser usada para medir atividades enzimáticas em diversos tipos de amostras. Para que a atividade extracelular fosse suficiente em ensaios industriais e biológicos, um estudo de indução da expressão da enzima foi realizado no presente trabalho, que consistiu em escolher o clone que melhor secreta a enzima e estudar o meio de crescimento (BMGY), a densidade inicial celular (0,05 mg/mL), o meio de indução enzimática (BMMY), a natureza do tampão (tampão fosfato), o pH (6,0), o tempo de produção da proteína (4 dias), a concentração da enzima através de membrana filtrante (120 vezes), a melhor fonte de peptona (Acumédia), o estudo de pré-indução celular por estresse osmótico (atividade de 0,477 ± 0,0 U/mL em 24 horas com NaCl 0,35M). O processo de produção da G3PDH mostrou que a máxima produtividade enzimática (795 U/mL e atividade específica de 44,49 U/mg) e biomassa final de 17,75 mg/mL foi obtida com as seguintes condições experimentais: 48 horas de indução com meio BMMY, utilizando 1% de metanol, 1% de glicerol, densidade inicial celular de 0,05 mg/mL, pH 5,0 e sobrenadante concentrado 120 vezes em membrana filtrante. / Abstract: The GPD2 gene from Saccharomyces cerevisiae, which encodes the enzyme glycerol-3-phosphate dehydrogenase (G3PDH, EC 1.1.1.8, NAD +: oxidoredutase) was cloned in the yeast Pichia pastoris to express the enzyme extracellularly in the culture medium. The enzyme G3PDH has practical application in various systems coupled to quantitative determination of triacylglycerol, glycerol, phosphatidic acid and other phospholipids. It can also be used to measure the enzymatic activities in diverse types of samples. For the application of the enzyme extracellular in industrial and biological tests, a study of induction of expression of the enzyme was accomplished in the present work, that consisted of to choose of clone that more expressing the enzyme, the growth medium (BMGY), the cellular initial density (0.05 mg/mL), the medium of enzymatic induction (BMMY), the buffer nature (phosphate potassium), pH (6.0), the time of production of the protein (4 days), the concentration of the protein (120-fold), the peptone source (Acumédia), the study of pre-induction cellular for osmotic stress (activity of 0.477 ± 0.0 U/mL in 24 hours with NaCl 0.35M). The study of the variable determinative in the process of production of the G3PDH it showed that the maximum enzymatic productivity (0.795 U/mL and 44.49 U/mg of specific activity) and final biomass of 17.75 mg/mL was obtained with the following experimental conditions: 48 hours of induction with medium BMMY, using 1% methanol, 1% glycerol, cellular initial density of 0.05mg/mL, pH 5.0 and the supernatant concentrated 120-fold in filter menbrane. / Orientador: Edwil Aparecida de Lucca Gattás / Coorientador: Maristela de Freitas Sanches Peres / Banca: Edwil Aparecida de Lucca Gattás / Banca: José Roberto Ernandes / Banca: Luiz Henrique Souza Guimarães / Mestre

Orientador: Eleni Gomes / Banca: Adalberto Pessoa Junior / Banca: Mario Tyago Murakami / Banca: Gustavo O. Bonilla Rodriguez / Banca: Aliandra Maura Gibertoni Malaman / Resumo: Os microrganismos termofilicos são de grande interesse biotecnológico, não apenas por tolerar altas temperaturas, mas também por secretarem proteínas de maior estabilidade, com características bioquímicas e estruturais importantes de múltiplas aplicações na indústria. A linhagem do fungo termofílico Myceliophthora heterothallica F.2.1.4, isolada de camas de frango, apresentou alta atividade da enzima β-1,4-endoglucanase (31 U/ml). O gene codificante da β-1,4-endoglucanase de F.2.1.4 foi identificado e expresso em leveduras Pichia pastoris e em bactéria Escherichia coli. A caracterização do gene permitiu identificar esta enzima como membro da família glicosídeo hidrolase 5 (GH5), nomeada neste estudo como Mh_GH5. A enzima apresenta na região N-terminal um módulo de ligação ao substrato (CBM) pertencente a família 1. As enzimas expressas em P. pastoris e em E. coli foram purificadas e submetidas à caracterização bioquímica. A caracterização bioquímica demonstrou que as enzimas recombinantes apresentaram ótimos de temperatura de 55°C (P. pastoris) e 60°C (E. coli) e pH ótimo de 4,0 para ambos os sistemas heterólogos. A análise da estabilidade das enzimas demonstrou que as enzimas expressas por Pichia pastoris apresentaram-se estáveis em temperaturas mais elevadas (70°C), indicando que a glicosilação teria papel essencial na termoestabilidade de enzimas recombinantes. A determinação estrutural em alta resolução foi realizada para o domínio catalítico desta nova β-1,4-endoglucanase GH5 de M. heterothallica expressa em E. coli. A estrutura foi determinada à 1.1 Å e apresenta uma arquitetura frequentemente encontrada dentre os membros da família de GH5, (β/α)8 TIMbarrel. Os dados obtidos neste estudo podem contribuir para a determinação de características envolvidas na termoestabilidade de enzimas provenientes de... / Abstract: Thermophilic microorganisms have gain attention in the last years because of the biotechnological interest. Essentially these organisms secrete thermostable proteins displaying suitable features, such as biochemical and structural, for industrial platforms. The thermophilic fungus Myceliophthora heterothallica F.2.1.4, isolated from poultry litter, showed high activity for β-1,4- endoglucanase (31 U/ml). A new sequence enconding for glycoside hydrolase was identified and expressed in Pichia pastoris and Escherichia coli. Sequence analysis allowed to classify this new β-1,4-endoglucanase as a member of the glycoside hydrolase 5 (GH5). The enzyme, named Mh_GH5, has N-terminal family 1 carbohydrate binding module (CBM1). The recombinant enzymes expressed in Pichia pastoris and Escherichia coli were purified and subjected to biochemical characterization. Recombinant endoglucanases showed optimal temperature of 55°C (P. pastoris) and 60°C (E. coli) and optimal pH of 4,0 for both heterologous systems. The thermostability of these new enzymes were evaluated and demonstrated that post-translational modifications, such as glycosylation, found on Pichia pastoris heterologous enzymes, contributed to higher thermostability. Structural analysis of the catalytic domain of the new GH5 from M. heterothallica expressed in E. coli was solved at 1.1 Å resolution and shows a common (β/α)8 TIM-barrel fold found in members of GH5 family. Based on the biochemical and structural information we have obtained for Mh_GH5 it is possible to suggest potential features involved in the thermostability of proteins from thermophilic microorganisms and might contribute for the development of new efficient biocatalysts required for biomass conversion to bioethanol and/or ... / Doutor

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-26Bitstream added on 2014-06-13T20:16:38Z : No. of bitstreams: 1 goncalves_mcm_me_jabo.pdf: 755871 bytes, checksum: 00a7ca290b99eaeb7742fdd22cdc3c6c (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A glicoproteína S1 do vírus da broquite infecciosa (VBI) além de ser altamente variável é responsável pela adsorção com os receptores das células hospedeiras e pela indução de anticorpos vírus-neutralizantes e inibidores da hemaglutinação em galinhas. A disponibilidade desse tipo de proteína pode contribuir com o imunodiagnóstico e com a imunoprofilaxia da bronquite infecciosa aviária. O presente estudo foi conduzido com a finalidade de fazer a clonagem e a expressão do gene da glicoproteína S1 derivada da estirpe M41 do VBI no sistema constituído pelo vetor pFLDa – Picchia pastoris. O cDNA completo do gene S1 foi preparado a partir do RNA extraído do líquido alantóide infectado com a estirpe M41 do VBI e o amplicon obtido pela PCR foi clonado no vetor de expressão pFLDa. O plasmídeo foi linearizado e usado na transformação da linhagem GS115 de Picchia pastoris por eletroporação. Foi avaliada a influência da composição do meio de cultura sobre a produção de biomassa e a expressão da proteína recombinante S1 durante a fase de indução. A produção da proteína S1 recombinante foi detectada somente no compartimento intracelular, embora o vetor pFLDa seja capaz de direcionar a síntese de uma proteína heteróloga para a via secretora. A proteína recombinante foi caracterizada imunoquimicamente como uma banda de aproximadamente 90 kDa e com reatividade específica para os anticorpos monoclonais anti-polihistidina. Concluindo, o sistema hospedeiro de células da levedura P. pastoris com o vetor pFLDa usado nesse estudo comprovou ser um método eficaz para a produção no compartimento intracelular da proteína recombinante S1 da estirpe M41 do VBI, com potencial para futura utilização em técnicas de imuno-diagnóstico da bronquite infecciosa aviária. / The glycoprotein (S1) of infectious bronchitis virus (IBV) has been shown to be highly variable and responsible for attachment to the receptor in host cellular membrane, induction of neutralizing and haemagglutination-inhibiting antibodies in chickens. Thus, the availability of such protein can contribute to the immuno-diagnosis and immunoprofilaxis of avian infectious bronchitis. The present study was carried out aiming to clone and to express the gene of S1 glycoprotein from M41 strain fo IBV in the system constituted by pFLDa vector – Picchia pastoris. Full length cDNA of IBV S1 glycoprotein was prepared from RNA extracted from infected allantoic fluid and the amplicon generated by PCR was cloned into yeast expression vector pFLDa to construct the plasmid of pFLDa - S1 - IBV. This plasmid was linearized and then transformed in Picchia pastoris GS115 by electroporation. The recombinant yeast clones were cultivated and selected. The influence of media composition on biomass and in the production of recombinant S1 during induction phase was studied. The production of recombinant S1 protein was detected only in the intra-cellular compartment, though the pFLDa vector would direct the synthesis for the secretor pathway. The recombinant protein was characterized by SDS-PAGE and Western-Blotting as a band of a molecular weight of approximately 90 kDa with specific reactivity against anti-Histidina monoclonal antibodies. The use of complex media promotes an increase in biomass, but no soluble S1 recombinant protein was produced. Concluding, the yeast system composed by pFLDa - P. pastoris was efficient to express intra-cellularly the recombinant S1 protein of M41 strain of IBV which has a potential to be used in the immuno-diagnosis of avian infectious bronchitis.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-06-10T19:08:01Z No. of bitstreams: 1 ArianeFerreiraLacerda_TESE.pdf: 2669795 bytes, checksum: 86dd6a13d7f026cc0544d3cb0e76e5f1 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-06-13T20:26:33Z (GMT) No. of bitstreams: 1 ArianeFerreiraLacerda_TESE.pdf: 2669795 bytes, checksum: 86dd6a13d7f026cc0544d3cb0e76e5f1 (MD5) / Made available in DSpace on 2016-06-13T20:26:34Z (GMT). No. of bitstreams: 1 ArianeFerreiraLacerda_TESE.pdf: 2669795 bytes, checksum: 86dd6a13d7f026cc0544d3cb0e76e5f1 (MD5) Previous issue date: 2015-06-30 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A inefic?cia de pesticidas qu?micos no controle de fungos fitopatog?nicos na agricultura e a frequente incid?ncia de doen?as humanas causadas por bact?rias resistentes a antibi?ticos levam ? busca por compostos antimicrobianos alternativos. Neste contexto, defensinas vegetais constituem uma promissora ferramenta no controle de agentes patog?nicos tanto de plantas quanto de humanos. Defensinas de plantas s?o pept?deos cati?nicos com aproximadamente 50 res?duos de amino?cidos, ricas em ciste?nas e cuja estrutura tridimensional ? bastante conservada entre as diferentes esp?cies vegetais. Estas mol?culas de a??o antimicrobiana representam um importante componente inato da resposta de defesa vegetal contra pat?genos e s?o expressas em diversos tecidos da planta, como folhas, tub?rculos, flores, vagens e sementes. O presente trabalho teve por finalidade a avalia??o da atividade antimicrobiana de duas defensinas vegetais contra diferentes esp?cies de fungos fitopatog?nicos e bact?rias patog?nicas ao homem. A defensina Drr230a, cujo gene foi isolado de ervilha (Pisum sativum) e a defensina CD1, cujo gene foi identificado no transcriptoma de caf? (Coffea arabica) foram subclonadas em vetor de express?o de levedura e expressas em Pichia pastoris. O gene cd1 foi subclonado em duas formas recombinantes: CD1tC, contendo uma sequ?ncia codificante para seis histidinas (6xHis) na regi?o C-terminal do pept?deo e CD1tN, contendo sequ?ncia codificante para 6xHis na regi?o N-terminal. No caso da defensina Drr230a, a sequ?ncia codificante para 6xHis foi inserida apenas na regi?o N-terminal da prote?na. Ensaios de atividade antimicrobiana das prote?nas recombinantes purificadas rDrr230a e rCD1 contra Phakopsora pachyrhizi, agente causal da ferrugem asi?tica da soja, foram realizados para analisar a inibi??o da germina??o de esporos in vitro e a severidade da doen?a causada pelo fungo in planta. As duas defensinas recombinantes testadas foram capazes de inibir a germina??o de uredosporos de P. pachyrhizi, n?o havendo diferen?a entre a a??o antimicrobiana de CD1tC e CD1tN. Ademais, rDrr230a e rCD1 reduziram drasticamente a severidade da ferrugem asi?tica da soja, conforme demonstrado em ensaios in planta. Apesar de rCD1 n?o ter sido capaz de inibir a prolifera??o das bact?rias patog?nicas humanas Staplylococcus aureus e Klebsiella pneumoniae, rCD1 mostrou-se capaz de inibir o crescimento do fungo fitopatog?nico Fusarium tucumaniae, causador da s?ndrome da morte s?bita da soja. Os resultados obtidos mostram que tais defensinas vegetais s?o candidatas ?teis para serem utilizadas em programas de engenharia gen?tica de plantas para controlar doen?as f?ngicas impactantes na agricultura. / The inefficiency of chemical pesticides to control phytopathogenic fungi in agriculture and the frequent incidence of human diseases caused by bacteria which are resistant to antibiotics lead to the search for alternative antimicrobial compounds. In this context, plant defensins are a promising tool for the control of both plant and human pathogenic agents. Plant defensins are cationic peptides of about 50 amino acid residues, rich in cysteine and whose tridimensional structure is considerably conserved among different plant species. These antimicrobial molecules represent an important innate component from plant defense response against pathogens and are expressed in various plant tissues, such as leaves, tubers, flowers, pods and seeds. The present work aimed at the evaluation of the antimicrobial activity of two plant defensins against different phytopathogenic fungi and pathogenic bacteria to humans. The defensin Drr230a, whose gene was isolated from pea (Pisum sativum), and the defensin CD1,whose gene was identified within coffee (Coffea arabica) transcriptome, were subcloned in yeast expression vector and expressed in Pichia pastoris. The gene cd1 was subcloned as two different recombinant forms: CD1tC, containing a six-histidine sequence (6xHis) at the peptide C-terminal region and CD1tN, containing 6xHis coding sequence at the N-terminal region. In the case of the defensin Drr230a, the 6xHis coding sequence was inserted only at the N-terminal region. Assays of the antimicrobial activity of the purified recombinant proteins rDrr230a and rCD1 against Phakopsora pachyrhizi, causal agent of soybean Asian rust, were performed to analyze the in vitro spore germination inhibition and disease severity caused by the fungus in planta. Both recombinant defensins were able to inhibit P. pachyrhizi uredospore germination, with no difference between the antimicrobial action of either CD1tC or CD1tN. Moreover, rDrr230a and rCD1 drastically reduced severity of soybean Asian rust, as demonstrated by in planta assays. In spite of the fact that rCD1 was not able to inhibit proliferation of the human pathogenic bacteria Staplylococcus aureus and Klebsiella pneumoniae, rCD1 was able to inhibit growth of the phytopathogenic fungus Fusarium tucumaniae, that causes soybean sudden death syndrome. The obtained results show that these plant defensins are useful candidates to be used in plant genetic engineering programs to control agriculture impacting fungal diseases.