In the AcMNPV genome, ~28% of the genes are arranged divergently on opposite strands with an intergenic region of <1 kbp. In this configuration, a bidirectional promoter generally drives expression of both genes. However, no baculovirus bidirectional promoters have been characterized in any detail. We chose the AcMNPV chiA/v-cath intergenic region to serve as a model to characterize transcriptional regulation of bidirectional gene pairs during AcMNPV infection. We sequentially truncated putative upstream regulatory regions of chiA and v-cath to identify sequences essential for transcriptional initiation. Forty bp of the chiA gene 5’-flanking region was sufficient to support chiA transcription at half the level of the AcΔCC+CC repair virus. Interestingly, v-cath transcription from viruses containing only 40 bp of their upstream 5’-flanking region was found to be higher by 4-fold relative to the level of native expression. Linker-scanning mutagenesis that inserted 5 bp linkers spanning the chiA/v-cath intergenic region identified nucleotides critical for the transcriptional activation of both genes. From this, nucleotides -36 to -45, of the v-cath gene were found to negatively regulate v-cath mRNA expression. Quantitative RT-PCR studies revealed a 2-4 fold higher chiA mRNA expression relative to v-cath possibly explaining why translation of CHIA can be detected 6 hours earlier than V-CATH. This study identifies upstream regions of viral chiA and v-cath required for initiation of transcription and provides the first insight into baculovirus mechanisms for transcriptional regulation of interdependent gene pairs.

Red kidney bean seeds (Phaseolus vulgaris) contain a variety of bioactive proteins including lectins, enzyme inhibitors, hydrolytic enzymes and antifungal proteins. The aim of this research was to investigate activities of selected low pH and heat-stable bioactive proteins extracted from different parts of red kidney bean seed, seedling and pod as well as seed and root exudates. Crude red kidney bean seed extracts inhibited growth of Alternaria alternata as well as its protease activity, but not its amylase activity. A protein with inhibitory activity against growth of A. alternata was purified from extracts of the red kidney bean cotyledons and embryonic axis. This purified bean protein was devoid of chitinase and β-1, 3- glucanase activities. Also, it did not inhibit porcine pancreatic α-amylase, bovine trypsin, amylase and protease of A. alternata suggesting that the antifungal activity of the protein is not related to these activities. Proteinaceous extracts of red kidney bean cotyledons induced melanin and conidia formation in mycelium of A. alternata. A protein responsible for this conidiation inducing effect was shown for the first time to be a mannose-binding lectin which is also known as PvFRIL (Phaseolus vulgaris fetal liver tyrosine kinase 3-receptor interacting lectin). An unexpected finding was that extracts of the embryonic axis stimulated rather than inhibited porcine α-amylase activity. Phytohemagglutinin (PHA-L in particular), co-extracted with α-amylase inhibitor from red kidney bean seeds, was implicated as an α-amylase stimulator with the potential of greatly assisting digestion of starch. In cotyledonary extracts, amylase stimulatory activity was masked by amylase inhibitory activity that was inactivated when the extracts were boiled for 10 min. An in-gel non-denaturing electrophoretic method was used to show presence of porcine α-amylase isoinhibitors in extracts of the cotyledons and embryonic axis. All other seedling parts as well as seed and root exudates had amylase stimulatory activity. Another improved non-denaturing electrophoretic method with immobilized azoalbumin was developed for in-gel detection of isoinhibitors of bovine trypsin in seed parts. It eliminates the need for both time-consuming and labourious staining, destaining or renaturation steps used in other methods. Accumulation of most of the selected bioactive proteins during seed development in different seed parts appeared to start at 20 days after flower abscission. The activities of these proteins decreased to lower levels after 11 days of germination. Besides these observed developmental changes, under abiotic (UV-C irradiation) and biotic (seedlings co-cultured with A. alternata) stress, increased activity of some of the selected bioactive proteins were detected. In conclusion, this study has contributed to a better understanding of antifungal activity and the selected bioactive proteins in extracts of red kidney bean.

LANDIM, Patrícia Gadelha de Castro. Produção em Pichia pastoris de uma quitinase de feijão-de-corda com atividade antifúngica. 2011. 155 f. Tese (Doutorado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2011. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-07-20T19:42:36Z No. of bitstreams: 1 2011_tese_pgclandim.pdf: 3599090 bytes, checksum: 2cf77e164a49e44c17edc739f713b302 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-07-26T18:17:14Z (GMT) No. of bitstreams: 1 2011_tese_pgclandim.pdf: 3599090 bytes, checksum: 2cf77e164a49e44c17edc739f713b302 (MD5) / Made available in DSpace on 2016-07-26T18:17:14Z (GMT). No. of bitstreams: 1 2011_tese_pgclandim.pdf: 3599090 bytes, checksum: 2cf77e164a49e44c17edc739f713b302 (MD5) Previous issue date: 2011 / Chitinases are enzymes that hydrolyze the β-(1,4) glycosidic bonds present in biopolymers of N-acety-β-D-glucosamine, mainly chitin, a structural polysaccharide which is found in cell walls of several fungi. In plants, chitinases play a role as defense proteins against the attack of pests and pathogens. In this work, a class I chitinase from cowpea (Vigna unguiculata) was expressed in heterologous systems. The recombinant protein (rVuChi) was purified, and characterized biochemically and in relation to its effects on mycelial growth and germination of spores/conidia of filamentous fungi. The DNA coding sequence of the cowpea chitinase was amplified by PCR and the products cloned in the expression vectors pET32a(+) and pPICZαA, for heterologous expression in Escherichia coli and Pichia pastoris, respectively. In E. coli cells, the recombinant fusion protein occurred mainly as inclusion bodies. On the other hand, in six strains of P. pastoris, the recombinant cowpea chitinase was secreted in a soluble form into the culture medium. The highest chitinase activity was detected in the extracellular fraction of KM71H strain, 72 hours after induction. The recombinant VuChi was detected by SDS-PAGE and Invision His-Tag stain kit, which identified two protein bands with apparent molecular masses of 30 and 33 kDa. These two protein bands showed the same N-terminal sequence, and an absence of N-glycosylation. Most recombinant chitinase secreted into the culture medium was recovered in the fraction F0/40, precipitated with ammonium sulfate. The expressed protein was purified to homogeneity by affinity chromatography on chitin matrix (yield of 18.31 mg per liter of culture medium), or by hydrophobic interactions chromatography on a column of Phenyl Sepharose CL-4B (yield = 13.2 mg/L), followed by ultrafiltration in a membrane with exclusion limit of 50 kDa. The purified rVuChi was able to hydrolyze colloidal chitin (in solution) as well as glycol chitin (in SDS-PAGE), although it did not show enzymatic activity against synthetic substrates containing p-nitrophenol. The purified chitinase showed molecular masses of 32 and 33.1 kDa by size exclusion chromatography on columns of Superose 12 HR and Superdex 200, respectively. When submitted to 2D electrophoresis, rVuChi presented a set of six spots with pI values between 4.44 and 5.15. The chitinase was thermostable at temperatures up to 50 ° C and the enzyme activity was highest at pH 5. In general, the presence of metal ions caused a reduction of its enzymatic activity. The chelating agent EDTA (0.5%) stimulated the enzyme activity, whereas in the presence of the detergent SDS (0.5%) the rVuChi activity was completely inhibited. The recombinant chitinase showed 37% of alpha helix and 26% of beta sheet, as determined by circular dichroism spectroscopy. Denaturing of 50% of the rVuChi molecules occurs at 54.41 ° C. The fluorescence spectra showed that the protein produced in P. pastoris was in its fully folded conformation. The recombinant cowpea chitinase was able to completely inhibit the germination of spores of Penicillium herquei, after 48 hours, at a dose of 100 mg, and caused 68% inhibition at doses of 50, 25 and 12.5 mg. At a dose of 150 mg, there was 55% inhibition on conidial germination of Rhizoctonia solani and a slight effect on spore germination of Colletotrichum lindemuthianum and C. musae. There was no effect of rVuChi on spore germination of C. gloeosporioides, Fusarium solani and F. oxysporum. In addition, the recombinant protein delayed the mycelial growth of P. herquei in approximately 50% (at the dose of 100 mg) but had no effect on mycelial growth of the other fungi. Therefore, the cowpea class I chitinase is a protein with anti-fungal activity. / As quitinases são enzimas capazes de hidrolisar as ligações β-(1,4)-glicosídicas presentes em biopolímeros de N-acetil-β-D-glucosamina, principalmente quitina, um polissacarídeo estrutural presente na parede celular de diversos fungos. No presente trabalho, uma quitinase de classe I de feijão-de-corda (Vigna unguiculata) foi expressa em sistemas heterólogos e a proteína recombinante (rVuChi) foi caracterizada bioquimicamente bem como em relação ao seu efeito sobre o crescimento micelial e germinação de esporos/conídios de fungos filamentosos. A seqüência de DNA codificando a proteína foi amplificada por PCR e clonada nos vetores de expressão pET32a(+) e pPICZαA, para expressão heteróloga em Escherichia coli e Pichia pastoris, respectivamente. A expressão de rVuChi em células de E. coli ArticExpress DE3 se deu em corpos de inclusão. Em seis estirpes de P. pastoris a proteína recombinante foi secretada, de forma solúvel, para o meio de cultura. Na fração extracelular da estirpe KM71H foi observada a maior atividade quitinolítica, após 72 horas de indução. A detecção de rVuChi foi feita por SDS-PAGE e com o kit Invision His-Tag stain, onde foram identificadas duas bandas protéicas de massas moleculares aparentes de 30 e 33 kDa. Ambas as bandas apresentaram a mesma sequência N-terminal e a ausência de N-glicosilação foi verificada. A quitinase recombinante estava presente principalmente na fração F0/40 precipitada com sulfato de amônio e foi purificada a homogeneidade tanto por cromatografia de afinidade em matriz de quitina (com rendimento de 18,31 mg por litro de meio de cultura), quanto por cromatografia de interações hidrofóbicas em coluna de Phenyl Sepharose CL-4B (rendimento de 13,2 mg/L), seguidas de ultrafiltração em membrana com limite de exclusão de 50 kDa. A rVuChi apresentou atividades endo e exo-quitinolítica frente a quitina coloidal e hidrolisou glicol-quitina em gel de SDS-PAGE, embora não tenha apresentado atividade contra substratos sintéticos contendo p-nitrofenol. A quitinase purificada apresentou massa molecular de 32 e 33,1 kDa por cromatografia de exclusão molecular em colunas de Superose 12 HR e Superdex 200, respectivamente. Em gel bidimensional, rVuChi apresentou um conjunto de seis ‘spots’ com pI entre 4,44 e 5,15. A quitinase mostrou-se ainda termoestável em temperaturas até 50 °C e sua atividade enzimática máxima ocorreu em pH 5. Em geral, a presença de íons metálicos causou uma redução de sua atividade enzimática. O agente quelante EDTA (0,5%) estimulou a atividade enzimática enquanto que o detergente SDS (0,5%) a inibiu totalmente. A quitinase recombinante apresentou 37% de hélice alfa e 26% de folha beta, como determinado por espectroscopia de dicroísmo circular. A desnaturação de 50% das moléculas de rVuChi ocorreu a 54,41 °C. Os espectros de fluorescência revelaram que a proteína produzida em P. pastoris estava em sua conformação totalmente enovelada. A quitinase recombinante de feijão-de-corda foi capaz de inibir totalmente a germinação de esporos de Penicillium herquei até 48 horas, na dose de 100 μg e causou inibição de 68%, nas doses de 50, 25 e 12,5 μg. Na dose de 150 μg, houve uma inibição de 55% na germinação dos conídios de Rhizoctonia solani e um leve efeito sobre a germinação dos esporos de Colletotrichum lindemuthianum e C. musae. Nenhum efeito da rVuChi foi observado sobre a germinação de esporos dos fungos C. gloeosporioides, Fusarium solani e F. oxysporum. Além disso, a proteína recombinante retardou o crescimento micelial de P. herquei em aproximadamente 50% (100 μg), porém não apresentou efeito sobre o crescimento micelial dos demais fungos. Desta forma, a quitinase classe I de V. unguiculata é uma proteína com atividade antifúngica.

Submitted by THIAGO TREVISOLI AGOSTINI null (ttrevisoli@gmail.com) on 2017-08-03T21:35:36Z No. of bitstreams: 1 Dissertação Thiago Trevisoli Agostini.doc: 4358656 bytes, checksum: e54359117ba09655fd5253a011dacfcb (MD5) / Rejected by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo as orientações abaixo: O arquivo submetido não contém o certificado de aprovação. A versão submetida por você é considerada a versão final da dissertação/tese, portanto não poderá ocorrer qualquer alteração em seu conteúdo após a aprovação. A versão final da dissertação/tese deve ser submetida no formato PDF (Portable Document Format). O arquivo PDF não deve estar protegido e a dissertação/tese deve estar em um único arquivo, inclusive os apêndices e anexos, se houver. Por favor, corrija estas informações e realize uma nova submissão. Agradecemos a compreensão. on 2017-08-04T17:14:48Z (GMT) / Submitted by THIAGO TREVISOLI AGOSTINI null (ttrevisoli@gmail.com) on 2017-08-04T17:30:14Z No. of bitstreams: 1 Versão Definitiva.pdf: 738534 bytes, checksum: 55f3aca1bc91c3ffe18c2eb2056d21ea (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-08-07T19:30:43Z (GMT) No. of bitstreams: 1 agostini_tt_me_jabo.pdf: 738534 bytes, checksum: 55f3aca1bc91c3ffe18c2eb2056d21ea (MD5) / Made available in DSpace on 2017-08-07T19:30:43Z (GMT). No. of bitstreams: 1 agostini_tt_me_jabo.pdf: 738534 bytes, checksum: 55f3aca1bc91c3ffe18c2eb2056d21ea (MD5) Previous issue date: 2017-07-04 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A exigência da sociedade por alimentos produzidos com baixos riscos de contaminação aos consumidores e ao meio ambiente tem aumentado constantemente. Nesse cenário, a identificação de novas formas eficazes e seguras para o controle de insetos-praga se mostra como uma tarefa contínua e as quitinases aparecem como uma interessante ferramenta. As quitinases são enzimas com ação de hidrólise sobre quitina e podem interferir no desenvolvimento de pragas visto que esse polímero constitui estruturas como a cutícula e a membrana peritrófica dos insetos. No presente trabalho, foi realizada a produção de um extrato quitinolítico a partir do cultivo da actinobactéria Streptomyces sp. ENT-21 em meio contendo quitina e avaliaram-se os efeitos desse extrato sobre o desenvolvimento de lagartas de Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae), importante praga da cultura do milho. O extrato quitinolítico produzido resultou na interferência do desenvolvimento larval de S. frugiperda, no aumento da mortalidade e na redução do ganho de peso das lagartas em relação a lagartas mantidas como controle. Após fervura, o extrato produzido teve sua atividade quitinolítica inativada e não exerceu influência sobre a mortalidade larval de S. frugiperda em relação ao controle na concentração avaliada. Os resultados indicam que o extrato quitinolítico produzido pelo cultivo de Streptomyces sp. ENT-21 possui atividade inseticida sobre lagartas de S. frugiperda e que a atividade quitinolítica é um fator importante para a atividade inseticida do extrato. / Society's demand for food production at low contamination risks to consumers and to the environment is in constant increase. In this scenario, the identification of new effective and safe forms for the control of insect pests come into sight as a continuous task and chitinases are considered an interesting promisse. Chitinases are enzymes that hydrolyze chitin and can disrupt the development of insect pests since this polymer constitutes vital structures such as the cuticle and peritrophic membrane of insects. In the present work, we produced a chitinolytic extract using the actinobacterium Streptomyces sp. ENT-21 and evaluated the effects of this extract on larval development of an important pest of maize, Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) larvae. The chitinolytic extract interfered in the larval development of S. frugiperda affecting parameters such as larval mortality and larval weight gain. After boiling, no chitinolytic activity was detected in the extract and no influence of boiled extract was observed over S. frugiperda larval development at the evaluated concentration. The results suggest that the evaluated chitinolitic extract has insecticidal activity against S. frugiperda larvae and indicate the chitinolytic activity as an important component of the insecticidal activity of the extract. / CNPq: 132363/2015-1

Orientador: Rosemeire Cristina Linhari Rodrigues Pietro / Banca: Suraia Said / Banca: Luiz Henrique Souza Guimarães / Banca: Tais Maria Bauab / Banca: Edwil Aparecida de Lucca Gattás / Resumo: Enzimas são catalisadores fundamentais para os sistemas biológicos sendo amplamente estudadas e aplicadas em todo o mundo, especialmente na área da saúde. Neste trabalho foi avaliada a aplicação de enzimas quitinolíticas e queratinolíticas em ensaios in vitro contra Pedicullus humanus capitis e na degradação de calos humanos, respectivamente. Foram selecionados os micro-organismos com maior potencial para produção dessas enzimas e determinados os parâmetros de produção, as condições de separação, purificação e caracterização das enzimas. Os micro-organismos com maior potencial para a produção de quitinases e queratinases foram os fungos Metarhizium anisopliae CG374 e Aspergillus oryzae, respectivamente. As maiores produções de quitinases foram obtidas em biorreator a 28°C, pH 7,0, 1,5 vvm e 200 rpm após 120 h. Quando produzida em biorreator hiperbárico sob pressão de 5 bar, apresentou um aumento de 21,3% na produtividade em relação ao biorreator convencional. Para queratinases em biorreator, os parâmetros de produção foram 28°C, pH 6, 200 rpm, 1,5 vvm e 120 h. A produção das quitinases e queratinases em biorreator foi 83 e 154% maior do que em frascos Erlenmeyer, respectivamente. A quitinase foi concentrada 5,12 vezes na purificação apresentando massas molares aproximadas de 24, 28 e 80 kDa. Fração purificada contendo quitinase apresentou estabilidade a 40°C por 60 minutos. A queratinase foi concentrada 4,52 vezes após purificação com massas molares de 40 e 50 kDa apresentando estabilidade térmica a 40°C por 60 min. A avaliação das enzimas quitinolíticas frente a piolhos humanos apontou para uma eficiência 9,1% maior quando comparadas com produtos comerciais. A avaliação da ação de enzimas queratinolíticas na degradação de calos humanos demonstrou resultados significativos quando comparados com produtos disponíveis no mercado... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Enzymes are catalysts for fundamental biological systems is extensively studied and applied worldwide, especially in health. In this study were evaluated the application of enzymes chitinolytic and keratinolytic in tests in vitro against Pedicullus humanus capitis and degradation of human callus, respectively. Were selected microorganisms with the greatest potential for production of these enzymes and determined the production parameters, conditions of separation, purification and characterization of enzymes. The microorganisms with the higher potential for the production of chitinases and keratinases were Metarhizium anisopliae CG374 and Aspergillus oryzae, respectively. The highest yields of chitinases obtained in fermentations in a bioreactor were 28°C, pH 7.0, 1.5 vvm and 200 rpm after 120 h. When produced in the hyperbaric bioreactor at a pressure of 5 bar, an increase of 21.3% in yield over conventional production in a conventional bioreactor was observed. To keratinases in a bioreactor, the production parameters were 28°C, pH 6.0, 200 rpm, and 1.5 vvm after 120h. The production of chitinases and keratinases in bioreactor was 83 and 154% higher than in Erlenmeyer flasks, respectively. The chitinase was 5.12 times concentred on the purification presenting molar weight of approximately 24, 28 and 78 kDa. Fraction containing purified chitinase showed thermal stability of 40°C for 60 min. Keratinase was concentred 4.52 times after purification and presents molar weight of 40 and 50 kDa. The purified fraction was thermal stability at 40°C for 60 min. Evaluation of chitinolytic enzymes against human lice showed an efficiency of 9.1% higher for the enzymatic extract when compared with commercial products. The evaluation of the action of keratinolytic enzyme in the degradation of human callus showed significative results when compared to products on the market less time reaching maximum degradation / Doutor

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / O cajueiro (Anacardium occidentale L.) à uma planta nativa do Brasil com grande valor comercial. Isso contribui com a geraÃÃo de milhares de empregos diretos e indiretos, especialmente na RegiÃo Nordeste, em Ãpoca de estiagem. Programas de melhoramento genÃtico vem selecionando cultivares de cajueiro melhores adaptados ao ambiente semiÃrido a fim de colocÃ-lo em um mercado cada vez mais competitivo. Entre os tipos de proteÃnas de sementes vÃrias tÃm funÃÃo de reserva, estrutural ou metabÃlica. AlÃm disso, as plantas sÃo dispostas de uma variedade de mecanismos de defesa contra o ataque de patÃgenos. Uma das respostas de defesa mais estudada diz respeito à expressÃo de proteÃnas relacionadas com a patogÃnese nas quais se inclui o grupo das quitinases. A enzima quitinase (EC 3.2.1.14) hidrolisa o polÃmero de quitina para a N-acetil glucosamina por qualquer uma das endo ou exo clivagens da ligaÃÃo &#946; (1-4). As respostas moleculares nos perfis das plantas a nÃvel transcricional tÃm demonstrado serem cruciais para o estabelecimento de um conjunto de mecanismos de defesa contra patÃgenos invasores. Usando ferramentas de bioinformÃtica foram identificados, no transcriptoma de cajueiro CCP076, dez contigs apresentando alto grau de semelhanÃa com quitinases, endoquitinases, e quitinases-like das famÃlias GH18 e GH 19 de Ricinus communis, Cicer arietinum, Mangifera indica, Citrus sinensis, Euonymus europaeus, Vitis vinifera, Aegilops tauschii e Hevea brasiliensis. Os ensaios enzimÃticos das proteÃnas da castanha confirmaram a presenÃa quitinases em seu proteoma. Ainda revelaram uma atividade catalÃtica Ãtima em pH 5. Um perfil de proteÃnas com sitio de ligaÃÃo à quitina tambÃm foi encontrado. Dessa forma, à demonstrado um grande potencial biotecnolÃgico nas quitinases provenientes da castanha de cajueiro CCP76. / The cashew (Anacardium occidentale L.) is a plant native to Brazil with high market value. This contributes to the generation of thousands of direct and indirect jobs, especially in the Northeast, in the dry season. Breeding program has been selecting the best cashew cultivars adapted to semi-arid environment in order to put it in an increasingly competitive market. Among the types of seed proteins have several reservation function, structural or metabolic. Furthermore, plants are arranged in an array of defense mechanisms against pathogen attack. One of the most studied defense response with respect to the expression of pathogenesis related proteins which are included in the group of chitinases. The enzyme chitinase (EC 3.2.1.14) hydrolyse the polymer chitin to N-acetyl glucosamine by either endo or exo cleavage of &#946; connection (1-4). The molecular profiles of responses in plants transcriptional level have been shown to be crucial for the establishment of a set of defense mechanisms against invading pathogens. Using bioinformatics tools were identified in the transcriptome cashew CCP076 ten contigs having high degree of similarity with chitinases, endoquitinases and chitinase-like the GH18 family and GH 19 of Ricinus communis, Cicer arietinum, Mangifera indica, Citrus sinensis, Euonymus europaeus , Vitis vinifera, Aegilops tauschii and Hevea brasiliensis. The enzyme assays of chestnut chitinase protein confirmed the presence in their proteome. Also revealed a great catalytic activity at pH 5. A protein profile with chitin-binding site was also found. Of these, it is demonstrated great potential in biotechnological chitinase from CCP76 cashew nuts.

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-06-10T12:50:24Z No. of bitstreams: 1 Talita Camila Evaristo da Silva Nascimento.pdf: 924627 bytes, checksum: e31eef0acccfe262c282d8536f058023 (MD5) / Made available in DSpace on 2016-06-10T12:50:24Z (GMT). No. of bitstreams: 1 Talita Camila Evaristo da Silva Nascimento.pdf: 924627 bytes, checksum: e31eef0acccfe262c282d8536f058023 (MD5) Previous issue date: 2012-07-27 / Sustainability is a theme that has been raised steadily in recent years, the production and use of enzymes in an efficient alternative recycling of industrial and agricultural waste. The chitinase acts in the hydrolysis of chitin, the substance can be found in large quantities in the shells of crustaceans. The objective of this research was to select Streptomyces spp. with the greatest potential in the production of chitinase, to characterize the crude and partially purified. For selection we used 30 strains of Streptomyces spp. isolated from lichen in the Amazon region. Parameters such as pH and temperature optima, stability to pH and temperature from the crude enzymatic extract. Partial purification of the enzyme was performed by precipitation in ammonium sulfate in the range of 0-80% and the electrophoresis profile by SDS-PAGE. Streptomyces sp. DPUA1581 chitinase produced by submerged fermentation with 1% of chitin, agitation 150 rpm, at 28 °C for 96 hours. The enzyme characterization showed the best activity in sodium phosphate buffer 100 mM, pH 7.0 and was stable after 180 minutes in all pHs tested. The optimum temperature was 80 °C chitinase, remained stable between 30 and 100 °C for 180 minutes. The activity was enhanced in the presence of Fe2+ (134%), Mn2+ (71%) and the anionic surfactant SDS (59%), however, Pb2+ (99%) and EDTA (62%) inhibited the enzyme function. After fractionation with ammonium sulfate showed the enzymatic extract purification factor equal to 7 and 108% yield. The chitinase produced by Streptomyces sp. DPUA1581 under the conditions described in this work, has a great industrial viability, demonstrating catalytic activity even when subjected to high temperatures for prolonged periods. / Sustentabilidade é um tema abordado constantemente nos últimos anos, sendo a produção e utilização de enzimas uma eficiente alternativa na reciclagem de resíduos industriais e agrícolas. A quitinase atua na hidrólise da quitina, esta substância pode ser encontrada em grande quantidade na carapaça de crustáceos. O objetivo desta pesquisa foi selecionar Streptomyces spp. com maior potencial na produção da quitinase, caracterizar o extrato bruto e purificar parcialmente. Para seleção foram utilizadas 30 linhagens de Streptomyces spp. isoladas de líquens da região Amazônica. Foram avaliados parâmetros como pH e temperatura ótimos, estabilidade ao pH e a temperatura a partir do extrato bruto enzimático. A purificação parcial da enzima foi realizada por precipitação em sulfato de amônio na faixa de 0-80% e o perfil eletroforético em SDS-PAGE. Streptomyces sp. DPUA1581 produziu quitinase por fermentação submersa com 1% de quitina, agitação de 150 rpm, a 28 °C por 96h. A caracterização enzimática demonstrou melhor atividade no tampão fosfato de sódio 100 mM, no pH 7,0 e manteve-se estável após 180 minutos em todas as variações de pH testadas. A temperatura ótima da quitinase foi 80 °C, mantendo-se estável entre 30 e 100 °C durante 180 minutos. A atividade foi potencializada na presença de Fe2+ (134%), Mn2+ (71%) e do surfactante aniônico SDS (59%), entretanto, Pb2+ (99%), e EDTA (62%) inibiram a função da enzima. Após o fracionamento com sulfato de amônio o extrato enzimático apresentou fator de purificação igual a 7 e rendimento de 108%. A quitinase produzida por Streptomyces sp. DPUA1581 nas condições descritas neste trabalho, apresenta grande viabilidade industrial, demonstrando atividade catalítica mesmo quando submetida a altas temperaturas por período prolongado.

Chitinase is an enzyme that catalyzes the hydrolysis of chitin, an essential component of the cell walls of many fungi. Plant chitinases have been implicated in plant defense against pathogens. In plants, chitinase often exists as isoforms and three classes of chitinases have been proposed. In this study, isolation, characterization and expression of chitinase genes in Kentucky bluegrass (Poa pratensis L.) were studied. With primers designed from conserved regions of chitinase genes from other plant species, a 710 bp fragment (CH710) containing a partial chitinase gene sequence was amplified from Kentucky bluegrass by PCR. Using cassette ligation mediated PCR, we amplified four 5’ and five 3’ unknown sequences flanking CH710. The sequence information of these flanking fragments led us to the amplification of three genomic sequences from Kentucky bluegrass by PCR, KBCH1, KBCH2 and KBCH3, which contain full coding regions of chitinase genes. The chitinase genes carrying KBCH1, KBCH2 and KBCH3 were designated as chi1, chi2 and chi3, respectively. Southern blot hybridization indicated the presence of more than seven chitinase genes in the Kentucky bluegrass genome. Chi1 and chi2 each contain an open reading frame with no introns, encoding polypeptides of 340 and 320 amino acids, respectively. Both CHI1 and CHI2, the predicted proteins encoded by chi7 and chi2, are class I chitinases and share 94% amino acid identity. CHI1 has a short C-terminal extension, implicating that this protein may be a vacuolar protein. Although chi3 has high sequence similarity to chi7 and chi2, the potential open reading frame of chi3 is interrupted by a translation termination codon at the 51st amino acid indicating that it does not encode a functional chitinase. In this study, the expression of chitinase genes in Kentucky bluegrass under various stress conditions was also investigated. RNA blot hybridization showed that stresses such as cold, heat, salicylic acid and ethephon all induced an increased accumulation of chitinase MRNA in Kentucky bluegrass leaves with ethephon leading to the highest induction. After ethephon treatment, the accumulation of chitinase transcripts at a high level was observed from 2 days to 5 days. The expression of two individual chitinase genes, chi1 and chi2, was shown to be stimulated, while being coordinately regulated, by ethephon. / Ph. D.

Bovine mastitis is a common disease among dairy cattle characterized by the inflammation of the udders and loss of milk production. Mastitis-associated Escherichia coli (MAEC) are frequent causes of the disease, but the features that distinguish them from other E. coli strains remain enigmatic. MAEC infections can range from sub-clinical to severe, acute cases that can be fatal. Historically, the severity of mastitis has been attributed to host factors but more recently, a few bacterial genes have been shown to contribute to virulence in mastitis infections. In a large-scale genomic analysis of >100 MAEC isolates the gene for Chitinase A (ChiA) was positively associated with robust growth in the mammary glands during a mouse model of mastitis. This correlation suggests the hypothesis that ChiA contributes directly to MAEC fitness. The regulation of chiA has not been documented in contexts relevant to bovine mastitis. In the lab strain K-12, chiA is not expressed during aerobic growth in rich media. However, previous work with enterotoxigenic E. coli strain H10407 indicated that expression may be induced by hypoxic environments and the presence of bile salts. To measure expression of chiA, I created a chiA-GFP reporter plasmid and measured changes in fluorescence using flow cytometry. My results indicate promoter activity of chiA in MAEC is significantly increased in hypoxic conditions and the presence of bile salts, but not both. Adhesion to host tissues is an important characteristic of successful pathogens. Since ChiA facilitates adhesion between adherent-invasive E. coli and intestinal epithelial cells, I investigated its role in adhesion to bovine mammary epithelial cells in four MAEC strain backgrounds. Isogenic mutants lacking chiA were made in 2 mild (M45 and M93) and 2 severe (M111 and G1) clinical isolates. Loss of chiA resulted in significant reduction of adherence of M45, M93, and G1 to epithelial cells, but not M111. Wild type levels of adhesion were restored upon reintroduction of chiA into mutants through a plasmid vector. Additionally, the genomes of each MAEC isolate were analyzed for the presence of genes that could possibly influence the adhesion and virulence. Strain M111 contained genes for 2 distinct fimbriae that were not present in the other MAEC strains, possibly reducing its reliance on ChiA. The interaction of ChiA with mammary epithelial cells in MAEC could possibly offer an advantage for certain strains to be better suited to colonize and persist in mammary glands. Increased understanding of the regulation of chiA and its role in adherence can lead to novel targets for more effective treatment and prevention of bovine mastitis.

Chitinase has been suggested to play a role in defense mechanisms. In this study, the activity and expression of chitinase in muskmelon seeds were investigated. Multiple chitinase isoforms were detected in muskmelon seeds from early development through radicle emergence. One acidic and three basic chitinase isoforms were detected in developing seeds at 40 days after anthesis (DAA). Both acidic and basic chitinase isoforms were detected in endosperm tissue during seed imbibition and after radicle emergence. Basic chitinase isoforms, but not acidic isoforms, were detected in embryo tissue. Basic chitinase isoforms were also detected in the embryonic axis or radicle tissue. Taken together, these observations indicate that chitinases are regulated developmentally and in a tissue-specific manner in muskmelon seeds. Therefore the potential function of chitinases in muskmelon seeds is discussed. Two complete cDNAs, Cmchi1 and Cmchi2, and a partial genomic clone of Cmchi2 have been isolated from muskmelon seeds. Cmchi2 gene has two introns in the coding region while Cmchi1 is intronless. Cmchi1 cDNA encodes a class III chitinase while Cmchi2 cDNA encodes a class II chitinase. Cmchi1 and Cmchi2 proteins might be targeted to secretory pathways because they possess signal peptides. Southern blotting suggested that there is at least one additional gene similar to Cmchi1 in the muskmelon seed genome, while there is only one copy of Cmchi2. Northern blotting analysis showed that both Cmchi1 and Cmchi2 are expressed in the radicle tissue at the time of radicle emergence. This indicates that the expression is regulated developmentally and in a tissue-specific manner. Salicylic acid (SA) and benzothiadiazole (BTH) stimulated the expression of Cmchi1 but not Cmchi2 in seeds after radicle emergence, indicating that SA might be involved in inducing the expression of Cmchi1, while a different signal might be involved in triggering the expression of Cmchi2. The protein encoded by Cmchi1cDNA was expressed in E.coli. It did not show any enzymatic activity. Western blotting using an antibody raised against the class III chitinase protein in cucumber was inconclusive, as this antibody recognized the purified Cmchi1 fusion protein and other unknown proteins isolated from the embryonic axis or the radicle tissue. / Ph. D.