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SILVA, Suzete Roberta da. Purificação e caracterização de CiL-2, uma nova lectina isolada da alga marinha verde Codium isthmocladum Vickers. 2013. 71 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Engenharia de Pesca, Fortaleza-CE, 2013 / Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-22T13:15:28Z No. of bitstreams: 1 2013_dis_srsilva.pdf: 1234725 bytes, checksum: 55b286fd2482006a3f365a7578cde4de (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-22T13:15:48Z (GMT) No. of bitstreams: 1 2013_dis_srsilva.pdf: 1234725 bytes, checksum: 55b286fd2482006a3f365a7578cde4de (MD5) / Made available in DSpace on 2016-07-22T13:15:48Z (GMT). No. of bitstreams: 1 2013_dis_srsilva.pdf: 1234725 bytes, checksum: 55b286fd2482006a3f365a7578cde4de (MD5) Previous issue date: 2013 / Lectins are ubiquitous proteins or glycoproteins with at least one non-catalytic domain binding reversibly to a specific mono- or oligosaccharide. Lectins are ubiquitously distributed in plants, animals and microorganisms. Marine algal lectins have been isolated and characterized from only a few species and at a much slower pace since the first report, more than 40 years ago, of the haemagglutinating activity in these organisms. This paucity is mainly due to difficulties in obtaining sufficient material for study. However, among characterized lectins, proteins observed with different biochemical characteristics. Lectins from genus Codium have been isolated and characterized in some detail. In general, have specificity for GalNAc and/or GlcNAc and glycoproteins mucin, fetuin and thyroglobulin, have no requirement for metal ions and were composed by low molecular subunits and may present oligomerization. The present work deals with the purification and characterization of two lectins (CiL-1 and CiL-2) from green marine alga Codium isthmocladum, compare their characteristics and evaluate the ability in agglutinate bacterial cells and toxicity against Artemia. The lectins was purified by a combination of ammonium sulphate precipitation and ion-exchange chromatography on a DEAE-Sephacel column. The main differences observed were the metal dependence, oligomerization state and thermostability. The amino acid sequence of CiL-2 showed no similarity with CiL-1 or other lectins from protein data bank. Only CiL-1 was able to agglutinate bacterial cells whereas CiL-2 showed toxicity against Artemia after 48 hours. In the evaluation of lectin interaction, the data suggest that the CiL-2 recognizes the glycoproteins present in the microcrustacean digest tract. The work has shown that the marine green alga has at least two different lectins with differences in amino acid sequences, biochemical characteristics and biological response. / Lectinas são (glico) proteínas com pelo menos um domínio não catalítico que podem se ligar específica e reversivelmente a carboidratos aglutinando células e/ou glicoconjugados. São ubíquas na natureza, presentes em todos os organismos. Comparando com lectinas de plantas terrestres, poucas lectinas de algas marinhas têm sido isoladas e caracterizadas, principalmente devido à dificuldade de obtenção de material suficiente para estudo. Entretanto, entre as lectinas de algas caracterizadas, observam-se proteínas com características bioquímicas diferentes. Várias lectinas do gênero Codium já foram isoladas e caracterizadas em algum detalhe. Em geral, possuem especificidade para GalNAc e/ou GlcNAc e glicoproteínas, mucina, fetuína e tiroglobulina, não dependem de cátions divalentes para exibir sua atividade e são compostas de subunidades de baixo peso molecular podendo apresentar formas oligoméricas. Neste trabalho objetivou-se isolar e caracterizar bioquimicamente duas lectinas isoladas da alga marinha verde Codium isthmocladum (CiL-1 e CiL-2), comparar suas características e avaliar a capacidade das duas lectinas de aglutinar células bacterianas e a toxicidade contra Artemia sp. As lectinas foram purificadas através da combinação de precipitação com sulfato de amônio e cromatografia de troca iônica. Entre as principais diferenças bioquímicas observadas foram: dependência de metais, oligomerização e estabilidade térmica. No entanto, ambas foram inibidas por Mucina e não por carboidratos simples. Quando comparada a sequência de aminoácidos de CiL-2, não houve similaridade com a sequência parcial da CiL-1 ou de qualquer outra sequência de lectinas de algas disponíveis nos bancos de dados. Somente CiL-1 foi capaz de aglutinar células bacterianas enquanto que a CiL-2 apresentou toxicidade contra Artemia sp. após 48 horas de contato. Na avaliação da interação da lectina, os dados sugerem que a proteína reconhece as glicoproteínas presentes no trato digestório do microcrustáceo. O trabalho evidenciou que alga marinha verde C. isthmocladum Vickers possui pelo menos duas lectinas com sequência de aminoácidos distinta, mostrando respostas biológicas diferentes, onde a CiL-1 teve capacidade de aglutinar células bacterianas e a foi CiL-2 tóxica contra Artemia sp.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Lectins are ubiquitous proteins or glycoproteins with at least one non-catalytic domain binding reversibly to a specific mono- or oligosaccharide. Lectins are ubiquitously distributed in plants, animals and microorganisms. Marine algal lectins have been isolated and characterized from only a few species and at a much slower pace since the first report, more than 40 years ago, of the haemagglutinating activity in these organisms. This paucity is mainly due to difficulties in obtaining sufficient material for study. However, among characterized lectins, proteins observed with different biochemical characteristics. Lectins from genus Codium have been isolated and characterized in some detail. In general, have specificity for GalNAc and/or GlcNAc and glycoproteins mucin, fetuin and thyroglobulin, have no requirement for metal ions and were composed by low molecular subunits and may present oligomerization. The present work deals with the purification and characterization of two lectins (CiL-1 and CiL-2) from green marine alga Codium isthmocladum, compare their characteristics and evaluate the ability in agglutinate bacterial cells and toxicity against Artemia. The lectins was purified by a combination of ammonium sulphate precipitation and ion-exchange chromatography on a DEAE-Sephacel column. The main differences observed were the metal dependence, oligomerization state and thermostability. The amino acid sequence of CiL-2 showed no similarity with CiL-1 or other lectins from protein data bank. Only CiL-1 was able to agglutinate bacterial cells whereas CiL-2 showed toxicity against Artemia after 48 hours. In the evaluation of lectin interaction, the data suggest that the CiL-2 recognizes the glycoproteins present in the microcrustacean digest tract. The work has shown that the marine green alga has at least two different lectins with differences in amino acid sequences, biochemical characteristics and biological response. / Lectinas sÃo (glico) proteÃnas com pelo menos um domÃnio nÃo catalÃtico que podem se ligar especÃfica e reversivelmente a carboidratos aglutinando cÃlulas e/ou glicoconjugados. SÃo ubÃquas na natureza, presentes em todos os organismos. Comparando com lectinas de plantas terrestres, poucas lectinas de algas marinhas tÃm sido isoladas e caracterizadas, principalmente devido à dificuldade de obtenÃÃo de material suficiente para estudo. Entretanto, entre as lectinas de algas caracterizadas, observam-se proteÃnas com caracterÃsticas bioquÃmicas diferentes. VÃrias lectinas do gÃnero Codium jà foram isoladas e caracterizadas em algum detalhe. Em geral, possuem especificidade para GalNAc e/ou GlcNAc e glicoproteÃnas, mucina, fetuÃna e tiroglobulina, nÃo dependem de cÃtions divalentes para exibir sua atividade e sÃo compostas de subunidades de baixo peso molecular podendo apresentar formas oligomÃricas. Neste trabalho objetivou-se isolar e caracterizar bioquimicamente duas lectinas isoladas da alga marinha verde Codium isthmocladum (CiL-1 e CiL-2), comparar suas caracterÃsticas e avaliar a capacidade das duas lectinas de aglutinar cÃlulas bacterianas e a toxicidade contra Artemia sp. As lectinas foram purificadas atravÃs da combinaÃÃo de precipitaÃÃo com sulfato de amÃnio e cromatografia de troca iÃnica. Entre as principais diferenÃas bioquÃmicas observadas foram: dependÃncia de metais, oligomerizaÃÃo e estabilidade tÃrmica. No entanto, ambas foram inibidas por Mucina e nÃo por carboidratos simples. Quando comparada a sequÃncia de aminoÃcidos de CiL-2, nÃo houve similaridade com a sequÃncia parcial da CiL-1 ou de qualquer outra sequÃncia de lectinas de algas disponÃveis nos bancos de dados. Somente CiL-1 foi capaz de aglutinar cÃlulas bacterianas enquanto que a CiL-2 apresentou toxicidade contra Artemia sp. apÃs 48 horas de contato. Na avaliaÃÃo da interaÃÃo da lectina, os dados sugerem que a proteÃna reconhece as glicoproteÃnas presentes no trato digestÃrio do microcrustÃceo. O trabalho evidenciou que alga marinha verde C. isthmocladum Vickers possui pelo menos duas lectinas com sequÃncia de aminoÃcidos distinta, mostrando respostas biolÃgicas diferentes, onde a CiL-1 teve capacidade de aglutinar cÃlulas bacterianas e a foi CiL-2 tÃxica contra Artemia sp.

&alpha / -Galactosidase is an exo-glycosidase that hydrolyses non-reducing, &alpha / -1,6-linked &alpha / -galactose units from oligosaccharides, galactomannans, and galactolipids. &alpha / -Galactosidase activity has biotechnological, industrial, and medical importance. &alpha / -Galactosidase from A. fumigatus IMI 385708, in particular, can catalyse unique hydrolysis and transgalactosylation reactions on polymeric substrates. In this study, &alpha / -galactosidase of the human pathogen A. fumigatus IMI 385708 was first produced in a GRAS organism, Aspergillus sojae. For this aim, &alpha / -galactosidase gene (aglB) of A. fumigatus IMI 385708 was ligated onto pAN52-4 vector (Acc. No: Z32699) and transformed into Aspergillus sojae ATCC11906, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (gpdA) of A. nidulans and the signal sequence of glucoamylase gene (glaA) of A. niger. This allowed high level of &alpha / -galactosidase production on glucose instead of locust bean gum (2.45 U/mL), corresponding to a 3-fold increase in volumetric production. Next, using response surface methodology, carbon and nitrogen sources and agitation speed were optimized (10.5% molasses (w/v) / 1.3% NH4NO3 (w/v) / 276 rpm). Compared to non-optimized cultivation, a further 4-fold increase in &alpha / -galactosidase production (10.4 U/mL) was achieved. Recombinant &alpha / -galactosidase was purified 18.7-fold using Anion Exchange and Hydrophobic Interaction Chromatography with an overall yield of 56% and 64.7 U/mg protein. The Vmax and Km values for the hydrolysis of p-nitrophenyl &alpha / -D-galactopyranoside were 78 U/mg protein and 0.45 mM, respectively. Optimum pH and temperature for &alpha / -galactosidase activity were between pH 4&ndash / 6 and 50&ndash / 60 &deg / C, respectively. Among the tested chemical agents, Ag+, Hg2+, and Fe2+ drastically decreased the activity, while biotin, I+1, Mn+2, Pb+2, Li+1, and Mg+2 enhanced between 12&ndash / 29%. To analyse the influence of osmotic stress as a means of further inducing &alpha / -galactosidase production, salt was added into the complete growth medium. In addition to enzyme production, fungal growth and morphology were analysed for both &lsquo / salt-adapted&rsquo / and &lsquo / salt non-adapted&rsquo / A. sojae Ta1 cells in the presence of KCl, MgCl2, MgSO4, NaCl, and Na2SO4 at 1 M and 2 M. Accordingly, 3-fold increase in &alpha / -galactosidase production was achieved by non-adapted cells in the presence of 1 M NaCl. Exposure of A. sojae Ta1 cells to salt resulted in predominantly mycelial form, rather than the pellet form observed under normal conditions. Finally, the transgalactosylation ability of &alpha / -galactosidase was studied. &alpha / -Galactosidase efficiently catalysed galactose transfer to different monosaccharides and disaccharides in the presence of pNP&alpha / Gal as monitored by TLC, ESI-MS, and HPLC.