Produção e purificação da enzima ciclodextrina glicosiltranferase: obtenção de fases sólidas e metálicas em escala nanométrica utilizando ciclodextrinas como nanorreatores

Pós-graduação em Ciências Biológicas (Microbiologia Aplicada) - IBRC

Planejamento, síntese e avaliação fotoprotetora de novos compostos híbridos derivados do resveratrol, cinamatos e avobenzona planejados como filtros solares

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Produção e obtenção de ciclodextrinas produzidas por CGTases bacterianas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Produção da enzima ciclodextrina glicosil transferase por Bacillus circulans

As ciclodextrinas (CDs) são oligossacarídeos cíclicos que solubilizam e modificam moléculas por possuir uma cavidade interna hidrofílica e região externa hidrofóbica, com uma estrutura tronco-cônica, conferindo a estes açúcares cíclicos propriedades físico-químicas para complexação de uma grande variedade de moléculas. A enzima ciclodextrina glicosiltransferase (CGTase) catalisa reações de conversão de amido em diferentes tipos de CDs, a sua produção é influenciada por vários fatores. As pesquisas dirigidas para a produção de CGTases de menor custo são importantes para viabilizar economicamente o uso das CDs em escala industrial. No presente estudo, as CDs foram produzidas a partir de diferentes fontes de carbono pelo micro-organismo Bacillus circulans ATCC 21783, estudou-se seu crescimento celular assim como sua produção enzimática, utilizando a ferramenta estatística de planejamento experimental. O micro-organismo Bacillus circulans ATCC 21783 mostrou-se ser eficiente na produção da enzima CGTase, os resultados indicaram a potencialidade do grão de sorgo como o melhor substrato para fermentação na produção de CGTase. Quanto maior o tamanho do grão de sorgo, maiores concentrações de glicose e amido estarão presentes, o que influencia diretamente a produção enzimática. Por meio do planejamento experimental foram proposto modelos matemáticos que expressam tanto a produção enzimática quanto a concentração das variáveis das concentrações da fonte de carbono, pH e a temperatura. A importância de desenvolver o modelo é demonstrar a sua aplicação bem-sucedida para determinação das condições ideais que representam o processo de alta produtividade enzimática da CGTase. A otimização das variáveis foram obtidas a partir de quatro planejamentos experimentais composto central (PCC) e seus resultados analisados pelas superfícies de resposta. Os melhores resultados do planejamento encontrados no...

Reuse of Cyclodextrin Glycosyltransferase through Immobilization on Magnetic Carriers

National Council for Scientific and Technological Development (CNPq)

Comparative analysis of three hyperthermophilic GH1 and GH3 family members with industrial potential

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystal structure of mature 2S albumin from Moringa oleifera seeds

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação de isolados de Trichoderma spp. para controle de Phytophthora nicotianae

Pós-graduação em Microbiologia Agropecuária - FCAV

Desenvolvimento de um bioprocesso utilizando-se resíduos para produção de amilases por Rhizopus oligosporus e etanol por Saccharomyces cerevisiae

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo comparativo das características bioquímicas funcionais e especificidade catalítica de aspartil, cisteíno e serino peptidases fúngicas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Induction of cellulase and hemicellulase activities of Thermoascus aurantiacus by xylan hydrolyzed products

Thermoascus aurantiacus is able to secrete most of the hemicellulolytic and cellulolytic enzymes. To establish the xylanase inducers of T. aurantiacus, the mycelia were first grown on glucose up until the end of the exponential growth phase, followed by washing and re-suspension in a basal medium without a carbon source. Pre-weighed amounts of xylose (final concentration of 3.5 mg/ml), xylobiose (7 mg/ml) and hydrolyzed xylan from sugarcane bagasse (HXSB) which contained xylose, xylobiose and xylotriose (6.8 mg/ml) were evaluated as inducers of xylanase. It was observed that xylose did not suppress enzyme induction of T. aurantiacus when used in low concentrations, regardless of whether it was inoculated with xylobiose. Xylobiose promoted fast enzyme production stopping after 10 h, even at a low consumption rate of the carbon source; therefore xylobiose appears to be the natural inducer of xylanase. In HXSB only a negligible xylanase activity was determined. Xylose present in HXSB was consumed within the first 10 h while xylobiose was partially hydrolyzed at a slow rate. The profile of alpha-arabinofuranosidase induction was very similar in media induced with xylobiose or HXSB, but induction with xylose showed some positive effects as well. The production profile for the xylanase was accompanied by low levels of cellulolytic activity. In comparison, growth in HXSB resulted in different profiles of both xylanase and cellulase production, excluding the possibility of xylanase acting as endoglucanases.

Development of enzymic time-temperature integrators with rapid detection for evaluation of continuous HTST pasteurization processes

The assessment of the thermal process impact in terms of food safety and quality is of great importance for process evaluation and design. This can be accomplished from the analysis of the residence time and temperature distributions coupled with the kinetics of thermal change, or from the use of a proper time-temperature integrator (TTI) as indicator of safety and quality. The objective of this work was to develop and test enzymic TTIs with rapid detection for the evaluation of continuous HTST pasteurization processes (70-85 degrees C, 10-60 s) of low-viscosity liquid foods, such as milk and juices. Enzymes peroxidase, lactoperoxidase and alkaline phosphatase in phosphate buffer were tested and activity was determined with commercial reflectometric strips. Discontinuous thermal treatments at various time-temperature combinations were performed in order to adjust a first order kinetic model of a two-component system. The measured time-temperature history was considered instead of assuming isothermal conditions. Experiments with slow heating and cooling were used to validate the adjusted model. Only the alkaline phosphatase TTI showed potential to be used for the evaluation of pasteurization processes. The choice was based on the obtained z-values of the thermostable and thermolabile fractions, on the cost and on the validation tests. (C) 2012 Elsevier Ltd. All rights reserved.

Computational methods for genome screening

Motivation An actual issue of great interest, both under a theoretical and an applicative perspective, is the analysis of biological sequences for disclosing the information that they encode. The development of new technologies for genome sequencing in the last years, opened new fundamental problems since huge amounts of biological data still deserve an interpretation. Indeed, the sequencing is only the first step of the genome annotation process that consists in the assignment of biological information to each sequence. Hence given the large amount of available data, in silico methods became useful and necessary in order to extract relevant information from sequences. The availability of data from Genome Projects gave rise to new strategies for tackling the basic problems of computational biology such as the determination of the tridimensional structures of proteins, their biological function and their reciprocal interactions. Results The aim of this work has been the implementation of predictive methods that allow the extraction of information on the properties of genomes and proteins starting from the nucleotide and aminoacidic sequences, by taking advantage of the information provided by the comparison of the genome sequences from different species. In the first part of the work a comprehensive large scale genome comparison of 599 organisms is described. 2,6 million of sequences coming from 551 prokaryotic and 48 eukaryotic genomes were aligned and clustered on the basis of their sequence identity. This procedure led to the identification of classes of proteins that are peculiar to the different groups of organisms. Moreover the adopted similarity threshold produced clusters that are homogeneous on the structural point of view and that can be used for structural annotation of uncharacterized sequences. The second part of the work focuses on the characterization of thermostable proteins and on the development of tools able to predict the thermostability of a protein starting from its sequence. By means of Principal Component Analysis the codon composition of a non redundant database comprising 116 prokaryotic genomes has been analyzed and it has been showed that a cross genomic approach can allow the extraction of common determinants of thermostability at the genome level, leading to an overall accuracy in discriminating thermophilic coding sequences equal to 95%. This result outperform those obtained in previous studies. Moreover, we investigated the effect of multiple mutations on protein thermostability. This issue is of great importance in the field of protein engineering, since thermostable proteins are generally more suitable than their mesostable counterparts in technological applications. A Support Vector Machine based method has been trained to predict if a set of mutations can enhance the thermostability of a given protein sequence. The developed predictor achieves 88% accuracy.

Die Isolierung und Aufreinigung der Metalloendopeptidase LysN und die Evaluierung der Spezifität der LysN E157D-Mutante und ihr Einsatz in der Proteomik und die Mechanismen der Bildung von “dendritic cell aggresome- like induced structures“ (DALIS)

Der proteolytische Verdau von Proteinen in Peptide ist ein wichtiger Schritt in der Tandem-Massenspektrometrie. Dabei werden Peptide fragmentiert und die sich ergebenden Fragmentionen geben Aufschluss über die Aminosäuresequenz des zu untersuchenden Proteins. Dabei sind für die Fragmentierung sowohl Länge und Sequenz, als auch der Ladungszustand des Peptids ungemein wichtig. Diese Parameter bedingen sich durch Endoproteasen, die für den proteolytischen Verdau eingesetzt werden. Eine Voraussetzung hierfür ist die Spezifität der Protease. Trypsin ist bei weitem die gebräuchlichste Protease zur massenspektrometrischen Probenvorbereitung. Allerdings bietet Trypsin keine Komplettlösung. Je nach Fragestellung und Applikation müssen weitere Proteasen eingesetzt werden, um eine komplette Sequenzabdeckung zu gewährleisten und möglichst alle posttranslationalen Modifikationen nachzuweisen, oder bestimmte Proteomklassen (z.B Phosphoproteom

Effects of increased CO2 on fish gill and plasma proteome

Ocean acidification and warming are both primarily caused by increased levels of atmospheric CO2, and marine organisms are exposed to these two stressors simultaneously. Although the effects of temperature on fish have been investigated over the last century, the long-term effects of moderate CO2 exposure and the combination of both stressors are almost entirely unknown. A proteomics approach was used to assess the adverse physiological and biochemical changes that may occur from the exposure to these two environmental stressors. We analysed gills and blood plasma of Atlantic halibut (Hippoglossus hippoglossus) exposed to temperatures of 12°C (control) and 18°C (impaired growth) in combination with control (400 µatm) or high-CO2 water (1000 µatm) for 14 weeks. The proteomic analysis was performed using two-dimensional gel electrophoresis (2DE) followed by Nanoflow LC-MS/MS using a LTQ-Orbitrap. The high-CO2 treatment induced the up-regulation of immune system-related proteins, as indicated by the up-regulation of the plasma proteins complement component C3 and fibrinogen beta chain precursor in both temperature treatments. Changes in gill proteome in the high-CO2 (18°C) group were mostly related to increased energy metabolism proteins (ATP synthase, malate dehydrogenase, malate dehydrogenase thermostable, and fructose-1,6-bisphosphate aldolase), possibly coupled to a higher energy demand. Gills from fish exposed to high-CO2 at both temperature treatments showed changes in proteins associated with increased cellular turnover and apoptosis signalling (annexin 5, eukaryotic translation elongation factor 1 gamma, receptor for protein kinase C, and putative ribosomal protein S27). This study indicates that moderate CO2-driven acidification, alone and combined with high temperature, can elicit biochemical changes that may affect fish health.