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Preparação de sílicas organofuncionalizadas para imobilização da lipase de Burkholderia capacia / Preparation of organofunctionalized silicas for the immobilization of the Burkholderia cepacia lipaseSilva, André Leonardo Patrício 19 September 2012 (has links)
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Previous issue date: 2012-09-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The preparation of stable immobilized lipases is one of the great challenges for modern biotechnology that involves the use of these enzymes in biocatalyzed processes. The commercial application of these biocatalysts depends of an efficient immobilization and the appropriate use of supports to ensure stability to enzymes. This work focused on the study of the preparation of chemically modified supports derived from silica gel. The organofunctionalized silicas were prepared by silylation through of the heteregeneous route using the compounds 3-aminepropyl- and 3-chloropropyl trimethoxysilane resulting in the solids named Sil-propil-NH2 and Sil-propil-Cl, respectively. The solids Sil-propil-NH2 and Sil-propil-Cl reacted subsequently with the cyanuric chloride and 1,6 diaminehexane as spacer, followed by covalent attack of cyanuric chloride resulting in the matrices Sil-propil-N-CC and Sil-propil-Hex-CC. Silica gel and the modified solid were characterized measures of adsorption / desorption of nitrogen, CHN elemental analysis, thermogravimetry (TG), Si29 and C13 NMR and FTIR spectroscopy. The modified support were used in the immobilization of the Burkholderia cepacia lipase, and the catalytic performance and stability of the immobilized enzyme derivatives were investigated in the hydrolysis reaction of p-nitrophenylpalmitate (pNPP) in five consecutive reaction cycles. The results of the preparation of matrizes showed the anchoring of the triazine molecule onto silanized surface. For material Sil-propil-N-CC was observed the increasing of the nitrogen content as indicated CNH elemental analysis, that corresponded to 1.82% of N due the introduction of amino group and 3.08% of N after the reaction of the triazine molecule. For the material Sil-propil-Cl, it was observed the increasing in the nitrogen percentage for the support with spacer (2.13% of N) and after the reaction with cyanuric chloride (3.6% of N). The tests of catalytic activity operational stability of the immobilized enzymes onto supports were 2910, 3000 e 3430 U/g, respectively, for supports with aminopropyl and triazine molecule, chloropropyl with the spacer and for the surface with spacer and triazine molecule. For catalytic test were observed a higher tendency for loss of stability for support without spacer. The obtained results showed that the chemical modification reactions of silica gel enabled the covalent anchoring of the cyanuric chloride and the use of spacer resulted in higher catalytic activity and stability for the immobilized bio catalysts. / A obtenção de lipases imobilizadas estáveis é um dos maiores desafios para a biotecnologia moderna que envolve o emprego destas enzimas em processos biocatalisados. A aplicação comercial desses biocatalisadores depende de métodos eficientes de imobilização e da utilização de um suporte apropriado para assegurar estabilidade às enzimas. Nessa direção, o escopo do presente trabalho envolveu a preparação de suportes quimicamente modificados a partir da sílica gel. As sílicas organofuncionalizadas foram preparadas por silanização pela rota heterogênea, utilizando os compostos 3-aminopropiltrimetoxissilano e 3-cloropropiltrimetoxissilano originando os sólidos denominados Sil-propil-NH2 e Sil-propil-Cl, respectivamente. Os sólidos Sil-propil-NH2 e Sil-propil-Cl reagiram subsequentemente com cloreto cianúrico e o com o espaçador 1,6 diaminohexano, seguido do ataque covalente do cloreto cianúrico resultando nas matrizes Sil-propil-N-CC e Sil-propil-Hex-CC. A sílica gel e os sólidos modificados foram caracterizados pelas técnicas de termogravimetria, medidas de adsorção/dessorção de nitrogênio, análises elementar de CHN, ressonância magnética nuclear de Si29 e C13 e espectroscopia de absorção na região do infravermelho. Os suportes modificados foram utilizados na imobilização da lipase de Burkholderia cepacia, sendo que o desempenho catalítico e estabilidade dos derivados enzimáticos imobilizados foram investigados em reações de hidrólise do éster palmitato de p-nitrofenila (p-NPP) em cinco ciclos reacionais consecutivos. Os resultados das preparações dos suportes mostraram o ancoramento da molécula triazínica nas superfícies silanizadas. No material Sil-propil-N-CC foi observado um aumento do teor de nitrogênio obtido da análise elementar, que correspondeu a 1,82% de N referente à introdução do grupo amino e 3,08% de N após a reação com o composto triazínico. No caso do material Sil-propil-Cl, observou-se um aumento na porcentagem de nitrogênio do suporte contendo espaçador (2,13% N) e após a reação com o cloreto cianúrico (3,6% N). Os testes de atividade hidrolítica e estabilidade das lipases imobilizadas mostraram 2910, 3000 e 3430 U/g, respectivamente para os suportes contendo aminopropil e o anel triazínico, cloropropil e o espaçador e a superfície com espaçador e o anel triazínico. Nos ensaios catalíticos foi observada uma maior tendência de perda de estabilidade na ausência do espaçador. Os resultados obtidos mostraram que as reações de modificação química da superfície da sílica possibilitaram o ancoramento covalente do cloreto cianúrico e a presença de um espaçador possibilitou maior atividade e estabilidade aos biocatalisadores imobilizados.
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Compréhension et prédiction de l'énantiosélectivité des lipases / Comprehension and prediction of lipases enantioselectivityLafaquière, Vincent 19 January 2010 (has links)
Cette étude a porté sur l’analyse de l’énantiosélectivité de la lipase de Burkholderia cepacia (BCL) pour les acides 2-substitués, synthons chiraux d’intérêt pharmaceutique, avec pour objectif d’examiner le rôle de l’accès au site actif enfoui de BCL sur l’énantiosélectivité et de développer une procédure d’ingénierie permettant de créer des mutants d’énantiosélectivité améliorée. Pour traiter le problème, une nouvelle approche de calcul, basée sur des algorithmes de planification de mouvements issus de la robotique a été développée. Elle permet l’exploration conformationnelle des espaces multi-dimensionnels contraints et a été appliquée au calcul des trajectoires de plusieurs racémiques dans le site actif de BCL et à l’identification de résidus pouvant potentiellement gêner le déplacement du substrat le long du site actif. Les résultats obtenus in silico ont révélé une corrélation qualitative avec les valeurs d’énantiosélectivité et ont permis de proposer des cibles de mutagénèse. Sur cette base, l’ingénierie du site actif de BCL a été entreprise pour moduler sélectivement l’accès des énantiomères R et S à la triade catalytique. Un système d’expression hétérologue de BCL chez E. coli compatible avec une expression en microplaque, a été développé. Une librairie de 57 (3x19) mono-mutants sur les positions : Leu17, Val266 et Leu287 a été construite par iPCR puis criblée en utilisant une procédure à moyen débit pour identifier les variants actifs pour l’hydrolyse du pNPB. L’énantiosélectivité de ces mutants a ensuite été évaluée pour l’hydrolyse du racémique (R,S)-2 bromophényl acétate de 2-chloro-éthyle, par utilisation d’une nouvelle procédure de criblage en deep-wells. Ce crible a permis de mettre en évidence plusieurs mutants dont les plus prometteurs ont été caractérisés. Ainsi les mutants Leu17Ser et Leu17Met présentent une augmentation de l’énantiosélectivité d’un facteur 10 accompagnée d’une augmentation de leur activité d’un facteur 4 à 5. Le mutant Val266Gly présente, quant à lui, une inversion de l’énantiosélectivité pour le substrat d’intérêt. L’étude des trajectoires par les techniques de planification combinée à une représentation sous la forme de carte de voxels a été réalisée en parallèle. Pour les mutants sélectionnés, une bonne corrélation a été observée entre les résultats obtenus in silico et expérimentalement. De plus, cela a permis de proposer de nouvelles combinaisons de mutations ayant conduit à l’identification de deux double-mutants Leu17Met/Val266Met et Leu17Ser/Leu287Ile d’énantiosélectivité supérieure à 150 pour le substrat modèle, révélant ainsi l’intérêt de l’approche semi-rationnelle proposée / This work has been focused on the understanding of the Burkholderia cepacia lipase (BCL) enantioselectivity towards 2-substituted acids which are chiral building blocks of pharmaceutical interest. The main objective of this work was the investigation of the potential role of substrate accessibility toward the buried active site of BCL on enantioselectivity and the development of an engineering procedure for the design of enantioselective mutants. To study further this hypothesis, a novel computational approach, based on motion-planning algorithms, originally used in robotics, was developed. It allows the conformational exploration of constrained high-dimensional spaces and was applied to the computation of trajectories for a set of racemates within the catalytic site. This methodology also enables the identification of residues potentially hindering substrates displacement along the active site. Results obtained in silico were correlated qualitatively with experimental values of enantioselectivity. On the basis of these results, engineering of the narrow active site of BCL has been undertaken to modulate selectively the access of R and S enantiomers to the catalytic triade. An heterologous expression system of BCL in E. coli compatible with production at microplate scale was developed. A library of 57 (3x19) variants targeted at positions Leu17, Val266 and Leu287 was built by iPCR and subsequently screened using a medium-throughput procedure to identify active variants against pNPB hydrolysis. Next, the enantioselectivity of these mutants was evaluated towards a given racemate, the (R,S)-2-chloro ethyl 2-bromophenylacetate, using a novel screening procedure developed in deep wells. Such screening enabled the identification of several variants amongst which the most promising were characterized. Mutants Leu17Ser and Leu17Met showed a remarkable 10-fold increase of their enantioselectivity and a 4- and 5-fold improvement of their specific activity. Compared to the wild-type enzyme, mutant Val266Gly displayed a reversed enantioselectivity for the substrate of interest. Investigation of the trajectories using motion-planning techniques combined to a voxel map representation was carried out. For selected variants, a fair correlation was observed between in silico and experimental results. Moreover, this enabled us to suggest novel combinations of mutations that led to the identification of two double-mutants Leu17Met/Val266Met and Leu17Ser/Leu287Ile showing an enantioselectivity value higher than 150 for the racemic substrate, revealing thus the effiency of the semi-rational strategy
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