Estudos experimentais e teórico-computacionais de novos complexos nanozeólitas-enzimas empregados na produção de biocombustíveis
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Data
2016-04-29
Autores
Miller, Alex Henrique [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A busca por fontes alternativas de energia e/ou a valorização das existentes (principalmente de caráter “limpo”) é tema amplamente discutido nos setores científicos e industriais, com ênfase aos processos catalíticos envolvidos na conversão de biomassa a biocombustíveis e/ou outros produtos de interesse de diversos setores industriais, e foi a base motivacional deste trabalho. O principal objetivo do presente trabalho foi a obtenção de biocatalisadores heterogêneos a partir da imobilização de lipases em nanozeólita faujasita (FAU) após troca iônica com o metal de transição Ni2+, e o uso dos complexos nanozeólita-enzima em reações de transesterificação do óleo de Microalgas em ésteres etílicos (FAEEs). Inicialmente, foi sintetizada a nanozeólita FAU (NaX-Na+), seguida da sua troca iônica com soluções de sulfato de níquel (0,5M) encubadas a 80°C por 3 dias (3D), formando o material denominado NaX-Ni2+(0,5M-3D). Os materiais obtidos tiveram suas estruturas cristalográficas confirmadas por Difração de Raios X (DRX). Após os experimentos de troca-iônica, os materiais foram utilizados para imobilização das lipases fungicas de T. lanuginosus (TLL), R. miehei (RML), C. rugosa (CRL) e de C. antarctica B (CALB), e foram denominados NaX-Ni2+(0,5M-3D)-TLL,Ni2+(0,5M-3D)-RML, Ni2+(0,5M-3D)-CRL e Ni2+(0,5M-3D)-CALB respectivamente. As atividades lipolíticas desses complexos foram determinadas a partir da hidrólise de azeite de oliva emulsificado, sendo encontrados os valores de 850, 550, 425 e 50 U/g respectivamente. Objetivando-se a otimização da produção de ésteres etílicos, os complexos foram aplicados em reações de transesterificação do óleo de microalgas em várias condições experimentais. O uso de 5% m/m (massa de catalisador/massa óleo) do complexo com TLL apresentou conversão de 85% de ésteres etílicos (12h/35°C) após adição única de etanol na proporção molar de 1:4 (óleo:etanol), enquanto 5% m/m do complexo com RML obteve 52 % de conversão (12h/45°C) após adição em 4 etapas do volume de álcool ( a cada duas horas), sendo as condições destes ensaios para estes dois complexos estabelecidas como condições ótimas para a reação. Os complexos com CRL e CALB apresentaram baixos rendimentos quando aplicados nas reações de transesterificação. Experimentalmente, foi observado apenas para o complexo Ni2+(0,5M-3D)-RML um aumento significativo da estabilidade térmica no intervalo de 25 a 80°C. Os complexos NaX-Ni2+(0,5M-3D)-TLL, e Ni2+(0,5M-3D)-CALB foram também estudados para a reação de esterificação de ácidos graxos livres. Em reações de esterificação (12h/45°C) de ácido oleico utilizando 5% m/m de complexo, na proporção molar 1:2 (ácido:etanol) produções de 89 e 83% de FAEEs foram obtidas para os complexos NaX-Ni2+(0,5M-3D)-TLL e Ni2+(0,5M-3D)-CALB, respectivamente. Ambos os complexos foram também aplicados nas reações de transesterificação/esterificação simultâneas de óleo de Macaúba com alto percentual de ácido graxos livres (56%) e índice de acidez de 112 mg KOH/g. Para 5% m/m de complexo, 67 e 52% de FAEEs foram produzidos (12h/45°C), para adição única do volume de etanol na proporção 1:6 (óleo:álcool). O complexo Ni2+(0,5M-3D)-CALB apesar de não atuar efetivamente nas reações de transesterificação do óleo de microalgas, apresentou bons resultados nas reações de esterificação do ácido oleico, e possivelmente de esterificação dos ácidos graxos livres presentes no óleo de macaúba, sendo essa enzima portanto qualificada como específica para esterificação no sistema estudado. Visando compreender os baixos rendimentos obtidos com as enzimas CRL e CALB nas reações de transesterificação quando comparadas com as lipases TLL e RML estudos teórico-computacionais foram realizados usando análises de modos normais e simulações de dinâmica molecular na presença de metanol. Para análise da abertura e exposição do sítio catalítico foram verificados os distanciamentos máximos entre carbonos betas presente nos resíduos Pro-74 (pertencente a lid) e Pro-443 para a CRL, e Ile-144 (pertencente a lid) e Leu-285 para a CALB, resultante das simulações em comparação com as distâncias encontradas em suas estruturas cristalográficas disponíveis no Protein Data Bank (PDB). Nas análises de modos normais, a distância entre Pro-74 e Pro-443 foi apenas 0,1 Ȧ superior a esta distância na lipase em conformação fechada (3,8 Ȧ). Após dinâmica molecular com metanol, uma distância máxima de 6,5 Ȧ foi encontrada. A distância calculada é superior à respectiva distância verificada na estrutura em conformação fechada e aproximadamente 3 vezes inferior à distância de 21,6 Ȧ verificada na estrutura cristalográfica em conformação aberta. Isto indica que o baixo potencial catalítico desta enzima quando aplicada experimentalmente estaria relacionado à restrição de seu sítio catalítico devido à abertura insuficiente de sua lid. Por outro lado, a simulação de dinâmica molecular para a lipase CALB, revelou uma abertura de 14,3 Ȧ maior que o dobro da abertura verificada na sua estrutura em conformação aberta (6Ȧ), o que em princípio pode ser atribuído a presença do solvente e está em concordância com as observações experimentais.
The attempt to find new sources of energy and/or the valorization of the existent (mainly those of “clean” sources) is an issue widely discussed at the scientific and industrial sectors, with emphasis to the catalytic processes involved in biomass conversion to biofuels and/or others products of industrial interest, motivated this work. The main goal of this work was the synthesis of heterogeneous biocatalysts by immobilization of several lipases onto nano sized faujasite zeolite (FAU) after transition metal Ni2+ ion exchange. These biocatalysts were used in the microalgae oil transesterification to fatty acid ethyl esters (FAEEs). At first, the nano sized zeolite FAU was synthetized (NaX-Na+), then the product was ion exchanged with nickel sulfate solution (0,5M) incubated under 80°C for 3 days (3D), yielding the denominated NaX-Ni2+(0,5M-3D) support. The obtained materials had their crystallographic structures confirmed by X-Ray Diffraction. After the ion exchange experiments, the materials were used to the fungal lipases from T. lanuginosus (TLL), R. miehei (RML), C. rugosa (CRL) and C. antarctica B (CALB) immobilization, and the complexes denominated NaX-Ni2+(0,5M-3D)-TLL,Ni2+(0,5M-3D)-RML, Ni2+(0,5M-3D)-CRL and Ni2+(0,5M-3D)-CALB respectively. The complexes’ activities were measured by the emulsified olive oil hydrolysis, its activities values being 850, 550, 425 e 50 U/g respectively. Aiming the FAEEs yielding optimization, the complexes were applied in the microalgae oil transesterification under several experimental conditions. Using 5% w/w (weight of catalyst/weight of oil) of the TLL’s complex 85% of FAEEs yield was achieved(12h/35°C) after one step ethanol addition in 1:4 molar ratio (oil:alcohol), while the RML’s complex yielded 52% (12h/45°C) after step-wise alcohol addition (every each 2 hours, total of 4 steps), and the assays conditions for these two complexes established as the reaction optimum conditions. The CRL and CALB complexes have shown low yield when applied in transesterification reactions. Experimentally it was observed for the RML’s complex a significantly thermal stability in the temperature range 25-80°C. The complexes NaX-Ni2+(0,5M-3D)-TLL and Ni2+(0,5M-3D)-CALB were also studied for the free fatty acids esterification. Esterification reactions (12h/35°C) using 5 % w/w of complex at molar ratio 1:2 (acid:ethanol) 89 and 83% FAEEs yields were achieved for the complexes NaX-Ni2+(0,5M-3D)-TLL and Ni2+(0,5M-3D)-CALB, respectively. Both complexes were also applied to the Macaw Palm oil, containing high free fatty acids percentage (56%) and acid value of 112 mg KOH/g, by simultaneous transesterification/esterification reactions. Using 5% w/w of complex in each assay, 67 and 52% of FAEEs were yielded (12h/45°C), for single step ethanol volume addition in molar rate 1:6 (oil:alcohol). Despite Ni2+(0,5M-3D)-CALB complex no efficiency in the microalgae oil transesterification, it has shown great results in the oleic acid esterification, and probably esterified the free fatty acids present in the Macaw Palm oil, being the enzyme then qualified as specific to the esterification reaction in this system. Aiming to understand the low yields obtained for the complexes with CRL and CALB in the transesterification reactions when compared with the TLL and RML ones, theoretical-computational studies were run using normal modes analysis and molecular dynamics simulations using methanol as solvent. For the catalytic site openness measurement the distance between carbons atoms present in the amino acids residues Pro-74 (belonging to the lid) and Pro-443 for CRL, and Ile-144 (belonging to the lid) and Leu-285 for CALB were monitored in the simulations and compared with the distances found in the enzymes crystallographic opened and closed structures available at the Protein Data Bank (PDB). After normal modes analysis, the maximum distance between Pro-74 and Pro-443 was only 0,1 Ȧ higher than this distance found in the CRL closed conformation (3,8 Ȧ). Later, after molecular dynamics with methanol, a 6,5 Ȧ maximum distance was verified, but even this distance being higher than the one found in the closed conformation, it is a lot lower than the one found in the opened structure (21,6 Ȧ), what makes possible to attribute the low catalytic potential of this enzyme when experimentally applied to its catalytic site restriction due insufficient lid opening. In the other hands, the molecular dynamics simulations to the CALB lipase exhibited a bigger openness more than two folds (14,3 Ȧ) the openness found in the opened lipase structure (6Ȧ), which can in principle be assigned to the solvent presence, and agrees with experimental data.
The attempt to find new sources of energy and/or the valorization of the existent (mainly those of “clean” sources) is an issue widely discussed at the scientific and industrial sectors, with emphasis to the catalytic processes involved in biomass conversion to biofuels and/or others products of industrial interest, motivated this work. The main goal of this work was the synthesis of heterogeneous biocatalysts by immobilization of several lipases onto nano sized faujasite zeolite (FAU) after transition metal Ni2+ ion exchange. These biocatalysts were used in the microalgae oil transesterification to fatty acid ethyl esters (FAEEs). At first, the nano sized zeolite FAU was synthetized (NaX-Na+), then the product was ion exchanged with nickel sulfate solution (0,5M) incubated under 80°C for 3 days (3D), yielding the denominated NaX-Ni2+(0,5M-3D) support. The obtained materials had their crystallographic structures confirmed by X-Ray Diffraction. After the ion exchange experiments, the materials were used to the fungal lipases from T. lanuginosus (TLL), R. miehei (RML), C. rugosa (CRL) and C. antarctica B (CALB) immobilization, and the complexes denominated NaX-Ni2+(0,5M-3D)-TLL,Ni2+(0,5M-3D)-RML, Ni2+(0,5M-3D)-CRL and Ni2+(0,5M-3D)-CALB respectively. The complexes’ activities were measured by the emulsified olive oil hydrolysis, its activities values being 850, 550, 425 e 50 U/g respectively. Aiming the FAEEs yielding optimization, the complexes were applied in the microalgae oil transesterification under several experimental conditions. Using 5% w/w (weight of catalyst/weight of oil) of the TLL’s complex 85% of FAEEs yield was achieved(12h/35°C) after one step ethanol addition in 1:4 molar ratio (oil:alcohol), while the RML’s complex yielded 52% (12h/45°C) after step-wise alcohol addition (every each 2 hours, total of 4 steps), and the assays conditions for these two complexes established as the reaction optimum conditions. The CRL and CALB complexes have shown low yield when applied in transesterification reactions. Experimentally it was observed for the RML’s complex a significantly thermal stability in the temperature range 25-80°C. The complexes NaX-Ni2+(0,5M-3D)-TLL and Ni2+(0,5M-3D)-CALB were also studied for the free fatty acids esterification. Esterification reactions (12h/35°C) using 5 % w/w of complex at molar ratio 1:2 (acid:ethanol) 89 and 83% FAEEs yields were achieved for the complexes NaX-Ni2+(0,5M-3D)-TLL and Ni2+(0,5M-3D)-CALB, respectively. Both complexes were also applied to the Macaw Palm oil, containing high free fatty acids percentage (56%) and acid value of 112 mg KOH/g, by simultaneous transesterification/esterification reactions. Using 5% w/w of complex in each assay, 67 and 52% of FAEEs were yielded (12h/45°C), for single step ethanol volume addition in molar rate 1:6 (oil:alcohol). Despite Ni2+(0,5M-3D)-CALB complex no efficiency in the microalgae oil transesterification, it has shown great results in the oleic acid esterification, and probably esterified the free fatty acids present in the Macaw Palm oil, being the enzyme then qualified as specific to the esterification reaction in this system. Aiming to understand the low yields obtained for the complexes with CRL and CALB in the transesterification reactions when compared with the TLL and RML ones, theoretical-computational studies were run using normal modes analysis and molecular dynamics simulations using methanol as solvent. For the catalytic site openness measurement the distance between carbons atoms present in the amino acids residues Pro-74 (belonging to the lid) and Pro-443 for CRL, and Ile-144 (belonging to the lid) and Leu-285 for CALB were monitored in the simulations and compared with the distances found in the enzymes crystallographic opened and closed structures available at the Protein Data Bank (PDB). After normal modes analysis, the maximum distance between Pro-74 and Pro-443 was only 0,1 Ȧ higher than this distance found in the CRL closed conformation (3,8 Ȧ). Later, after molecular dynamics with methanol, a 6,5 Ȧ maximum distance was verified, but even this distance being higher than the one found in the closed conformation, it is a lot lower than the one found in the opened structure (21,6 Ȧ), what makes possible to attribute the low catalytic potential of this enzyme when experimentally applied to its catalytic site restriction due insufficient lid opening. In the other hands, the molecular dynamics simulations to the CALB lipase exhibited a bigger openness more than two folds (14,3 Ȧ) the openness found in the opened lipase structure (6Ȧ), which can in principle be assigned to the solvent presence, and agrees with experimental data.
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Palavras-chave
Nanozeólita, Lipase, Imobilização, Biocombustíveis, Bioinformática, Immobilization, Biofuels, Bioinformatics