Nanozeólitas como suporte para imobilização de lacases: aplicação dos biocatalisadores na reação de oxidação mediada do glicerol
Carregando...
Data
2020-09-24
Autores
Miller, Alex Henrique
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
A oxidação do glicerol tem sido objeto de diversos estudos no mundo todo; em geral, o principal objetivo destes trabalhos é o encontro de um catalisador apropriado para converter, seletivamente, esta molécula bloco em produtos de alto valor agregado. Lacases imobilizadas em suportes sólidos são catalisadores heterogêneos não prejudiciais ao meio ambiente, que podem ser aplicados para a oxidação do glicerol num sistema lacase-mediador. Estas enzimas contêm átomos de cobre em sua estrutura e catalisam a oxidação de muitos compostos fenólicos com a concomitante redução de água a oxigênio molecular. Apesar da sua especificidade, quando apropriadamente combinada com mediadores, as lacases podem também agir na oxidação de compostos não fenólicos. Este trabalho abordou a síntese e caracterização de zeólitas em nano escala – morfologias FAU, BEA, LTA e MFI –, a aplicação destes materiais como suporte para a imobilização de diferentes lacases comerciais – dos organismos A. bisporus, Aspergillus sp. e P. ostreatus –, e o uso dos complexos obtidos para oxidação do glicerol mediada pelo composto N-oxil-2,2,6,6-tetrametilpiperidina (TEMPO). Todas as zeólitas foram sintetizadas de acordo com a literatura, e a forma sódica da zeólita FAU modificadas por troca iônica com Cu2+ para geração de um suporte extra. Os materiais sintetizados foram amino-funcionalizados para permitir a imobilização das lacases covalentemente. Os suportes foram caracterizados por XRD, SEM-EDX, HRTEM, FTIR, e as enzimas livres ou complexos espectroscopicamente avaliados através da oxidação do composto 2,2´-azino-bis(ácido 3-etilbenzotiazolina-6-sulfônico) (ABTS). Com base nas atividades de oxidação do composto ABTS, os complexos FAU/Cu2+/APTMS/GA/LPO, FAU/Cu2+/APTMS/ GA/LAB, FAU/Cu2+/APTMS/GA/LAsp, e BEAc/APTMS/GA/LAsp foram selecionados para aplicação nas reações de oxidação do glicerol. Para comparação, as enzimas LPO, LAB e LAsp na forma livre e os suportes FAU/Cu2+/APTMS/GA e BEAc/APTMS/GA foram aplicadas em condições reacionais similares. Todas as reações utilizando os complexos lacase/nanozeólitas apresentaram, após 48 h de reação, baixa conversão do glicerol – inferiores a 5%. No entanto, uma alta seletividade foi observada para gliceraldeído, acima de 88 % em todos os casos. Não houve conversão do glicerol utilizando apensas os suportes sem enzima. Por outro lado, quando as enzimas LPO e LAsp foram aplicadas nas suas formas livres, as conversões do glicerol obtidas foram significativamente superiores em comparação aos complexos – ~29% para a LAsp e mais de 80% para a LPO após 48 h de reação. Seletividades de 57,10% a gliceraldeído e 25,75% a ácido glicérico, foram quantificadas das reações com a enzimas livre LPO, enquanto da reação com LAsp, a seletividade a gliceraldeído foi de 78%, e o segundo produto mais obtido foi ácido glioxílico, 11,47%. A lacase LAB na forma livre apresentou conversão muito menor quando comparada as demais – ~3,2% após 48 h. Visando compreender quais razões levaram a redução da atividade enzimática após imobilização nos materiais nanozeolíticos, um estudo sistemático utilizando voltametria cíclica foi empregado para a LAsp. Este estudo apontou que o potencial de redução desta enzima é um fator limitante na sua aplicação, e que em meio ácido, a taxa de oxidação do mediador TEMPO é bastante reduzida. Com o uso de ressonância paramagnética eletrônica (EPR), foi possível inferir que a imobilização enzimática causou distorções estruturais na LAsp, em específico, nos centros de cobre do seu sítio catalítico, pois variações significativas nos Hamiltonianos de spin (tensores g, e constantes de acoplamento hiperfino A) foram observadas quando comparadas enzimas em solução ou imobilizadas. Os espectros revelaram também uma alta dependência da acidez do meio na interação dos sítios de cobre com os ligantes de coordenação. Além disso, os Hamiltonianos de spin paralelos do cobre T2 da enzima imobilizada em zeólita BEAc/APTMS/GA (g|| = 2,275, A|| = 173,4x10-4cm-1) são mais próximos da enzima livre em pH 7 (g|| = 2,275, A|| = 170,1x10-4 cm-1) do que em pH 4 (g|| = 2,275, A|| = 220,2x10-4 cm-1). Isso indica que o pH dos microambientes enzimáticos após imobilização é neutro, e isso é a provável razão pela redução na atividade enzimática após imobilização nos suportes zeolíticos.
The oxidation of glycerol has been the subject of several studies worldwide; in general, these studies main goal is to find an appropriate catalyst to selectively convert this building-block molecule into value-added products. Laccases immobilized on solid supports are environment-friendly heterogeneous catalysts, which can be applied for the oxidation of glycerol in a laccase-mediated system. These enzymes contain copper atoms in their structure and catalyze the oxidation of many phenolic compounds with the concomitant reduction of water to molecular oxygen. Despite their specificity, when properly combined with mediators, laccases can also act on the oxidation of non-phenolic compounds. This study addressed the synthesis and characterization of nanoscale zeolites – FAU, BEA, LTA and MFI morphologies – , the application of these materials as support for the immobilization of different commercial laccases – from A. bisporus, Aspergillus sp. and P. ostreatus –, and the use of the complexes obtained for 2,2,6,6-tetramethylpiperidine-N-oxyl(TEMPO) mediated glycerol oxidation. All zeolites were synthesized according to the literature, and the sodium form of the FAU zeolite modified by ion exchange with Cu2+ to generate an extra support. The synthesized materials were amino-functionalized to allow the laccases to be immobilized covalently. The supports were characterized by XRD, SEM-EDX, HRTEM, FTIR, and the free enzymes or complexes spectroscopically evaluated through the oxidation of the compound 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS). Based on the ABTS oxidation activities, the complexes FAU/Cu2+/APTMS/GA/LPO, FAU/Cu2+/APTMS/GA/LAB, FAU/Cu2+/APTMS/GA/ LAsp, and BEAc/APTMS/GA/LAsp were selected to be applied in the glycerol oxidation reactions. For comparison, the enzymes LPO, LAB and LAsp in free form and the supports FAU/Cu2+/APTMS/GA and BEAc/APTMS/GA were applied under similar reaction conditions. All reactions using the laccases/nanozeolites complexes showed, after 48 h of reaction, low glycerol conversion – less than 5%. However, a high selectivity was observed for glyceraldehyde, above 88% in all cases. There was no conversion of glycerol using only supports without enzyme. On the other hand, when the enzymes LPO and LAsp were applied in their free forms, the glycerol conversions obtained were significantly superior in comparison to the complexes – ~29% for LAsp and more than 80% for LPO, after 48 h of reaction. Selectivities of 57.10% to glyceraldehyde and 25.75% to glyceric acid, were quantified from the reactions with the free LPO enzymes, while from the reaction with free LAsp, the selectivity to glyceraldehyde was 78%, and the second most yielded product was glyoxylic acid, 11.47%. The LAB laccase in the free form showed a much lower conversion when compared to the others – ~3.2% after 48 h. In order to understand what reasons led to the reduction of enzyme activity after immobilization on nanozeolitic materials, a systematic study using cyclic voltammetry was employed for LAsp. This study pointed out that the redox potential of this enzyme is a limiting factor in its application, and that in an acid environment, the oxidation rate of the TEMPO mediator is quite low. With the use of electronic paramagnetic resonance (EPR), it was possible to infer that the enzymatic immobilization caused structural distortions in the LAsp, in particular, in the copper centers of its catalytic site, since significant variations in the spin Hamiltonians (tensors g, and hyperfine coupling constants A) were observed when enzymes in solution or immobilized were compared. The spectra also revealed a high pH dependence on the interaction of the copper sites with the coordination ligands. In addition, the parallel spin Hamiltonians of copper T2 of the enzyme immobilized in zeolite BEAc/APTMS/GA (g|| = 2.275, A|| = 173.4x10-4cm-1) are closer to the free enzyme at pH 7 (g|| = 2.275, A|| = 170.1x10-4cm-1) than at pH 4 (g|| = 2.275, A|| = 220.2x10-4cm-1). This indicates that the pH of the enzymatic microenvironments after immobilization is neutral, and this is likely to be the reason for the reduction in enzymatic activity after immobilization on the zeolitic supports.
The oxidation of glycerol has been the subject of several studies worldwide; in general, these studies main goal is to find an appropriate catalyst to selectively convert this building-block molecule into value-added products. Laccases immobilized on solid supports are environment-friendly heterogeneous catalysts, which can be applied for the oxidation of glycerol in a laccase-mediated system. These enzymes contain copper atoms in their structure and catalyze the oxidation of many phenolic compounds with the concomitant reduction of water to molecular oxygen. Despite their specificity, when properly combined with mediators, laccases can also act on the oxidation of non-phenolic compounds. This study addressed the synthesis and characterization of nanoscale zeolites – FAU, BEA, LTA and MFI morphologies – , the application of these materials as support for the immobilization of different commercial laccases – from A. bisporus, Aspergillus sp. and P. ostreatus –, and the use of the complexes obtained for 2,2,6,6-tetramethylpiperidine-N-oxyl(TEMPO) mediated glycerol oxidation. All zeolites were synthesized according to the literature, and the sodium form of the FAU zeolite modified by ion exchange with Cu2+ to generate an extra support. The synthesized materials were amino-functionalized to allow the laccases to be immobilized covalently. The supports were characterized by XRD, SEM-EDX, HRTEM, FTIR, and the free enzymes or complexes spectroscopically evaluated through the oxidation of the compound 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS). Based on the ABTS oxidation activities, the complexes FAU/Cu2+/APTMS/GA/LPO, FAU/Cu2+/APTMS/GA/LAB, FAU/Cu2+/APTMS/GA/ LAsp, and BEAc/APTMS/GA/LAsp were selected to be applied in the glycerol oxidation reactions. For comparison, the enzymes LPO, LAB and LAsp in free form and the supports FAU/Cu2+/APTMS/GA and BEAc/APTMS/GA were applied under similar reaction conditions. All reactions using the laccases/nanozeolites complexes showed, after 48 h of reaction, low glycerol conversion – less than 5%. However, a high selectivity was observed for glyceraldehyde, above 88% in all cases. There was no conversion of glycerol using only supports without enzyme. On the other hand, when the enzymes LPO and LAsp were applied in their free forms, the glycerol conversions obtained were significantly superior in comparison to the complexes – ~29% for LAsp and more than 80% for LPO, after 48 h of reaction. Selectivities of 57.10% to glyceraldehyde and 25.75% to glyceric acid, were quantified from the reactions with the free LPO enzymes, while from the reaction with free LAsp, the selectivity to glyceraldehyde was 78%, and the second most yielded product was glyoxylic acid, 11.47%. The LAB laccase in the free form showed a much lower conversion when compared to the others – ~3.2% after 48 h. In order to understand what reasons led to the reduction of enzyme activity after immobilization on nanozeolitic materials, a systematic study using cyclic voltammetry was employed for LAsp. This study pointed out that the redox potential of this enzyme is a limiting factor in its application, and that in an acid environment, the oxidation rate of the TEMPO mediator is quite low. With the use of electronic paramagnetic resonance (EPR), it was possible to infer that the enzymatic immobilization caused structural distortions in the LAsp, in particular, in the copper centers of its catalytic site, since significant variations in the spin Hamiltonians (tensors g, and hyperfine coupling constants A) were observed when enzymes in solution or immobilized were compared. The spectra also revealed a high pH dependence on the interaction of the copper sites with the coordination ligands. In addition, the parallel spin Hamiltonians of copper T2 of the enzyme immobilized in zeolite BEAc/APTMS/GA (g|| = 2.275, A|| = 173.4x10-4cm-1) are closer to the free enzyme at pH 7 (g|| = 2.275, A|| = 170.1x10-4cm-1) than at pH 4 (g|| = 2.275, A|| = 220.2x10-4cm-1). This indicates that the pH of the enzymatic microenvironments after immobilization is neutral, and this is likely to be the reason for the reduction in enzymatic activity after immobilization on the zeolitic supports.
Descrição
Palavras-chave
Glicerol, Lacase, Nanozeólita, Imobilização, Oxidação, Voltametria cíclica, EPR, Glycerol, Lacase, Nanozeolite, Immobilization, Oxidation, Cyclic voltammetry