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Nanozeolites as support for laccases immobilization: application of the biocatalysts to the mediated glycerol oxidation reaction

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Alex Henrique Miller
Total Authors: 1
Document type: Doctoral Thesis
Press: São José do Rio Preto. 2020-10-23.
Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências Letras e Ciências Exatas. São José do Rio Preto
Defense date:
Advisor: José Geraldo Nery

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. (AU)

FAPESP's process: 16/24303-0 - Synthesis and characterization of new biocatalysts formed by nanozeolite and laccase enzymes complexes: their applications in oxidation reactions of glycerol to tartronic acid and mesoxalic acid compounds
Grantee:Alex Henrique Miller
Support Opportunities: Scholarships in Brazil - Doctorate