Development of enzymatic processes for carbon dioxide and organic waste valorization: co-immobilization of FDH and GlyDH over hierarchical zeolites

In this thesis, the enzymatic reduction of carbon dioxide (CO2) into formic acid, conducted by enzyme formate dehydrogenase (FDH), was investigated. In order to have a profitable implementation, some issues need to be solved. First of all, it is necessary to recover the enzyme at the end of the reaction, separating it from the products, with simple and cost-effective operations. Then, oxidoreductases need cofactors as electron donors, in this case NADH, which have to be regenerated to work in continuous. The solutions studied in this work are the employment of glycerol dehydrogenase (GlyDH) as cofactor restoring agent, and hierarchical zeolites as immobilization carriers. This enzyme has the additional benefit of promoting the valorization of another organic waste, which is glycerol. Hierarchization of zeolites is finalized at reproducing an optimal environment for the two enzymes, which are characterized by different dimensions and catalytic features. It was carried out over natural and synthetic zeolites, namely clinoptilolite and ZSM-5, by alkaline leaching. Later, the materials were completely characterized by several standard tests (nitrogen physisorption at 77 K, XRD, FE-SEM and HR-TEM), achieving enhanced surface area and well-distributed multimodal pore size, and besides maintaining their characteristic morphology and crystallinity. Multipoint covalent immobilization was conducted over the materials, functionalized with amino and glyoxyl groups. Single-enzyme immobilization was quantified by Bradford assay, giving an overall yield of 60-80%, while co-immobilization of the two enzymes was qualitatively confirmed by fluorescence labelling and microscopy imaging. Thermal stability of the enzymes was significantly improved by supporting them over hierarchical clinoptilolite (H-Clino), which also exhibited the best performances in terms of retained activity. In addition, immobilized enzymes showed widely optimal conditions with respect to free enzymes. In particular, it brought the optimal pH value of GlyDH (from 10 to 8) way closer to the one of FDH (7), boosting their synergistic action. All the activity test involved the spectrophotometric measurement of NADH production, at the wavelength of 340 nm, from the corresponding oxidation reactions: conversion of sodium formate into CO2 by FDH and conversion of glycerol into DHA by GlyDH. Fed-batch reduction of CO2 provided satisfying concentrations of formic acid, analysed by HPLC technique, for both the single reaction mediated by FDH (up to 53% of yield), and the chain-reaction of the two enzymes (21%). The first one was performed by feeding initially NADH and by bubbling CO2 in continuous, while for the second one, NADH was replaced by NAD+ and glycerol. However, since GlyDH was way slower than FDH, these two were employed in a 4:1 ratio. These results provide considerable insight into the effective employment of meso-macroporous hierarchical zeolites as enzyme carriers, in particular concerning good immobilization performances, and the improvement of both single and synergic activity.