Immobilization of enzymes on SBA-15 and ZIF-8 for biocatalytic applications

This PhD work deals with the immobilization of different enzymes, namely lysozyme, glucose oxidase, horseradish peroxidase, and lipase on either SBA-15 mesoporous silica or the metal organic framework ZIF-8. Enzyme immobilization was carried out through different methods, as outlined by the following three tasks: 1. Physical adsorption of lysozyme (LYZ) on mesoporous silica SBA-15 and amino functionalized SBA-15-NH2. 2. Covalent binding of glucose oxidase (GOx) and horseradish peroxidase (HRP) on SBA-15-NH2 to obtain a bienzymatic system. 3. Encapsulation of lipase AK from Pseudomonas fluorescens (AKL) and lipase RM from Rhizomucor miehei (RML) on ZIF-8 by means of encapsulation. Task 1. Specific buffer effects on the physical adsorption of LYZ on SBA-15 and SBA-15-NH2 The synthesis of mesoporous silica SBA-15 and the amino-functionalization were carried out according to standard protocols. LYZ from hen egg white was physically adsorbed onto both supports at a fixed pH (7.15), and the loading was quantified. Although it is known that pH affects the loading of the physisorbed enzyme, the effect of the type of the buffer used to fix pH is less investigated. To this purpose LYZ immobilization was carried out in four different buffers (TRIS [tris(hydroxymethyl) aminomethane], BES [N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid], phosphate and citrate) at the same nominal pH 7.15. A specific buffer effect on LYZ loading was observed on SBA-15, while this effect was negligible for SBA-15-NH2. The presence of strong electrolytes was found to significantly affect the loading according to the Hofmeister series. The effects of buffer and salts were also investigated towards electrophoretic mobility of the free LYZ, SBA-15 and SBA-15-NH2. Specific buffer effects and the synergistic action of salts were observed, suggesting the occurrence of Hofmeister phenomena. Task 2. Covalent binding of GOx and HRP on SBA-15-NH2 The covalent immobilization of GOx and HRP on amino-functionalized SBA-15 was investigated. These two enzymes catalyze a tandem cascade reaction where the oxidation of glucose to gluconolactone and the reduction of oxygen to hydrogen peroxide is carried out by GOx. Then, HRP uses the hydrogen peroxide to oxidize different kind of substrates, such as phenolic compounds. The “in situ” generation of H2O2, provided by GOx, is useful to avoid a possible irreversible inactivation of HRP. The silica support was synthesized, functionalized and characterized. The enzymes were anchored on SBA-15-NH2 using glutaraldehyde. The activities, were significantly influenced by the drying process. Storage stability and recycling tests were performed. In order to test the possible effectiveness of the biocatalysts on the degradation of phenolic pollutants, the oxidation of ferulic acid and caffeic acid was investigated. Task 3. Encapsulation of AKL and RML on ZIF-8. Lipases AKL and RML were encapsulated in the Zeolite Imidazolate Framework ZIF-8, in a “one pot” synthesis. Two Zinc:2-methyl imidazole molar ratios (Zn:L ratio1:4 vs 1:40) were compared. The structural and textural parameters were investigated by means of X-rays diffraction and N2 physisorption and the biocatalysts displayed different crystal structure and surface area. A sodalite-like crystal structure of ZIF-8 was obtained for the Zn:L1:40, while a mixed phase was likely obtained at Zn:L1:4. The activity of the immobilized lipases was tested by means of the p-nitrophenyl butyrate assay. The Zn:L1:4 biocatalysts displayed a higher specific activity than the Zn:L1:40. Thus, in order to improve the activity of the Zn:L1:40, a series of synthesis with a control of pH was carried out. The decrease of pH ligand solution led to the reduction of surface areas of the lipase@ZIF-8 biocatalysts. The most active biocatalysts were obtained at Zn:L1:4. The storage stability at 15 days showed a good retention of activity for both AK@ZIF-81:4 and RM@ZIF-81:4.