In this thesis, three trimesic acid-based MOFs: Fe-BTC, Tb-BTC, Gd-BTC, an imidazolate-based MOF: ZIF- zni and on functionalised pure-silica hierarchical (microporous-macroporous) MFI zeolite (ZMFI) to screen which, among the chosen supports, is more suitable for Laccase (LC) immobilisation. Aspergillus sp. Laccase immobilisation within MOFs occurred in situ under mild conditions, e.g. aqueous solution, neutral pH, and at room temperature.The immobilisation of the laccase on MFI-Type Zeolite particles with embedded macropores was instead carried out post synthesis. All Biocatalysts were characterised through XRD, SEM, FTIR, N2 adsorption/desorption isotherms and TGA. The kinetic parameters (KM and Vmax) and the specific activity of the immobilised biocatalysts were determined. The effect of enzyme loading was studied for Fe-BTC and ZIF-zni supports. LC@FeBTC had an optimal loading of 45.2 mg g-1, at higher enzyme loadings the specific activity decreased. In contrast, the specific activity of LC@ZIF-zni increased linearly over the loading range investigated. LC@GdBTC showed the highest specific activity compared with other carriers investigated. Nevertheless, a drastic decrease in specific activity was found due to enzyme immobilisation. Laccase from Aspergillus sp. (LC) was immobilised on functionalised pure-silica hierarchical (microporous-macroporous) MFI zeolite (ZMFI). The optimal pH, kinetic parameters (KM and Vmax), specific activity, as well as both storage and operational stability of LC@ZMFI were determined. The dependence of specific activity on the pH for free and immobilised LC was investigated in the pH range of 2 to 7. Immobilisation of laccase on hierarchical pure-silica MFI zeolite allows to carry out the reaction under acidic pH values without affecting the support structure. Although enzymatic immobilisation is gaining an increasing attention, especially regarding of enzyme encapsulated within MOFs, the enzymatic location and its change in conformation after immobilisation within MOFs are still poorly investigated. Commercial enzymes often show a very low grade of purity, and their enzymatic structure is still unknown, these aspects make tricky the investigation of their location and conformation. In this regards Bovine serum albumin (BSA) with high purity (> 98%?) with a known secondary and tertiary structure was used as a model protein. BSA was immobilised within two different zeolitic imidazolate frameworks (ZIF-zni and ZIF-8) through a one-pot synthesis carried out under mild conditions (room temperature and aqueous solution). The ZIF-zni, ZIF-8 and the BSA@ZIF-zni, BSA@ZIF-8 samples were characterised by X-ray diffraction, scanning electron microscopy, confocal laser scanning microscopy, thermogravimetric analysis, micro-FTIR and confocal Raman spectroscopy to characterize the MOF structures and evaluate the protein location in the materials. Moreover, the secondary structure and conformation changes of BSA due to its immobilisation. on both ZIF-zni and ZIF-8 were studied. Results showed that BSA seems to concentrate in domains of 5-40 μm, which form an extended network across the MOF. Additional information on changes in the BSA structure upon immobilisation was extracted by the deconvolution of the the amide I band in the reflectance spectra. Data showed that the crystalline content of BSA increases significantly when the protein is immobilised on the MOFs in BSA@ZIF-zni and ZIF-8. Before interaction with the MOF, amide I deconvolution indicates that BSA has a strong content in β -turns (~89%), with limited contribution from β-sheets (~4%). Whilst the crystalline content of BSA increases significantly when the protein is immobilised on both ZIF-zni and ZIF -8 resulting in increased up to ~25% (β -sheets + α-helices), and ~40 % (β -sheets + α-helices) respectively with a consequent drastic reduction of β -turns.

The importance of Laccase immobilisation in Biocatalysis: towards biomass pretreatment / Tocco, Davide. - (2022 Dec 01).

The importance of Laccase immobilisation in Biocatalysis: towards biomass pretreatment

TOCCO, Davide
2022-12-01

Abstract

In this thesis, three trimesic acid-based MOFs: Fe-BTC, Tb-BTC, Gd-BTC, an imidazolate-based MOF: ZIF- zni and on functionalised pure-silica hierarchical (microporous-macroporous) MFI zeolite (ZMFI) to screen which, among the chosen supports, is more suitable for Laccase (LC) immobilisation. Aspergillus sp. Laccase immobilisation within MOFs occurred in situ under mild conditions, e.g. aqueous solution, neutral pH, and at room temperature.The immobilisation of the laccase on MFI-Type Zeolite particles with embedded macropores was instead carried out post synthesis. All Biocatalysts were characterised through XRD, SEM, FTIR, N2 adsorption/desorption isotherms and TGA. The kinetic parameters (KM and Vmax) and the specific activity of the immobilised biocatalysts were determined. The effect of enzyme loading was studied for Fe-BTC and ZIF-zni supports. LC@FeBTC had an optimal loading of 45.2 mg g-1, at higher enzyme loadings the specific activity decreased. In contrast, the specific activity of LC@ZIF-zni increased linearly over the loading range investigated. LC@GdBTC showed the highest specific activity compared with other carriers investigated. Nevertheless, a drastic decrease in specific activity was found due to enzyme immobilisation. Laccase from Aspergillus sp. (LC) was immobilised on functionalised pure-silica hierarchical (microporous-macroporous) MFI zeolite (ZMFI). The optimal pH, kinetic parameters (KM and Vmax), specific activity, as well as both storage and operational stability of LC@ZMFI were determined. The dependence of specific activity on the pH for free and immobilised LC was investigated in the pH range of 2 to 7. Immobilisation of laccase on hierarchical pure-silica MFI zeolite allows to carry out the reaction under acidic pH values without affecting the support structure. Although enzymatic immobilisation is gaining an increasing attention, especially regarding of enzyme encapsulated within MOFs, the enzymatic location and its change in conformation after immobilisation within MOFs are still poorly investigated. Commercial enzymes often show a very low grade of purity, and their enzymatic structure is still unknown, these aspects make tricky the investigation of their location and conformation. In this regards Bovine serum albumin (BSA) with high purity (> 98%?) with a known secondary and tertiary structure was used as a model protein. BSA was immobilised within two different zeolitic imidazolate frameworks (ZIF-zni and ZIF-8) through a one-pot synthesis carried out under mild conditions (room temperature and aqueous solution). The ZIF-zni, ZIF-8 and the BSA@ZIF-zni, BSA@ZIF-8 samples were characterised by X-ray diffraction, scanning electron microscopy, confocal laser scanning microscopy, thermogravimetric analysis, micro-FTIR and confocal Raman spectroscopy to characterize the MOF structures and evaluate the protein location in the materials. Moreover, the secondary structure and conformation changes of BSA due to its immobilisation. on both ZIF-zni and ZIF-8 were studied. Results showed that BSA seems to concentrate in domains of 5-40 μm, which form an extended network across the MOF. Additional information on changes in the BSA structure upon immobilisation was extracted by the deconvolution of the the amide I band in the reflectance spectra. Data showed that the crystalline content of BSA increases significantly when the protein is immobilised on the MOFs in BSA@ZIF-zni and ZIF-8. Before interaction with the MOF, amide I deconvolution indicates that BSA has a strong content in β -turns (~89%), with limited contribution from β-sheets (~4%). Whilst the crystalline content of BSA increases significantly when the protein is immobilised on both ZIF-zni and ZIF -8 resulting in increased up to ~25% (β -sheets + α-helices), and ~40 % (β -sheets + α-helices) respectively with a consequent drastic reduction of β -turns.
Laccase; Zeolite; Metal Frameworks; Enzyme; Biocatalysis
Biocatalysis
The importance of Laccase immobilisation in Biocatalysis: towards biomass pretreatment / Tocco, Davide. - (2022 Dec 01).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11388/299947
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