Enzyme-catalyzed cascade reactions with (nano)-biocatalytic systems
Φόρτωση...
Ημερομηνία
Συγγραφείς
Giannakopoulou, Archontoula
Τίτλος Εφημερίδας
Περιοδικό ISSN
Τίτλος τόμου
Εκδότης
Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Βιολογικών Εφαρμογών και Τεχνολογιών
Περίληψη
Τύπος
Είδος δημοσίευσης σε συνέδριο
Είδος περιοδικού
Είδος εκπαιδευτικού υλικού
Όνομα συνεδρίου
Όνομα περιοδικού
Όνομα βιβλίου
Σειρά βιβλίου
Έκδοση βιβλίου
Συμπληρωματικός/δευτερεύων τίτλος
Περιγραφή
The present PhD thesis focuses on the design, development, and application of innovative multienzymatic biocatalytic systems in cascade reactions of biotechnological interest. These systems consist of more than one enzyme originating either from different organisms or from different classes of enzymes (e.g. hydrolases, oxodoreductases, etc.) and can be either free or co-immobilized on nanocarriers bearing characteristics of particular interest. This thesis examines the different approaches for the design and development of multienzymatic systems and investigates their potential in different biocatalytic applications. Specifically, the thesis presents the scientific studies obtained and are related to the development and optimization of multienzymatic systems for the hydrolysis of cellulose, the bioconversion of oleuropein to hydroxytyrosol and the enzymatic modification of various polymers. The characterization of the above systems in terms of their structure, activity and functional stability was based on a combined approach of various techniques, such as Ultraviolet-visible spectroscopy, Fluorescence Spectroscopy and Circular Dichroism among others. Overall, this thesis offers new insights and innovative approaches for the design of multienzymatic (nano)assemblies to improve the performance of various biocatalytic processes and to develop innovative biomaterials with improved properties. In the context of this PhD thesis, various methods for the design of multienzymatic systems were approached, such as the combined use of inorganic and organic support materials, the combined use of enzymes deriving from different classes, as well as the combined use of different reaction conditions to achieve the highest yields.
The first research topic of this thesis focuses on the preparation, characterization and application of a novel four-enzyme magnetic nanobiocatalyst developed through the simultaneous covalent co-immobilization of cellulase (CelDZ1), β-glucosidase (bgl), glucose oxidase (GOx) and horseradish peroxidase (HRP) on the surface of magnetic nanoparticles (MNPs) modified with (3-aminopropyl)triethoxysilane (APTES). The prepared nanobiocatalyst was characterized by various spectroscopic techniques. The co-immobilization procedure yielded the maximum immobilization efficiency (CelDZ1: 42%, bgl: 66%, GOx: 94% and HRP: 78%) at a concentration of glutaraldehyde (chemical reagent that acts as linker between enzyme and material) 10% v/v, after 2 h incubation and at a 1:1 ratio of total enzyme mass to nanomaterial. Co-immobilization led to an increase in the apparent affinity constant Km and a concomitant decrease in the maximum velocity Vmax of the co-immobilized enzymes. Studies on the thermal stability of the co-immobilized enzymes revealed an up to a 2-fold increase in the half-life constant and up to a 1.5-fold increase in the deactivation energy compared to the free enzymes. The enhancement of the thermodynamic parameters of the four enzymes co-immobilized on MNPs also indicates an increase in their thermal stability. Furthermore, the co-immobilized enzymes retained a significant percentage of their catalytic activity (up to 50%) after 5 reaction cycles at 50 °C and even after 24 days of incubation at 5 °C. Finally, the nanobiocatalyst was successfully applied to a four-step cascade reaction involving the hydrolysis of cellulose.
An innovative multi-enzyme nanobiocatalytic system was then developed consisting of the enzymes lipase A from Candida antarctica (CalA) and β-glucosidase from Thermotoga maritima (bgl) which were covalently co-immobilized on the surface of magnetic nanoparticles coated with chitosan (CS-MNPs). Several parameters affecting the co-immobilization process (glutaraldehyde concentration, incubation time, CS-MNPs to enzyme mass ratio and bgl to CalA mass ratio) were evaluated and optimized. The developed nanobiocatalyst was characterized by various spectroscopic techniques. Biochemical parameters such as the apparent kinetic constants and the thermal stability of both free and co-immobilized enzymes were also evaluated. The co-immobilized enzymes exhibited an increase in the apparent kinetic constant Km followed by a decrease in the Vmax value compared to the free enzymes, while a significant increase (>5-fold) in the thermal stability of immobilized CalA, both in individually immobilized and co-immobilized form, was observed after 24 h incubation at 60 °C. Finally, the nanobiocatalyst was effectively applied in the bioconversion of oleuropein to hydroxytyrosol, one of the most potent naturally occurring antioxidants, and could be recycled for up to 10 reaction cycles (240 hours of steady operation) at 60 °C, retaining more than 50% of of its initial activity.
In the third study of this thesis a series of polymers, including chitosan (CS), carboxymethylcellulose (CMC) and a chitosan-gelatin hybrid polymer (CS-GEL), were functionalized with ferulic acid (FA) obtained from the enzymatic treatment of arabinoxylan through the synergistic action of two enzymes, xylanase and feruloyl esterase. Subsequently, ferulic acid served as substrate for laccase from Agaricus bisporus (AbL) in order to enzymatically modify the aforementioned polymers. The successful incorporation of the ferulic acid oxidation products onto the various polymers was confirmed through various techniques such as ultraviolet-visible (UV-Vis) spectroscopy and attenuated total reflection (ATR) spectroscopy. In addition, enhancement of the antioxidant properties of the modified polymers was observed with two protocols. Finally, the modified polymers exhibited strong antimicrobial activity against bacterial populations of Escherichia coli strain BL21DE3, suggesting their potential application in the pharmaceutical, cosmetology and food industries.
The results of this thesis constitute an important contribution to the research field of biocatalysis, as they demonstrate the importance of developing innovative multi-enzymatic biocatalytic systems for a multitude of applications. The utilization of agro-industrial by-products and the production of biofuels are just some of the applications that can be achieved using such systems. The results of this thesis also demonstrate the possibility of developing innovative biological systems in vitro with improved characteristics, a necessary condition for the competitiveness of industry in the modern world. Finally, this work highlights the importance of nanotechnology in biocatalytic research, paving the way for further future research in this specific research field.
Περιγραφή
Λέξεις-κλειδιά
Cascade reactions, Enzyme co-immobilization, Nanomaterials, Cellulose hydrolysis, Bioconversion of oleuropein, Enzymatic modification of biopolymers, Ενζυματική τροποποίηση βιοπολυμερών, Eνζυμική συν-ακινητοποίηση, Νανοϋλικά, Υδρόλυση κυτταρίνης, Βιομετατροπή ελευρωπαΐνης
Θεματική κατηγορία
Biotechnology, Nanobiotechnology, Βιοτεχνολογία, Νανοβιοτεχνολογία
Παραπομπή
Σύνδεσμος
Γλώσσα
en
Εκδίδον τμήμα/τομέας
Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Βιολογικών Εφαρμογών και Τεχνολογιών
Όνομα επιβλέποντος
Trangas, Theoni
Εξεταστική επιτροπή
Kara, Selin
Avalos, José L.
Avgeropoulos, Apostolos
Albanis, Triantafyllos
Politou, Anastasia
Friligos, Efstathios
Avalos, José L.
Avgeropoulos, Apostolos
Albanis, Triantafyllos
Politou, Anastasia
Friligos, Efstathios
Γενική Περιγραφή / Σχόλια
Ίδρυμα και Σχολή/Τμήμα του υποβάλλοντος
Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Tμήμα Βιολογικών Εφαρμογών και Τεχνολογιών
Πίνακας περιεχομένων
Χορηγός
Βιβλιογραφική αναφορά
Ονόματα συντελεστών
Αριθμός σελίδων
375 σ.
Λεπτομέρειες μαθήματος
Συλλογές
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Άδεια Creative Commons
Άδεια χρήσης της εγγραφής: Attribution-NonCommercial-NoDerivs 3.0 United States