Laccase-Functionalized Graphene Oxide Assemblies as Efficient Nanobiocatalysts for Oxidation Reactions

Multi-layer graphene oxide-enzyme nanoassemblies were prepared through the multi-point covalent immobilization of laccase from Trametes versicolor (TvL) on functionalized graphene oxide (fGO). The catalytic properties of the fGO-TvL nanoassemblies were found to depend on the number of the graphene oxide-enzyme layers present in the nanostructure. The fGO-TvL nanoassemblies exhibit an enhanced thermal stability at 60 degrees C, as demonstrated by a 4.7-fold higher activity as compared to the free enzyme. The multi-layer graphene oxide-enzyme nanoassemblies can efficiently catalyze the oxidation of anthracene, as well as the decolorization of an industrial dye, pinacyanol chloride. These materials retained almost completely their decolorization activity after five reaction cycles, proving their potential as efficient nano- biocatalysts for various applications

Hybrid Nanomaterials of Magnetic Iron Nanoparticles and Graphene Oxide as Matrices for the Immobilization of beta-Glucosidase:Synthesis, Characterization, and Biocatalytic Properties

Hybrid nanostructures of magnetic iron nanoparticles and graphene oxide were synthesized and used as nanosupports for the covalent immobilization of β-glucosidase. This study revealed that the immobilization efficiency depends on the structure and the surface chemistry of nanostructures employed. The hybrid nanostructure-based biocatalysts formed exhibited a two to four-fold higher thermostability as compared to the free enzyme, as well as an enhanced performance at higher temperatures (up to 70°C) and in a wider pH range. Moreover, these biocatalysts retained a significant part of their bioactivity (up to 40%) after 12 repeated reaction cycles

Development of a Multi-Enzymatic Biocatalytic System through Immobilization on High Quality Few-Layer bio-Graphene

In this work, we report the green production of few-layer bio-Graphene (bG) through liquid exfoliation of graphite in the presence of bovine serum albumin. Microscopic characterization evaluated the quality of the produced nanomaterial, showing the presence of 3–4-layer graphene. Moreover, spectroscopic techniques also confirmed the quality of the resulted bG, as well as the presence of bovine serum albumin on the graphene sheets. Next, for the first time, bG was used as support for the simultaneous covalent co-immobilization of three enzymes, namely β-glucosidase, glucose oxidase, and horseradish peroxidase. The three enzymes were efficiently co-immobilized on bG, demonstrating high immobilization yields and activity recoveries (up to 98.5 and 90%, respectively). Co-immobilization on bG led to an increase of apparent K(M) values and a decrease of apparent V(max) values, while the stability of the nanobiocatalysts prevailed compared to the free forms of the enzymes. Co-immobilized enzymes exhibited high reusability, preserving a significant part of their activity (up to 72%) after four successive catalytic cycles at 30 °C. Finally, the tri-enzymatic nanobiocatalytic system was applied in three-step cascade reactions, involving, as the first step, the hydrolysis of p-Nitrophenyl-β-D-Glucopyranoside and cellobiose

A facile approach to hydrophilic oxidized fullerenes and their derivatives as cytotoxic agents and supports for nanobiocatalytic systems

A facile, environment-friendly, versatile and reproducible approach to the successful oxidation of fullerenes (oxC60) and the formation of highly hydrophilic fullerene derivatives is introduced. This synthesis relies on the widely known Staudenmaier’s method for the oxidation of graphite, to produce both epoxy and hydroxy groups on the surface of fullerenes (C60) and thereby improve the solubility of the fullerene in polar solvents (e.g. water). The presence of epoxy groups allows for further functionalization via nucleophilic substitution reactions to generate new fullerene derivatives, which can potentially lead to a wealth of applications in the areas of medicine, biology, and composite materials. In order to justify the potential of oxidized C60 derivatives for bio-applications, we investigated their cytotoxicity in vitro as well as their utilization as support in biocatalysis applications, taking the immobilization of laccase for the decolorization of synthetic industrial dyes as a trial case.Peer ReviewedPostprint (published version

Carbon nanostructures derived through hypergolic reaction of conductive polymers with fuming nitric acid at ambient conditions

Hypergolic systems rely on organic fuel and a powerful oxidizer that spontaneously ignites upon contact without any external ignition source. Although their main utilization pertains to rocket fuels and propellants, it is only recently that hypergolics has been established from our group as a new general method for the synthesis of different morphologies of carbon nanostructures depending on the hypergolic pair (organic fuel-oxidizer). In search of new pairs, the hypergolic mixture described here contains polyaniline as the organic source of carbon and fuming nitric acid as strong oxidizer. Specifically, the two reagents react rapidly and spontaneously upon contact at ambient conditions to afford carbon nanosheets. Further liquid-phase exfoliation of the nanosheets in dimethylformamide results in dispersed single layers exhibiting strong Tyndall effect. The method can be extended to other conductive polymers, such as polythiophene and polypyrrole, leading to the formation of different type carbon nanostructures (e.g., photolumincent carbon dots). Apart from being a new synthesis pathway towards carbon nanomaterials and a new type of reaction for conductive polymers, the present hypergolic pairs also provide a novel set of rocket bipropellants based on conductive polymers.Web of Science266art. no. 159

Synthesis, characterization and assessment of hydrophilic oxidized carbon nanodiscs in bio-related applications

Oxidation of industrially prepared carbon nanodiscs using a simple, versatile, and reproducible approach based on the Staudenmaier method yields a new hydrophilic form of nanocarbon. As a result of the strong acid treatment, which also enables the separation of carbon nanodiscs from the mixed starting material, the graphene planes detach from the discs, while the surface of the carbon nanodiscs is decorated with various oxygen-containing functional polar groups. Thus, the completely insoluble carbon nanodiscs are converted to a hydrophilic derivative dispersable in many polar solvents, including water. The new carbon structure is expected to have a wide range of applications in several fields including bioapplications. To this end, the functionalized carbon nanodiscs exhibit very low cytotoxicity, while they achieve high drug loadings, enabling their application as an effective drug nanocarrier. Furthermore, the carbon disks were evaluated as supports in nanobiocatalytic applications, increasing significantly the stability of the systems, due to carbon disks' nano-sized dimensions

Ανάπτυξη νέων βιοκαταλυτικών συστημάτων μέσω της ακινητοποίησης ενζύμων σε νανοδομικά υλικά

The aim of this thesis is the development of novel nanobiocatalytic systems by means of immobilization of enzymes onto carbon-based nanostructured-supports, through the understanding of the correlation between structure and function of enzymes with these nanomaterials. Carbon-based nanomaterials such as carbon nanotubes and graphene oxide, functionalized with different length of alkyl chains and functional groups, were used to study the effect of their presence on the catalytic and structural characteristics of cytochrome c. The presence of these functionalized nanomaterials increases the catalytic efficiency of cytochrome c, as well the stability of the protein, as they protect it from thermal denaturation and hydrogen peroxide deactivation. Cytochrome c preserves its secondary structure in the presence of the functionalized nanomaterials, while the observed changes in the heme microenvironment suggest that the heme plane reorients in the active site pocket, possibly making the heme more accessible to the substrates and thus leading to higher peroxidase activity. Cytochrome c was further immobilized on functionalized derivatives of graphene oxide through physical adsorption and covalent binding. The immobilization efficiency and the catalytic behavior of the immobilized protein are affected by the surface chemistry, the alkyl chain length and the terminal functional group of the nanomaterials, as well as the immobilization procedure. The experimental results show that functionalized graphene oxide derivatives are excellent supports for protein immobilization. The thermostability of cytochrome c is improved upon immobilization, while the nanomaterials seem to offer a protective role against denaturing agents, such as methanol and hydrogen peroxide. The immobilization of cytochrome c on the functionalized nanomaterials results in changes in the secondary structure of the protein. More specific, a loss in the α-helical content is observed, while the content of β-sheets is increased, indicating that the protein undergoes a conformational transition to a more rigid structure, which could explain the increased stability of the immobilized protein. Finally, the development of multi-layer nanomaterial-enzyme nanoassemblies, through multi-point covalent immobilization was achieved for the synthesis of novel biocatalysts with improved properties which can be used in numerous industrial applications. The prepared nanobiocatalysts consist of alternate layers of laccase and amino-functionalized graphene oxide and present excellent thermal stability compared to the free enzyme. In addition, the multi-layer nanoassemblies present excellent oxidation activity against polycyclic aromatic hydrocarbons and dyes, while they are able to retain up to 94% of their initial activity after 5 uses. In conclusion, the results of this study demonstrate the significant benefits arising from the implementations of nanosturctured materials as supports for enzyme immobilization, and form the basis for the development of numerous applications in the field of nanobiotechnology.Στόχος της παρούσας διατριβής είναι η ανάπτυξη νέων βιοκαταλυτικών συστημάτων μέσω της ακινητοποίησης ενζύμων σε νανοδομικά υλικά, μελετώντας και κατανοώντας τη σχέση δομής-λειτουργίας των ακινητοποιημένων ενζύμων με τα νανοϋλικά. Νανοσωλήνες άνθρακα και οξείδιο του γραφενίου, τροποποιημένα με διάφορου μήκους αλκυλικές αλυσίδες και διαφορετικές λειτουργικές ομάδες, χρησιμοποιήθηκαν για τη μελέτη της επίδρασης της παρουσίας τους στα καταλυτικά και δομικά χαρακτηριστικά του κυτοχρώματος c. Η παρουσία των νανοϋλικών αυτών αυξάνει τη δραστικότητα της πρωτεΐνης, καθώς και τη σταθερότητά της, προστατεύοντάς την από τη θερμική μετουσίωση και την απενεργοποίηση από υπεροξείδιο του υδρογόνου. Το κυτόχρωμα c διατηρεί τη δευτεροταγή δομή του παρουσία των νανοϋλικών, ενώ παρατηρούνται αλλαγές στο μικροπεριβάλλον της αίμης, που οδηγούν σε ένα πιο προσβάσιμο ενεργό κέντρο, με αποτέλεσμα την αυξημένη καταλυτική δραστικότητα της πρωτεΐνης. Στη συνέχεια, το κυτόχρωμα c ακινητοποιήθηκε σε τροποποιημένα παράγωγα οξειδίου του γραφενίου μέσω φυσικής προσρόφησης και ομοιοπολικής ακινητοποίησης. Η απόδοση της ακινητοποίησης και η δραστικότητα της πρωτεΐνης επηρεάζονται από τη σύσταση του νανοϋλικού, το μήκος της αλκυλικής αλυσίδας, τη λειτουργική ομάδα και τον τρόπο της ακινητοποίησης. Τα πειραματικά αποτελέσματα υποδεικνύουν ως κατάλληλους φορείς ακινητοποίησης τα τροποποιημένα νανοϋλικά. Η θερμική σταθερότητα της ακινητοποιημένης πρωτεΐνης βελτιώνεται σε σχέση με την ελεύθερη, ενώ ταυτόχρονα τα νανοϋλικά προστατεύουν το κυτόχρωμα c από παράγοντες όπως η μεθανόλη και το υπεροξείδιο του υδρογόνου. Η ακινητοποίηση του κυτοχρώματος c έχει ως αποτέλεσμα την αλλαγή της δευτεροταγούς δομής του. Διαπιστώθηκε μείωση της περιεκτικότητας σε α-έλικα με ταυτόχρονη αύξηση της περιεκτικότητας σε β-φύλλα, γεγονός που υποδηλώνει πως η πρωτεΐνη υιοθετεί μια πιο άκαμπτη διαμόρφωση που εξηγεί την αυξημένη σταθερότητα της ακινητοποιημένης πρωτεΐνης. Τέλος, επιτεύχθηκε η δημιουργία πολυστρωματικών νανοσυστοιχιών νανοϋλικού-ενζύμου, μέσω ομοιοπολικής ακινητοποίησης πολλαπλών σημείων, που οδηγεί στη δημιουργία καινοτόμων βιοκαταλυτών με βελτιωμένες ιδιότητες και πλήθος βιομηχανικών εφαρμογών. Οι νανοβιοκαταλύτες που παρασκευάσθηκαν αποτελούνται από εναλλασσόμενα στρώματα λακάσης-τροποποιημένου οξειδίου του γραφενίου, και παρουσιάζουν εξαιρετική σταθερότητα έναντι του ελεύθερου ενζύμου. Επιπλέον, παρουσιάζουν αυξημένη καταλυτική δραστικότητα κατά την οξείδωση πολυαρωματικών υδρογονανθράκων και χρωστικών, ενώ διατηρούν μέχρι και 94% της αρχικής τους δραστικότητας ύστερα από 5 κύκλους χρήσης. Εν κατακλείδι, τα νανοδομικά υλικά με βάση τον άνθρακα βελτιώνουν σημαντικά την καταλυτική δράση των οξειδοναγωγικών πρωτεϊνών, οδηγώντας στη δημιουργία βιοκαταλυτικών συστημάτων με ενδιαφέρουσες ιδιότητες. Τα αποτελέσματα της παρούσας διατριβής καταδεικνύουν τα σημαντικά πλεονεκτήματα που προκύπτουν από την εφαρμογή των νανοϋλικών ως φορείς ακινητοποίησης ενζύμων, και τα οποία αποτελούν τη βάση για την ανάπτυξη πλήθους εφαρμογών στο πεδίο της νανοβιοτεχνολογίας

Magnetic Microreactors with Immobilized Enzymes—From Assemblage to Contemporary Applications

Microfluidics, as the technology for continuous flow processing in microscale, is being increasingly elaborated on in enzyme biotechnology and biocatalysis. Enzymatic microreactors are a precious tool for the investigation of catalytic properties and optimization of reaction parameters in a thriving and high-yielding way. The utilization of magnetic forces in the overall microfluidic system has reinforced enzymatic processes, paving the way for novel applications in a variety of research fields. In this review, we hold a discussion on how different magnetic particles combined with the appropriate biocatalyst under the proper system configuration may constitute a powerful microsystem and provide a highly explorable scope

Biocatalytic Performance of <i>β</i>-Glucosidase Immobilized on 3D-Printed Single- and Multi-Channel Polylactic Acid Microreactors

Microfluidic devices have attracted much attention in the current day owing to the unique advantages they provide. However, their application for industrial use is limited due to manufacturing limitations and high cost. Moreover, the scaling-up process of the microreactor has proven to be difficult. Three-dimensional (3D) printing technology is a promising solution for the above obstacles due to its ability to fabricate complex structures quickly and at a relatively low cost. Hence, combining the advantages of the microscale with 3D printing technology could enhance the applicability of microfluidic devices in the industrial sector. In the present work, a 3D-printed single-channel immobilized enzyme microreactor with a volume capacity of 30 μL was designed and created in one step via the fused deposition modeling (FDM) printing technique, using polylactic acid (PLA) as the printing material. The microreactor underwent surface modification with chitosan, and β-glucosidase from Thermotoga maritima was covalently immobilized. The immobilized biocatalyst retained almost 100% of its initial activity after incubation at different temperatures, while it could be effectively reused for up to 10 successful reaction cycles. Moreover, a multi-channel parallel microreactor incorporating 36 channels was developed, resulting in a significant increase in enzymatic productivity