Germanane Monolayer Films as Antibacterial Coatings

Germanane (GeH), a graphane analogue, has attracted significant interest because of its optoelectronic properties; however, the environmental and biological effects of GeH have scarcely been investigated so far. Here we report a facile approach based on the Langmuir-Schaefer deposition to produce homogeneous and dense GeH monolayer films on various substrates. In view of possible applications and to extend the use of GeH to unexplored fields, we investigated its antibacterial activity for the first time and found that this promising 2D structure exhibits remarkable antibacterial activity against both Gram-negative and Gram-positive bacterial strains

Development of enzyme-catalyzed processes for the targeted biotransformation of natural products with the purpose to improve their properties

The current PhD thesis aims at developing enzyme-catalyzed processes for the targeted biotransformation of natural products with the purpose to improve their properties. Carbon-based nanostructured materials play an important role in the fields of nanobiocatalysis and enzyme biotechnology. Their special properties have attracted considerable research interest. In this study, nanostructured hierarchically porous carbon materials (HPCs) and porous carbon cuboids (PCCs) were employed. These types of nanomaterials were used as carriers for the immobilization of different enzymes - laccase, an oxidoreductase and two hydrolases, namely lipase and β-glucosidase. The nanomaterials used were either in their bare form or chemically modified, in order to obtain functional groups or to gain magnetic properties. Enzyme immobilization was carried out in two methods – by adsorption or by covalent linkage. Finally, different spectroscopic and microscopic techniques were employed for the nanobiocatalysts characterization in terms of their structure and catalytic behavior. Surface chemistry of nanomaterials is of fundamental importance regarding their interaction with biomolecules, affecting both immobilization yield and catalytic activity of immobilized enzymes. Similarly, the immobilization techniques that were utilized seem to affect the catalytic activity, as well as the thermal and operational stability of the immobilized enzymes. The developed nanobiocatalysts exhibited remarkable catalytic activity and stability. The nanobiocatalytic systems developed under the scope of the present thesis were subsequently utilized for the biotransformation of various natural products aiming at the improvement of their biological activity. Immobilized laccase was successively used for the modification of tyrosol and hydroxytyrosol, two phenolic compounds with various biological activities. The modification process led mainly to dimeric derivatives, which demonstrated increased biological activities. Following, the immobilized β-glucosidases were successfully employed for the production of hydroxytyrosol from the standard oleuropein compound, while they were also applied for the enrichment of olive leaves extracts in hydroxytyrosol with the purpose to improve their biological activity. The hydroxytyrosol enriched olive leaves extracts demonstrated improved antioxidant, antimicrobial and anticancer activity. Furthermore, scale-up of the chemoenzymatic modification process was carried out, moving from 1 mg to 20 g by using batch bioreactor systems. Lastly, an “NMR-tube bioreactor” system was developed, as a three-step method carried out entirely in a 5 mm NMR tube. This simple procedure enables: a) the prediction of the ability of a natural product or a mixture of products to perform as a substrate for particular enzymes, b) the real time monitoring of the biotransformation, and d) the investigation of interaction of different products with a target protein of pharmaceutical interest. As a conclusion, carbon-based nanostructured materials consist efficient carriers for enzyme immobilization, leading to the formation of robust biocatalytic systems. On the other hand, modification of natural compounds with biotechnology tools is a much promising guideline for the synthesis of compounds with enriched biological activity. The results of the present thesis indicate the benefits which arise from the incorporation of nanotechnology in the development of biocatalytic systems, directing the research to the field of nanobiotechnology, with a plethora of new and alternative applications.Σκοπό της διατριβής αποτελεί η ανάπτυξη ενζυμικά καταλυόμενων διεργασιών για τον στοχευμένο βιομετασχηματισμό φυσικών προϊόντων, με στόχο την αναβάθμιση των ιδιοτήτων τους. Τα νανοδομημένα υλικά με βάση τον άνθρακα διαδραματίζουν σημαντικό ρόλο στα πεδία της νανοβιοκατάλυσης και της ενζυμικής βιοτεχνολογίας. Οι ιδιαίτερες ιδιότητες που εμφανίζουν έχουν προσελκύσει το ερευνητικό ενδιαφέρον. Τα νανοϋλικά που χρησιμοποιήθηκαν ήταν τα νανοδομημένα υλικά ιεραρχημένου πορώδους άνθρακα (hierarchically porous carbon materials, HPCs) και οι κυβοειδείς πορώδεις άνθρακες (porous carbon cuboids, PCCs). Τα υλικά αυτά μελετήθηκαν ως φορείς για την ακινητοποίηση ενός οξειδωαναγωγικού ενζύμου, της λακάσης και δύο υδρολυτικών ενζύμων, της λιπάσης και της β-γλυκοσιδάσης. Χρησιμοποιήθηκαν στην απλή τους μορφή και μετά από χημική επεξεργασία, είτε για να αποκτήσουν λειτουργικές ομάδες, είτε για να αποκτήσουν μαγνητικές ιδιότητες. Η ακινητοποίηση των ενζύμων πραγματοποιήθηκε με δύο τρόπους, με απλή προσρόφηση ή με τη δημιουργία ομοιοπολικού δεσμού. Τέλος, χρησιμοποιήθηκαν διάφορες φασματοσκοπικές και μικροσκοπικές μέθοδοι, για τον χαρακτηρισμό των νανοβιοκαταλυτών ως προς τη δομή τους και την καταλυτική τους συμπεριφορά. Η χημεία της επιφάνειας των νανοϋλικών φάνηκε να παίζει πολύ σημαντικό ρόλο στην αλληλεπίδραση τους με τις πρωτεΐνες, επηρεάζοντας τόσο το ποσοστό ακινητοποίησης όσο και την καταλυτική δραστικότητα των ακινητοποιημένων ενζύμων. Ομοίως, οι προσεγγίσεις ακινητοποίησης που χρησιμοποιήθηκαν, φάνηκε να επιδρούν στην καταλυτική δραστικότητα, καθώς και στη θερμική και λειτουργική σταθερότητα των ακινητοποιημένων ενζύμων. Οι νανοβιοκαταλύτες που αναπτύχθηκαν επέδειξαν υψηλή καταλυτική δραστικότητα και σταθερότητα. Αυτά τα νανοβιοκαταλυτικά συστήματα, χρησιμοποιήθηκαν στη συνέχεια για την βιομετατροπή διάφορων φυσικών προϊόντων με σκοπό την βελτίωση της βιολογικής τους δράσης. Η ακινητοποιημένη λακάση χρησιμοποιήθηκε επιτυχώς για την τροποποίηση της τυροσόλης και της υδροξυτυροσόλης, δύο φαινολικές ενώσεις με ποικίλες βιολογικές δράσεις. Η τροποποίηση αυτή οδήγησε στη δημιουργία κυρίως διμερών παραγώγων, τα οποία εμφάνισαν βελτιωμένες βιολογικές δράσεις. Στη συνέχεια, οι ακινητοποιημένες β-γλυκοσιδάσες χρησιμοποιήθηκαν επιτυχώς για την παραγωγή υδροξυτυροσόλης από πρότυπη ένωση ελευρωπαΐνης, καθώς και για τον εμπλουτισμό εκχυλισμάτων φύλλων ελιάς σε υδροξυτυροσόλη. Τα εμπλουτισμένα εκχυλίσματα φύλλων ελιάς εμφάνισαν καλύτερη αντιοξειδωτική, αντιμικροβιακή και αντικαρκινική δράση. Επιπλέον, πραγματοποιήθηκε κλιμάκωση (Scale-up) της χημειοενζυμικής τροποποίησης των εκχυλισμάτων φύλλων ελιάς από 1 mg σε 20 g σε βιοαντιδραστήρες διαλείποντος έργου. Τέλος, αναπτύχθηκε ο ‘NMR-tube βιοαντιδραστήρας’, μία μέθοδος τριών σταδίων που έχει διεξαχθεί ολοκληρωτικά μέσα σε ένα σωλήνα NMR 5 mm. Αυτή η απλή μεθοδολογία επιτρέπει: α) την πρόβλεψη της ικανότητας ενός φυσικού προϊόντος να λειτουργήσει ως υπόστρωμα για συγκεκριμένα ένζυμα, β) την καταγραφή της εξέλιξης της αντίδρασης βιοτροποποίησης προϊόντος/ών σε πραγματικό χρόνο και γ) τον έλεγχο της αλληλεπίδρασης των προϊόντων με πρωτεΐνες στόχους φαρμακευτικού ενδιαφέροντος. Συμπερασματικά, τα νανοδομημένα υλικά με βάση τον άνθρακα αποτελούν αποτελεσματικούς φορείς ακινητοποίησης ενζύμων για τη δημιουργία ισχυρών βιοκαταλυτικών συστημάτων. Παράλληλα, η τροποποίηση φυσικών προϊόντων με βιοτεχνολογικά εργαλεία μπορεί να οδηγήσει στη σύνθεση ενώσεων με πλούσια βιολογική δράση. Τα αποτελέσματα της παρούσας διατριβής καταδεικνύουν τα πλεονεκτήματα που προκύπτουν από την εφαρμογή της νανοτεχνολογίας στην ανάπτυξη βιοκαταλυτικών συστημάτων, κατευθύνοντας την έρευνα προς τον τομέα της νανοβιοτεχνολογίας, έναν κλάδο που μπορεί να προσφέρει μία πληθώρα νέων και εναλλακτικών εφαρμογών

Mycoprotein Production by Submerged Fermentation of the Edible Mushroom <i>Pleurotus ostreatus</i> in a Batch Stirred Tank Bioreactor Using Agro-Industrial Hydrolysate

The demand for cheap, healthy, and sustainable alternative protein sources has turned research interest into microbial proteins. Mycoproteins prevail due to their quite balanced amino acid profile, low carbon footprint and high sustainability potential. The goal of this research was to investigate the capability of Pleurotus ostreatus to metabolize the main sugars of agro-industrial side streams, such as aspen wood chips hydrolysate, to produce high-value protein with low cost. Our results indicate that P. ostreatus LGAM 1123 could be cultivated both in a C-6 (glucose)- and C-5(xylose)-sugar-containing medium for mycoprotein production. A mixture of glucose and xylose was found to be ideal for biomass production with high protein content and rich amino acid profile. P. ostreatus LGAM 1123 cultivation in a 4 L stirred-tank bioreactor using aspen hydrolysate was achieved with 25.0 ± 3.4 g L−1 biomass production, 1.8 ± 0.4 d−1 specific growth rate and a protein yield of 54.5 ± 0.5% (g/100 g sugars). PCA analysis of the amino acids revealed a strong correlation between the amino acid composition of the protein produced and the ratios of glucose and xylose in the culture medium. The production of high-nutrient mycoprotein by submerged fermentation of the edible fungus P. ostreatus using agro-industrial hydrolysates is a promising bioprocess in the food and feed industry

Laccase-Mediated Oxidation of Phenolic Compounds from Wine Lees Extract towards the Synthesis of Polymers with Potential Applications in Food Packaging

Laccase from Trametes versicolor was applied to produce phenolic polymeric compounds with enhanced properties, using a wine lees extract as the phenolic source. The influence of the incubation time on the progress of the enzymatic oxidation and the yield of the formed polymers was examined. The polymerization process and the properties of the polymeric products were evaluated with a variety of techniques, such as high-pressure liquid chromatography (HPLC) and gel permeation chromatography (GPC), Fourier-transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies, differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The enzymatic polymerization reaction resulted in an 82% reduction in the free phenolic compounds of the extract. The polymeric product recovery (up to 25.7%) and the molecular weight of the polymer depended on the incubation time of the reaction. The produced phenolic polymers exhibited high antioxidant activity, depending on the enzymatic oxidation reaction time, with the phenolic polymer formed after one hour of enzymatic reaction exhibiting the highest antioxidant activity (133.75 and 164.77 μg TE mg−1 polymer) towards the ABTS and DPPH free radicals, respectively. The higher thermal stability of the polymeric products compared to the wine lees phenolic extract was confirmed with TGA and DSC analyses. Finally, the formed phenolic polymeric products were incorporated into chitosan films, providing them with increased antioxidant activity without affecting the films’ cohesion

Chest CT findings in patients with inflammatory myopathy and Jo1 antibodies

Thoracic high-resolution computed tomography scans (HRCT) of 17 patients with inflammatory muscle disorders (IMD) and positive Jo1 antibodies were retrospectively reviewed regarding presence, extension, and distribution of pathological findings. Abnormal findings were found in 14 (82.3%) patients. The predominant CT abnormality was ground glass attenuation, which was present in seven patients (41.1%), having a bilateral and diffuse distribution. In general, lesions tended to appear in the lower lobes and more specifically in the lung bases. lnterlobular septal thickening was found in six patients (35.3%); it was seen in the upper and lower lobes with peripheral distribution and bilateral localization in five out of six patients. Bronchiectases, reticular opacities, and honeycombing were found in six patients (35.3%). Air space consolidation was seen in about 17% of the patients. Lung involvement is a frequent feature of IMD patients with positive Jo1 antibodies and its most common radiological pattern is that of nonspecific interstitial pneumonia. (c) 2007 Elsevier Ireland Ltd. All rights reserved

Enzymatic Conversion of Oleuropein to Hydroxytyrosol Using Immobilized β-Glucosidase on Porous Carbon Cuboids

In the present study, we developed novel &beta;-glucosidase-based nano-biocatalysts for the bioconversion of oleuropein to hydroxytyrosol. Using non-covalent or covalent immobilization approaches, &beta;-glucosidases from almonds and Thermotoga maritima were attached for the first time on oxidized and non-oxidized porous carbon cuboids (PCC). Various methods were used for the characterization of the bio-nanoconjugates, such as Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and fluorescence spectroscopy. The oxidation state of the nan&omicron;-support and the immobilization procedure play a key role for the immobilization efficiency or the catalytic activity of the immobilized &beta;-glucosidases. The nano-biocatalysts were successfully used for the hydrolysis of oleuropein, which leads to the formation of its bioactive derivative, hydroxytyrosol (up to 2.4 g L&minus;1), which is a phenolic compound with numerous health benefits. The bio-nanoconjugates exhibited high thermal and operational stability (up to 240 h of repeated use), which indicated that they are efficient tools for various bio-transformations

Green Synthesized Magnetic Nanoparticles as Effective Nanosupport for the Immobilization of Lipase: Application for the Synthesis of Lipophenols

In this work, hybrid zinc oxide–iron oxide (ZnOFe) magnetic nanoparticles were synthesized employing Olea europaea leaf aqueous extract as a reducing/chelating and capping medium. The resulting magnetic nanoparticles were characterized by basic spectroscopic and microscopic techniques, namely, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), fourier-transform infrared (FTIR) and atomic force microscopy (AFM), exhibiting a spherical shape, average size of 15–17 nm, and a functionalized surface. Lipase from Thermomyces lanuginosus (TLL) was efficiently immobilized on the surface of ZnOFe nanoparticles through physical absorption. The activity of immobilized lipase was found to directly depend on the enzyme to support the mass ratio, and also demonstrated improved pH and temperature activity range compared to free lipase. Furthermore, the novel magnetic nanobiocatalyst (ZnOFe-TLL) was applied to the preparation of hydroxytyrosyl fatty acid esters, including derivatives with omega-3 fatty acids, in non-aqueous media. Conversion yields up to 90% were observed in non-polar solvents, including hydrophobic ionic liquids. Different factors affecting the biocatalyst performance were studied. ZnOFe-TLL was reutilized for eight subsequent cycles, exhibiting 90% remaining esterification activity (720 h of total operation at 50 °C). The green synthesized magnetic nanoparticles, reported here for the first time, are excellent candidates as nanosupports for the immobilization of enzymes with industrial interest, giving rise to nanobiocatalysts with elevated features

Green Synthesized Magnetic Nanoparticles as Effective Nanosupport for the Immobilization of Lipase: Application for the Synthesis of Lipophenols

In this work, hybrid zinc oxide–iron oxide (ZnOFe) magnetic nanoparticles were synthesized employing Olea europaea leaf aqueous extract as a reducing/chelating and capping medium. The resulting magnetic nanoparticles were characterized by basic spectroscopic and microscopic techniques, namely, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), fourier-transform infrared (FTIR) and atomic force microscopy (AFM), exhibiting a spherical shape, average size of 15–17 nm, and a functionalized surface. Lipase from Thermomyces lanuginosus (TLL) was efficiently immobilized on the surface of ZnOFe nanoparticles through physical absorption. The activity of immobilized lipase was found to directly depend on the enzyme to support the mass ratio, and also demonstrated improved pH and temperature activity range compared to free lipase. Furthermore, the novel magnetic nanobiocatalyst (ZnOFe-TLL) was applied to the preparation of hydroxytyrosyl fatty acid esters, including derivatives with omega-3 fatty acids, in non-aqueous media. Conversion yields up to 90% were observed in non-polar solvents, including hydrophobic ionic liquids. Different factors affecting the biocatalyst performance were studied. ZnOFe-TLL was reutilized for eight subsequent cycles, exhibiting 90% remaining esterification activity (720 h of total operation at 50 °C). The green synthesized magnetic nanoparticles, reported here for the first time, are excellent candidates as nanosupports for the immobilization of enzymes with industrial interest, giving rise to nanobiocatalysts with elevated features

NGIWY-Amide: A Bioinspired Ultrashort Self-Assembled Peptide Gelator for Local Drug Delivery Applications

Fibrillar structures derived from plant or animal origin have long been a source of inspiration for the design of new biomaterials. The Asn-Gly-Ile-Trp-Tyr-NH2 (NGIWY-amide) pentapeptide, isolated from the sea cucumber Apostichopus japonicus, which spontaneously self-assembles in water to form hydrogel, pertains to this category. In this study, we evaluated this ultra-short cosmetic bioinspired peptide as vector for local drug delivery applications. Combining nuclear magnetic resonance, circular dichroism, infrared spectroscopy, X-ray diffraction, and rheological studies, the synthesized pentapeptide formed a stiff hydrogel with a high β-sheet content. Molecular dynamic simulations aligned well with scanning electron and atomic-force microscopy studies, revealing a highly filamentous structure with the fibers adopting a helical-twisted morphology. Model dye localization within the supramolecular hydrogel provided insights on the preferential distribution of hydrophobic and hydrophilic compounds in the hydrogel network. That was further depicted in the diffusion kinetics of drugs differing in their aqueous solubility and molecular weight, namely, doxorubicin hydrochloride, curcumin, and octreotide acetate, highlighting its versatility as a delivery vector of both hydrophobic and hydrophilic compounds of different molecular weight. Along with the observed cytocompatibility of the hydrogel, the NGIWY-amide pentapeptide may offer new approaches for cell growth, drug delivery, and 3D bioprinting tissue-engineering applications