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.

Biocatalytic Performance of ß-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 ß-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.

Tyrosinase Magnetic Cross-Linked Enzyme Aggregates: Biocatalytic Study in Deep Eutectic Solvent Aqueous Solutions.

In the field of biocatalysis, the implementation of sustainable processes such as enzyme immobilization or employment of environmentally friendly solvents, like Deep Eutectic Solvents (DESs) are of paramount importance. In this work, tyrosinase was extracted from fresh mushrooms and used in a carrier-free immobilization towards the preparation of both non-magnetic and magnetic cross-linked enzyme aggregates (CLEAs). The prepared biocatalyst was characterized and the biocatalytic and structural traits of free tyrosinase and tyrosinase magnetic CLEAs (mCLEAs) were evaluated in numerous DES aqueous solutions. The results showed that the nature and the concentration of the DESs used as co-solvents significantly affected the catalytic activity and stability of tyrosinase, while the immobilization enhanced the activity of the enzyme in comparison with the non-immobilized enzyme up to 3.6-fold. The biocatalyst retained the 100% of its initial activity after storage at -20 °C for 1 year and the 90% of its activity after 5 repeated cycles. Tyrosinase mCLEAs were further applied in the homogeneous modification of chitosan with caffeic acid in the presence of DES. The biocatalyst demonstrated great ability in the functionalization of chitosan with caffeic acid in the presence of 10% v/v DES [Bet:Gly (1:3)], enhancing the antioxidant activity of the films.

Does Green Exfoliation of Graphene Produce More Biocompatible Structures?

Graphene has been studied thoroughly for its use in biomedical applications over the last decades. A crucial factor for a material to be used in such applications is its biocompatibility. Various factors affect the biocompatibility and toxicity of graphene structures, including lateral size, number of layers, surface functionalization, and way of production. In this work, we tested that the green production of few-layer bio-graphene (bG) enhances its biocompatibility compared to chemical-graphene (cG). When tested against three different cell lines in terms of MTT assays, both materials proved to be well-tolerated at a wide range of doses. However, high doses of cG induce long-term toxicity and have a tendency for apoptosis. Neither bG nor cG induced ROS generation or cell cycle modifications. Finally, both materials affect the expression of inflammatory proteins such as Nrf2, NF-kB and HO-1 but further research is required for a safe result. In conclusion, although there is little to choose between bG and cG, bG's sustainable way of production makes it a much more attractive and promising candidate for biomedical applications.

Is graphene the rock upon which new era continuous glucose monitors could be built?

Diabetes mellitus' (DM) prevalence worldwide is estimated to be around 10% and is expected to rise over the next decades. Monitoring blood glucose levels aims to determine whether glucose targets are met to minimize the risk for the development of symptoms related to high or low blood sugar and avoid long-term diabetes complications. Continuous glucose monitoring (CGMs) systems emerged almost two decades ago and have revolutionized the way diabetes is managed. Especially in Type 1 DM, the combination of a CGM with an insulin pump (known as a closed-loop system or artificial pancreas) allows an autonomous regulation of patients' insulin with minimal intervention from the user. However, there is still an unmet need for high accuracy, precision and repeatability of CGMs. Graphene was isolated in 2004 and found immediately fertile ground in various biomedical applications and devices due to its unique combination of properties including its high electrical conductivity. In the last decade, various graphene family nanomaterials have been exploited for the development of enzymatic and non-enzymatic biosensors to determine glucose in biological fluids, such as blood, sweat, and so on. Although great progress has been achieved in the field, several issues need to be addressed for graphene sensors to become a predominant material in the new era of CGMs.

Structure-Function Studies of Glucose Oxidase in the Presence of Carbon Nanotubes and Bio-Graphene for the Development of Electrochemical Glucose Biosensors.

In this work, we investigated the effect of multi-walled carbon nanotubes (MWCNTs) and bio-graphene (bG) on the structure and activity of glucose oxidase (GOx), as well as on the performance of the respective electrochemical glucose biosensors. Various spectroscopic techniques were applied to evaluate conformational changes in GOx molecules induced by the presence of MWCNTs and bG. The results showed that MWCNTs induced changes in the flavin adenine dinucleotide (FAD) prosthetic group of GOx, and the tryptophan residues were exposed to a more hydrophobic environment. Moreover, MWCNTs caused protein unfolding and conversion of α-helix to ß-sheet structure, whereas bG did not affect the secondary and tertiary structure of GOx. The effect of the structural changes was mirrored by a decrease in the activity of GOx (7%) in the presence of MWCNTs, whereas the enzyme preserved its activity in the presence of bG. The beneficial properties of bG over MWCNTs on GOx activity were further supported by electrochemical data at two glucose biosensors based on GOx entrapped in chitosan gel in the presence of bG or MWCNTs. bG-based biosensors exhibited a 1.33-fold increased sensitivity and improved reproducibility for determining glucose over the sweat-relevant concentration range of glucose.

A Four-enzyme Nanoassembly Consisting of Hydrolases and Oxidoreductases for Multi-step Cascade Reactions.

Cascade reactions catalyzed by multi-enzymatic systems have attracted enormous scientific interest over the last decade. They are an emerging technology that significantly expands the applicability of biocatalysts in several biotechnological processes, such as the synthesis of high value-added products. Immobilization of enzymes on a solid carrier is a commonly used strategy to improve the stability and reuse of multiple enzyme systems. Magnetic nanoparticles have been applied as promising nanocarriers for either the immobilization of one enzyme or the co-immobilization of multiple enzymes. In this chapter, we describe the preparation of magnetic iron oxide nanoparticles γ-Fe2O3 modified with 3-(aminopropyl)-triethoxysilane (APTES), for the simultaneous covalent co-immobilization of oxidoreductases and hydrolytic enzymes, such as cellulase, ß-glucosidase (bgl), glucose oxidase (GOx), and horseradish peroxidase (HRP). Several spectroscopic techniques that are used to characterize the structure and the catalytic performance of such systems are also described.

Graphene Oxide-Cytochrome c Multilayered Structures for Biocatalytic Applications: Decrypting the Role of Surfactant in Langmuir-Schaefer Layer Deposition.

Graphene, a two-dimensional single-layer carbon allotrope, has attracted tremendous scientific interest due to its outstanding physicochemical properties. Its monatomic thickness, high specific surface area, and chemical stability render it an ideal building block for the development of well-ordered layered nanostructures with tailored properties. Herein, biohybrid graphene-based layer-by-layer structures are prepared by means of conventional and surfactant-assisted Langmuir-Schaefer layer deposition techniques, whereby cytochrome c molecules are accommodated within ordered layers of graphene oxide. The biocatalytic activity of the as-developed nanobio-architectures toward the enzymatic oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and decolorization of pinacyanol chloride is tested. The results show that the multilayer structures exhibit high biocatalytic activity and stability in the absence of surfactant molecules during the deposition of the monolayers.

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 ß-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 KM values and a decrease of apparent Vmax 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-ß-D-Glucopyranoside and cellobiose.

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.

Trends in the development of innovative nanobiocatalysts and their application in biocatalytic transformations.

The ever-growing demand for cost-effective and innocuous biocatalytic transformations has prompted the rational design and development of robust biocatalytic tools. Enzyme immobilization technology lies in the formation of cooperative interactions between the tailored surface of the support and the enzyme of choice, which result in the fabrication of tremendous biocatalytic tools with desirable properties, complying with the current demands even on an industrial level. Different nanoscale materials (organic, inorganic, and green) have attracted great attention as immobilization matrices for single or multi-enzymatic systems. Aiming to unveil the potentialities of nanobiocatalytic systems, we present distinct immobilization strategies and give a thorough insight into the effect of nanosupports specific properties on the biocatalysts' structure and catalytic performance. We also highlight the development of nanobiocatalysts for their incorporation in cascade enzymatic processes and various types of batch and continuous-flow reactor systems. Remarkable emphasis is given on the application of such nanobiocatalytic tools in several biocatalytic transformations including bioremediation processes, biofuel production, and synthesis of bioactive compounds and fine chemicals for the food and pharmaceutical industry.

Trends of nanotechnology in type 2 diabetes mellitus treatment.

There are several therapeutic approaches in type 2 diabetes mellitus (T2DM). When diet and exercise fail to control hyperglycemia, patients are forced to start therapy with antidiabetic agents. However, these drugs present several drawbacks that can affect the course of treatment. The major disadvantages of current oral modalities for the treatment of T2DM are mainly depicted in the low bioavailability and the immediate release of the drug, generating the need for an increase in frequency of dosing. In conjugation with the manifestation of adverse side effects, patient compliance to therapy is reduced. Over the past few years nanotechnology has found fertile ground in the development of novel delivery modalities that can potentially enhance anti-diabetic regimes efficacy. All efforts have been targeted towards two main vital steps: (a) to protect the drug by encapsulating it into a nano-carrier system and (b) efficiently release the drug in a gradual as well as controllable manner. However, only a limited number of studies published in the literature used in vivo techniques in order to support findings. Here we discuss the current disadvantages of modern T2DM marketed drugs, and the nanotechnology advances supported by in vivo in mouse/rat models of glucose homeostasis. The generation of drug nanocarriers may increase bioavailability, prolong release and therefore reduce dosing and thus, improve patient compliance. This novel approach might substantially improve quality of life for diabetics. Application of metal nanoformulations as indirect hypoglycemic agents is also discussed.

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.

Use of functionalized carbon nanotubes for the development of robust nanobiocatalysts.

This chapter deals with the use of functionalized carbon nanotubes (fCNTs) as supports for the development of nanobiocatalytic systems through the immobilization of enzymes. The surface characteristics, properties and production of carbon nanotubes are described, while an analysis in their biological applications is also presented. The results presenting within the text are giving insights to the effect of carbon nanotubes on the catalytic and structural characteristics of different proteins, such as cytochrome c from equine heart (cyt c) and laccase from Trametes versicolor (TvL), either when they are used as additives in the reaction medium or as supports for protein immobilization. A variety of biochemical and spectroscopic techniques is applied to investigate the interactions between the protein biomolecules and carbon nanotubes. The results showed that the presence of fCNTs enables cyt c to maintain both its secondary structure and heme microenvironment. Non-covalent and covalent immobilization approaches are also described, while the immobilized biocatalysts are characterized with respect to their catalytic and structural characteristics. Immobilized TvL was found to exhibit higher catalytic activity when non-specific binding was used as immobilization procedure (up to 0.85Uµg-1), compared to covalent immobilization (up to 0.7Uµg-1), while the increase of the alkyl chain of the functionalized CNTs also seems to affect the catalytic efficiency of the immobilized enzymes. The nanobiocatalytic systems that are presented here demonstrated exceptional stability (up to 31% of their initial activity is maintained after 24h incubation at 60°C) and reusability (up to 58% remaining activity after 8 successive catalytic cycles) compared to the native enzymes, leading to robust biocatalytic systems appropriate for various applications of biotechnological and industrial interest.

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

In the present study, we developed novel ß-glucosidase-based nano-biocatalysts for the bioconversion of oleuropein to hydroxytyrosol. Using non-covalent or covalent immobilization approaches, ß-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 nanο-support and the immobilization procedure play a key role for the immobilization efficiency or the catalytic activity of the immobilized ß-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-1), which is a phenolic compound with numerous health benefits. The bio-nanoconjugates exhibited high thermal and operational stability (up to 240 hours of repeated use), which indicated that they are efficient tools for various bio-transformations.

Development of Effective Lipase-Hybrid Nanoflowers Enriched with Carbon and Magnetic Nanomaterials for Biocatalytic Transformations.

In the present study, hybrid nanoflowers (HNFs) based on copper (II) or manganese (II) ions were prepared by a simple method and used as nanosupports for the development of effective nanobiocatalysts through the immobilization of lipase B from Pseudozyma antarctica. The hybrid nanobiocatalysts were characterized by various techniques including scanning electron microscopy (SEM), energy dispersion spectroscopy (EDS), X-ray diffraction (XRD), Raman spectroscopy, and Fourier transform infrared spectroscopy (FTIR). The effect of the addition of carbon-based nanomaterials, namely graphene oxide and carbon nanotubes, as well as magnetic nanoparticles such as maghemite, on the structure, catalytic activity, and operational stability of the hybrid nanobiocatalysts was also investigated. In all cases, the addition of nanomaterials during the preparation of HNFs increased the catalytic activity and the operational stability of the immobilized biocatalyst. Lipase-based magnetic nanoflowers were effectively applied for the synthesis of tyrosol esters in non-aqueous media, such as organic solvents, ionic liquids, and environmental friendly deep eutectic solvents. In such media, the immobilized lipase preserved almost 100% of its initial activity after eight successive catalytic cycles, indicating that these hybrid magnetic nanoflowers can be applied for the development of efficient nanobiocatalytic systems.

Stabilization of Laccase Through Immobilization on Functionalized GO-Derivatives.

This chapter deals with the use of functionalized derivatives of graphene oxide as nanoscaffolds for the immobilization and stabilization of laccase from Trametes versicolor. Covalent and noncovalent immobilization approaches are described, while a novel method for the development of laccase-based multilayer nanoassemblies is also presented. Various biochemical, spectroscopic, and microscopic techniques were applied to characterize the nanobiocatalytic systems in respect to their microstructure and catalytic performance. Laccase-GO nanosystems were characterized with FTIR spectroscopy in order to confirm the functionalization of the nanomaterials, as well as to interpret the nanomaterial-enzyme interactions, while the multilayer structure of laccase-based multilayer nanoassemblies was confirmed by atomic force microscopy. The nanobiocatalytic systems presented here demonstrated exceptional stability and reusability compared with the free enzyme form, leading to robust biocatalytic systems appropriate for various applications of industrial interest.

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 °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.

Graphene oxide derivatives with variable alkyl chain length and terminal functional groups as supports for stabilization of cytochrome c.

In this study we report the ability of reduced and non-reduced graphene oxide-based nanomaterials (GONs), modified with variable alkyl chain length and terminal functional groups, to act as effective scaffolds for the immobilization of cytochrome c (cyt c) using different immobilization procedures. The GONs/cyt c conjugates are characterized by a combination of techniques, namely atomic force microscopy, X-ray photoelectron and FT-IR spectroscopies as well as thermo-gravimetric and differential thermal analysis. The effect of the structure of functional groups and the surface chemistry of GONs on the immobilization efficiency, the peroxidase activity and the stability of the cyt c was investigated and correlated with conformational changes on the protein molecule upon immobilization. The enhanced thermal stability (up to 2-fold) and increased tolerance (up to 25-fold) against denaturing agents observed for immobilized cyt c, indicates that these functionalized GONs are suitable as nanoscaffolds for the development of robust nanobiocatalysts.

Graphene-based nanobiocatalytic systems: recent advances and future prospects.

Graphene-based nanomaterials are particularly useful nanostructured materials that show great promise in biotechnology and biomedicine. Owing to their unique structural features, exceptional chemical, electrical, and mechanical properties, and their ability to affect the microenvironment of biomolecules, graphene-based nanomaterials are suitable for use in various applications, such as immobilization of enzymes. We present the current advances in research on graphene-based nanomaterials used as novel scaffolds to build robust nanobiocatalytic systems. Their catalytic behavior is affected by the nature of enzyme-nanomaterial interactions and, thus, the availability of methods to couple enzymes with nanomaterials is an important issue. We discuss the implications of such interactions along with future prospects and possible challenges in this rapidly developing area.