Designing Antioxidant and Antimicrobial Polyethylene Films with Bioactive Compounds/Clay Nanohybrids for Potential Packaging Applications.

In the present work, direct incorporation of bioactive compounds onto the surface and interlayer of nanoclays before their incorporation into the final polymeric film was conducted, based on a green methodology developed by our group that is compatible with food packaging. This will lead to the higher thermal stability and the significant reduction of the loss of activity of the active ingredients during packaging configuration. On this basis, the essential oil (EO) components carvacrol (C), thymol (T) as well as olive leaf extract (OLE), which is used for the first time, were incorporated onto organo-modified montmorillonite (O) or inorganic bentonite (B) through the evaporation/adsorption method. The prepared bioactive nanocarriers were further mixed with low-density polyethylene (LDPE), via melt compounding, in order to prepare films for potential use as fresh fruit and vegetable packaging material. Characterization of the bioactive nanocarriers and films were performed through XRD, TGA, tensile, antimicrobial and antioxidant tests. Films with organically modified montmorillonite loaded with carvacrol (OC), thymol (OT) and olive leaf extract (OOLE) at 5% wt. showed better results in terms of mechanical properties. The films with polyethylene and organically modified montmorillonite loaded with carvacrol or thymol at 20% wt. (PE_OC20 and PE_OT20), as well as with olive leaf extract at 5 or 10 %wt., clay:bioactive substance ratio 1:0.5 and 10% compatibilizer (PE_OOLE5_MA10 and PE_OOLE10_MA10) exhibited the highest antioxidant activity. The resulting films displayed outstanding antimicrobial properties against Gram-negative Escherichia coli (E. coli) with the best results appearing in the films with 10% OC and OT.

Development of a Multi-Enzymatic Approach for the Modification of Biopolymers with Ferulic Acid.

A series of polymers, including chitosan (CS), carboxymethylcellulose (CMC) and a chitosan-gelatin (CS-GEL) hybrid polymer, were functionalized with ferulic acid (FA) derived from the enzymatic treatment of arabinoxylan through the synergistic action of two enzymes, namely, xylanase and feruloyl esterase. Subsequently, the ferulic acid served as the substrate for laccase from Agaricus bisporus (AbL) in order to enzymatically functionalize the above-mentioned polymers. The successful grafting of the oxidized ferulic acid products onto the different polymers was confirmed through ultraviolet-visible (UV-Vis) spectroscopy, attenuated total reflectance (ATR) spectroscopy, scanning electron microscopy (SEM) and nuclear magnetic resonance (NMR) spectroscopy. Additionally, an enhancement of the antioxidant properties of the functionalized polymers was observed according to the DDPH and ABTS protocols. Finally, the modified polymers exhibited strong antimicrobial activity against bacterial populations of Escherichia coli BL21DE3 strain, suggesting their potential application in pharmaceutical, cosmeceutical and food industries.

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.

A Bi-enzymatic Immobilized Nanobiocatalyst for the Biotransformation of Oleuropein to Hydroxytyrosol.

Multi-enzymatic assemblies offer the opportunity of bringing in proximity several enzymes that are enabled to work together for the catalysis of multi-step reactions. Especially, the development of robust nanobiocatalytic systems comprising of several enzymes has gained considerable attention over the last few years for the catalysis of complex reactions and the production of high added-value products. In the present chapter, we describe the methodology for the development of a bi-enzymatic nanobiocatalyst consisting of the enzymes ß-glucosidase from Thermotoga maritima and lipase A from Candida antarctica (CalA) co-immobilized on chitosan-coated magnetic nanoparticles. This nanobiocatalyst can be efficiently applied for the biotransformation of oleuropein to hydroxytyrosol, a reaction of increased biotechnological interest. Several techniques, as well as methodologies that are required for the characterization of the structure and the activity of such systems are also comprehensively described.

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.

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.

Multienzymatic Nanoassemblies: Recent Progress and Applications.

Biotechnological research has turned to multienzymatic nanoassemblies as a promising concept to host multiple applications. Here, we consider important aspects around the development and optimization of such biocatalytic systems and present current advances in utilizing bi- and multienzymatic cascade reactions in diverse fields, including ultrasensitive biosensing, development of pharmaceuticals, and conversion of natural biopolymers to valuable products, highlighting their future potential in the chemical, biotechnological, and pharmaceutical industries. Diverse co-immobilization techniques and different parameters affecting the performance of multienzymatic cascade reactions are discussed. Continuous flow processes incorporating multienzymatic nanoassemblies in different reactor configurations are also presented. This technology provides an arsenal of tools for the development of innovative and effective multienzymatic systems offering new possibilities for biocatalysts applications.