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

Development of a Four-Enzyme Magnetic Nanobiocatalyst for Multi-Step Cascade Reactions

We report the preparation, characterization and application of a novel magnetic four-enzyme nanobiocatalyst prepared by the simultaneous covalent co-immobilization of cellulase (CelDZ1), β-glucosidase (bgl), glucose oxidase (GOx) and horseradish peroxidase (HRP) onto the surface of amino-functionalized magnetic nanoparticles (MNPs). This nanobiocatalyst was characterized by various spectroscopic techniques. The co-immobilization process yielded maximum recovered enzymatic activity (CelDZ1: 42%, bgl: 66%, GOx: 94% and HRP: 78%) at a 10% v/v cross-linker concentration, after 2 h incubation time and at 1:1 mass ratio of MNPs to total enzyme content. The immobilization process leads to an increase of Km and a decrease of Vmax values of co-immobilized enzymes. The thermal stability studies of the co-immobilized enzymes indicated up to 2-fold increase in half-life time constants and up to 1.5-fold increase in their deactivation energies compared to the native enzymes. The enhanced thermodynamic parameters of the four-enzyme co-immobilized MNPs also suggested increment in their thermal stability. Furthermore, the co-immobilized enzymes retained a significant part of their activity (up to 50%) after 5 reaction cycles at 50 °C and remained active even after 24 d of incubation at 5 °C. The nanobiocatalyst was successfully applied in a four-step cascade reaction involving the hydrolysis of cellulose

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