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 K_m and a decrease of V_max 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.

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.

Biomass derived from green marine macroalgae Ulva sp. is considered a rich source of bioactive compounds. In the present study, we demonstrate the increased bioactivity of a phenolic-rich extract derived from green marine macroalgae Ulva intestinalis after its treatment with fungal laccases from Trametes versicolor and Agaricus bisporus. The phenolic composition of the extract, before and after its enzymatic treatment, was determined through several analytical methods. Furthermore, the antioxidant, enzyme-inhibitory, antimicrobial and cytotoxic activity of enzymatically modified and non-modified extracts was comparatively investigated. Depending on the laccase used, the enzyme-modified extracts exhibited different phenolic content and enhanced bioactivities compared to the non-treated ones. The enzymatically modified extracts presented enhanced antimicrobial, anti-lipoxygenase and anti-collagenase activities, and mild cytotoxicity. These results indicate that the proposed biocatalytic process could be applied to produce green macroalgal natural extracts with enhanced bioactivities paving the way for their implementation in the food, pharmaceutical, and cosmeceutical industries.

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.

Lipase A from Candida antarctica (CalA) and β-glucosidase from Thermotoga maritima (bgl) were covalently co-immobilized onto the surface of chitosan-coated magnetic nanoparticles (CS-MNPs). Several parameters regarding the co-immobilization procedure (glutaraldehyde concentration, incubation time, CS-MNPs to enzyme mass ratio and bgl to CalA mass ratio) were evaluated and optimized. The developed nanobiocatalyst was characterized by various spectroscopic techniques. Biochemical parameters such as kinetic constants and thermal stability were also evaluated. The nanobiocatalytic system revealed an increase in the K_m constant followed by a decrease in V_max value compared with the native enzymes, while a significant increase (>5-fold higher) of the thermal stability of the immobilized CalA, both in individual and in co-immobilized form, was observed after 24 h incubation at 60 °C. Finally, the nanobiocatalyst was efficiently applied for the bioconversion of oleuropein to hydroxytyrosol, one of the most powerful naturally derived antioxidants, and it could be recycled for up to 10 reaction cycles (240 h of constant operation) at 60 °C, retaining more than 50% of its initial activity.

The aim of this study was the biochemical and kinetic characterization of the gentisate 1,2-dioxygenase (GDO) from Pseudarthrobacter phenanthrenivorans Sphe3 and the development of a nanobiocatalyst by its immobilization on Ni²⁺-functionalized Fe₃O₄-polydopamine magnetic nanoparticles (Ni²⁺-PDA-MNPs). This is the first GDO to be immobilized. The gene encoding the GDO was cloned with an N-terminal His-tag and overexpressed in E. coli. The nanoparticles showed a high purification efficiency of GDO from crude cell lysates with a maximum activity recovery of 97%. The immobilized enzyme was characterized by Fourier transform infrared spectroscopy (FTIR). The reaction product was identified by ¹H NMR. Both free and immobilized GDO exhibited Michaelis–Menten kinetics with K_m values of 25.9 ± 4.4 and 82.5 ± 14.2 μM and V_max values of 1.2 ± 0.1 and 0.03 ± 0.002 mM·s⁻¹, respectively. The thermal stability of the immobilized GDO was enhanced at 30 °C, 40 °C, and 50 °C, compared to the free GDO. Stored at −20 °C, immobilized GDO retained more than 60% of its initial activity after 30 d, while the free enzyme completely lost its activity after 10 d. Furthermore, the immobilized nanoparticle–enzyme conjugate retained more than 50% enzyme activity up to the fifth cycle.