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.

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.

High Prevalence of Klebsiella pneumoniae in Greek Meat Products: Detection of Virulence and Antimicrobial Resistance Genes by Molecular Techniques.

Background: The presence of antimicrobial-resistant pathogens such as Klebsiella pneumoniae strains in the food supply is dangerous. The aim of this study was to assess the prevalence of Klebsiella pneumonia strains in Greek meat products and evaluate their phenotypes and genotypes. Methods: One hundred and ten meat specimens were cultured for the isolation of K. pneumoniae. In positive specimens, PCR (Polymerase Chain Reaction) analysis was performed to confirm the presence of K. pneumoniae. Genotypic and phenotypic evaluation of the isolated strains included multiplex immunoassay for the detection of carbapenemases, and PCR screening for the detection of resistance and virulence genes. Results:K. pneumoniae strains were recovered in 90 (81.8%) meat samples. The ecpA gene was identified in 30 (33.3%) isolates, while the fimH-1 and mrkA genes were present in 15 (16.7%) and 65 (72.2%) isolates, respectively. Sixty-five K. pneumoniae isolates (72.2%) were found to carry at least one resistance gene; of these, the blaNDM-like was the most commonly identified gene in 40 (61.5%) isolates, followed by the blaOXA-48 like gene in 20 isolates (30.8%). Conclusions: A high frequency of foodborne K. pneumoniae in Greece was found. Our results indicate that most strains carried resistance and virulence genes, indicating a high pathogenic potential and a significant risk to human health.

Production of hydroxytyrosol rich extract from Olea europaea leaf with enhanced biological activity using immobilized enzyme reactors.

As olive leaves constitute the main by-product of the olive oil industry with important environmental and economic impact, there is an increasing demand for its valorization. In the present work, we report the development and application of immobilized enzyme batch bioreactors for the chemo-enzymatic treatment of an aqueous Olea europaea leaf extract rich in oleuropein to produce an extract enriched in hydroxytyrosol and other oleuropein hydrolysis products. To this end, a robust biocatalyst was developed through the immobilization of ß-glucosidase on chitosan-coated magnetic beads which exhibited high hydrolytic stability after 240 h of incubation at 37 °C. The biocatalyst was successfully used in both a rotating bed-reactor and a stir-tank reactor for the modification of the olive leaf extract leading to high conversion yields of oleuropein (exceeding 90%), while an up to 2.5 times enrichment in hydroxytyrosol was achieved. Over 20 phenolic compounds (from different classes of phytochemicals such as flavonoids, secoiridoids, and their derivatives) were identified, in the extract before and after its modification through various chromatographic and spectroscopic techniques. Finally, the biological activity of both extracts was evaluated. Compared to the non-modified extract, the modified one demonstrated 20% higher antioxidant activity, seven-fold higher antibacterial activity, and enhanced cytotoxicity against leiomyosarcoma cells.

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.

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 biotransformations catalysed by enzyme-inorganic hybrid nanoflowers in environmentally friendly ionic solvents.

Environmentally friendly ionic solvents such as (a) ionic liquids (ILs) formulated with hydroxyl ammonium cations and various carboxylic acid anions and (b) choline chloride or ethyl ammonium chloride-based deep eutectic solvents (DES) were tested as media for hydrolytic and synthetic reactions catalysed by lipase-inorganic hybrid nanoflowers. The nature of ionic solvents used has a significant effect on the hydrolytic and synthetic activity of the immobilized lipase, as well as on its stability and reusability. In choline chloride-based DES, the activity and especially the operational stability of the biocatalyst are significantly increased compared to those observed in buffer, indicating the potential application of these solvents as green media for various biocatalytic processes of industrial interest.

Enzymatic hybridization of α-lipoic acid with bioactive compounds in ionic solvents.

The lipase-catalyzed molecular hybridization of α-lipoic acid (LA) with bioactive compounds pyridoxine, tyrosol and tyramine was performed in ionic solvents and deep eutectic solvents. The biocatalytic reactions were catalyzed by Candida antarctica lipase B immobilized onto various functionalized multi-walled carbon nanotubes (f-CNTs-CaLB), as well as by commercial Novozym 435. The use of f-CNTs-CaLB leads, in most cases, to higher conversion yields as compared to Novozym 435. The nature and ion composition of ionic solvents affect the performance of the biocatalytic process. The highest conversion yield was observed in (mtoa)NTf2. The high enzyme stability and the relatively low solubility of substrates in specific media account for the improved biocatalytic synthesis of molecular hybrids of LA. Principal component analysis was used to screen for potential lipoxygenase inhibitors. In vitro studies showed that the synthesized compounds exhibit up to 10-fold increased inhibitory activity on lipoxygenase mediated lipid peroxidation as compared to parent molecules.

Efficient enzymatic preparation of hydroxycinnamates in ionic liquids enhances their antioxidant effect on lipoproteins oxidative modification.

Biocatalytic lipophilization of hydroxycinnamic acids was performed in several BF(4)(-) and PF(6)(-) imidazolium ionic liquids using immobilized lipases. The influence of various reaction parameters on the performance of the biocatalytic process was pointed out, using as model reaction the esterification of ferulic acid. The biocatalytic lipophilization strongly depended on the ion composition of ionic liquids used. Conversions and initial reaction rates were significantly higher in PF(6)(-) as compared with BF(4)(-) ionic liquids and commonly used organic solvents. The high enzyme stability and the relative solubility of substrate versus product in PF(6)(-) ionic liquids can account for the improved synthesis of lipophilic ferulates. These lipophilic derivatives, when used at a concentration of up to 400-fold lower than the parental compound, efficiently inhibited the oxidation of isolated LDL, HDL and total serum in vitro. Moreover, it has been shown for the first time that the lipophilic ferulates improve the antioxidant efficiency of the HDL(3c) towards LDL in vitro oxidation.