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This study aimed to explore the capacity of immobilized lipases on the acetylation of six aglycon flavonoids, namely myricetin, quercetin, luteolin, naringenin, fisetin and morin. For this purpose, lipase B from Candida antarctica (CaLB) and lipase from Thermomyces lanuginosus (TLL) were immobilized onto the surface of ZnOFe nanoparticles derived from an aqueous olive leaf extract. Various factors affecting the conversion of substrates and the formation of monoesterified and diesterified products, such as the amount of biocatalyst and the molar ratio of the substrates and reaction solvents were investigated. Both CaLB and TLL-ZnOFe achieved 100% conversion yield of naringenin to naringenin acetate after 72 h of reaction time, while TLL-ZnOFe achieved higher conversion yields of quercetin, morin and fisetin (73, 85 and 72% respectively). Notably, CaLB-ZnOFe displayed significantly lower conversion yields for morin compared with TLL-ZnOFe. Molecular docking analysis was used to elucidate this discrepancy, and it was revealed that the position of the hydroxyl groups of the B ring on morin introduced hindrances on the active site of CaLB. Finally, selected flavonoid esters showed significantly higher antimicrobial activity compared with the original compound. This work indicated that these lipase-based nanobiocatalysts can be successfully applied to produce lipophilic derivatives of aglycon flavonoids with improved antimicrobial activity. [ABSTRACT FROM AUTHOR]

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 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-β-D-Glucopyranoside and cellobiose. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 β-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this work, neoteric and biodegradable ionic liquids (ILs) based on various hydroxyl ammonium cations and formic acid anions have been used as media for biocatalytic oxidoreductions catalyzed by different metalloproteins. The effect of these ILs on the biocatalytic behavior and structure of solubilized enzymes was investigated using cytochrome c (cyt c) as a model protein. The use of IL-based media enhances the tolerance of cyt c against the denaturing effect of H2O2 and increases (up to 20 fold) its catalytic efficiency compared to that observed in buffer. This beneficial effect strongly correlates with the concentration of ILs used, as well as the chaotropicity of their cations. UV–vis, circular dichroism and Fourier transform infrared (FT-IR) spectroscopic studies indicated that, the effect of ILs on the catalytic behavior of cyt c could be correlated with slight structural changes on the protein molecule and/or perturbations of the heme microenvironment. The use of hydroxyl ammonium-based ILs as reaction media increased (up to 4-fold) the decolorization activity of cyt c. All ILs used were recycled and successfully reused three times indicating the potential application of these novel ILs as environmentally friendly media for biocatalytic processes of industrial interest. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The effect of storage on the antioxidant activity of pasteurized reconstituted orange juice was studied. Either heat or high pressure was used for pasteurization and total antioxidant activity and ascorbic acid loss were measured during storage at different temperatures (0, 5, 10, 15 °C). The total antioxidant activity of orange juice, calculated as the sum of the activities of different antioxidant compounds, decreased during storage. This was mainly because of loss of ascorbic acid. However, rates of degradation of ascorbic acid were lower for orange juice treated with high pressure and this led to a better retention of its antioxidant activity when compared with juice pasteurized in a conventional way. [ABSTRACT FROM AUTHOR]

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 Km constant followed by a decrease in Vmax 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]