Laccase-Mediated Oxidation of Phenolic Compounds from Wine Lees Extract towards the Synthesis of Polymers with Potential Applications in Food Packaging.

Laccase from Trametes versicolor was applied to produce phenolic polymeric compounds with enhanced properties, using a wine lees extract as the phenolic source. The influence of the incubation time on the progress of the enzymatic oxidation and the yield of the formed polymers was examined. The polymerization process and the properties of the polymeric products were evaluated with a variety of techniques, such as high-pressure liquid chromatography (HPLC) and gel permeation chromatography (GPC), Fourier-transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies, differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The enzymatic polymerization reaction resulted in an 82% reduction in the free phenolic compounds of the extract. The polymeric product recovery (up to 25.7%) and the molecular weight of the polymer depended on the incubation time of the reaction. The produced phenolic polymers exhibited high antioxidant activity, depending on the enzymatic oxidation reaction time, with the phenolic polymer formed after one hour of enzymatic reaction exhibiting the highest antioxidant activity (133.75 and 164.77 µg TE mg-1 polymer) towards the ABTS and DPPH free radicals, respectively. The higher thermal stability of the polymeric products compared to the wine lees phenolic extract was confirmed with TGA and DSC analyses. Finally, the formed phenolic polymeric products were incorporated into chitosan films, providing them with increased antioxidant activity without affecting the films' cohesion.

Enhancement of the biological activity of hydroxytyrosol through its oxidation by laccase from Trametes versicolor.

The laccase-catalyzed oxidation of hydroxytyrosol (HT) towards the formation of its bioactive oligomer derivatives was investigated. The biocatalytic oligomerization was catalyzed by laccase from Trametes versicolor in aqueous or various water-miscible organic solvents and deep eutectic solvent (DES)-based media. Mass Spectroscopy and Nuclear Magnetic Resonance were used for the characterization of the products. The solvent system used significantly affects the degree of HT oligomerization. The use of 50 % v/v methanol favored the production of the HT dimer, while other organic solvents as well as DESs led to the formation of hydroxytyrosol trimer and other oligomers. In vitro studies showed that the HT dimer exhibits 3- to 4-fold enhanced antibacterial activity against Gram-positive and Gram-negative bacteria compared to the parent compound. Moreover, the ability of HT dimer to inhibit the activity of soybean lipoxygenase and Candida rugosa lipase was 1.5-fold higher than HT, while molecular docking supported these results. Furthermore, HT dimer showed reduced cytotoxicity against HEK293 cells and exhibited a strong ability to inhibit ROS formation. The enhanced bioactivity of HT dimer indicates that this compound could be considered for use in cosmetics, skin-care products, and nutraceuticals.

Mycoprotein Production by Submerged Fermentation of the Edible Mushroom Pleurotus ostreatus in a Batch Stirred Tank Bioreactor Using Agro-Industrial Hydrolysate.

The demand for cheap, healthy, and sustainable alternative protein sources has turned research interest into microbial proteins. Mycoproteins prevail due to their quite balanced amino acid profile, low carbon footprint and high sustainability potential. The goal of this research was to investigate the capability of Pleurotus ostreatus to metabolize the main sugars of agro-industrial side streams, such as aspen wood chips hydrolysate, to produce high-value protein with low cost. Our results indicate that P. ostreatus LGAM 1123 could be cultivated both in a C-6 (glucose)- and C-5(xylose)-sugar-containing medium for mycoprotein production. A mixture of glucose and xylose was found to be ideal for biomass production with high protein content and rich amino acid profile. P. ostreatus LGAM 1123 cultivation in a 4 L stirred-tank bioreactor using aspen hydrolysate was achieved with 25.0 ± 3.4 g L-1 biomass production, 1.8 ± 0.4 d-1 specific growth rate and a protein yield of 54.5 ± 0.5% (g/100 g sugars). PCA analysis of the amino acids revealed a strong correlation between the amino acid composition of the protein produced and the ratios of glucose and xylose in the culture medium. The production of high-nutrient mycoprotein by submerged fermentation of the edible fungus P. ostreatus using agro-industrial hydrolysates is a promising bioprocess in the food and feed industry.

Substrate Specificity of the Highly Thermostable Esterase EstDZ3.

Esterases are among the most studied enzymes, and their applications expand into several branches of industrial biotechnology. Yet, despite the fact that information on their substrate specificity is crucial for selecting or designing the best fitted biocatalyst for the desired application, it cannot be predicted from their amino acid sequence. In this work, we studied the substrate scope of the newly discovered hydrolytic extremozyme, EstDZ3, against a library of esters with variable carbon chain lengths in an effort to understand the crucial amino acids for the substrate selectivity of this enzyme. EstDZ3 appears to be active against a wide range of esters with high selectivity towards medium- to long-carbon chain vinyl esters. In-silico studies of its 3D structure revealed that the selectivity might arise from the mainly hydrophobic nature of the active site's environment.

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.

NGIWY-Amide: A Bioinspired Ultrashort Self-Assembled Peptide Gelator for Local Drug Delivery Applications.

Fibrillar structures derived from plant or animal origin have long been a source of inspiration for the design of new biomaterials. The Asn-Gly-Ile-Trp-Tyr-NH2 (NGIWY-amide) pentapeptide, isolated from the sea cucumber Apostichopus japonicus, which spontaneously self-assembles in water to form hydrogel, pertains to this category. In this study, we evaluated this ultra-short cosmetic bioinspired peptide as vector for local drug delivery applications. Combining nuclear magnetic resonance, circular dichroism, infrared spectroscopy, X-ray diffraction, and rheological studies, the synthesized pentapeptide formed a stiff hydrogel with a high ß-sheet content. Molecular dynamic simulations aligned well with scanning electron and atomic-force microscopy studies, revealing a highly filamentous structure with the fibers adopting a helical-twisted morphology. Model dye localization within the supramolecular hydrogel provided insights on the preferential distribution of hydrophobic and hydrophilic compounds in the hydrogel network. That was further depicted in the diffusion kinetics of drugs differing in their aqueous solubility and molecular weight, namely, doxorubicin hydrochloride, curcumin, and octreotide acetate, highlighting its versatility as a delivery vector of both hydrophobic and hydrophilic compounds of different molecular weight. Along with the observed cytocompatibility of the hydrogel, the NGIWY-amide pentapeptide may offer new approaches for cell growth, drug delivery, and 3D bioprinting tissue-engineering applications.

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.

Germanane Monolayer Films as Antibacterial Coatings.

Germanane (GeH), a graphane analogue, has attracted significant interest because of its optoelectronic properties; however, the environmental and biological effects of GeH have scarcely been investigated so far. Here we report a facile approach based on the Langmuir-Schaefer deposition to produce homogeneous and dense GeH monolayer films on various substrates. In view of possible applications and to extend the use of GeH to unexplored fields, we investigated its antibacterial activity for the first time and found that this promising 2D structure exhibits remarkable antibacterial activity against both Gram-negative and Gram-positive bacterial strains.

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.

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.

The NMR tube bioreactor.

Enzymes are pliable systems and core cellular components allowing the performance of several processes. They can also be utilized as "green" synthetic factories to generate bioactive therapeutic, diagnostic or theranostic compounds. Methods to enable the mapping of enzyme substrates as well as the understanding of the interactions of the formed products with target proteins could be of importance. This chapter describes the utilization of the "NMR tube bioreactor" method. This method, carried out inside an NMR tube, can allow for the prediction of compounds that are able to serve as potential enzyme substrates, and also exploit the regioselectivity of the enzymatic reactions. Furthermore, it enables the real time monitoring of multiple-biotransformation products in the NMR tube without the need of fractionation or isolation of each individual component. Finally, it allows for the screening of the resulting biotransformation products as ligands for protein targets.

Enzymatic Conversion of Oleuropein to Hydroxytyrosol Using Immobilized ß-Glucosidase on Porous Carbon Cuboids.

In the present study, we developed novel ß-glucosidase-based nano-biocatalysts for the bioconversion of oleuropein to hydroxytyrosol. Using non-covalent or covalent immobilization approaches, ß-glucosidases from almonds and Thermotoga maritima were attached for the first time on oxidized and non-oxidized porous carbon cuboids (PCC). Various methods were used for the characterization of the bio-nanoconjugates, such as Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and fluorescence spectroscopy. The oxidation state of the nanο-support and the immobilization procedure play a key role for the immobilization efficiency or the catalytic activity of the immobilized ß-glucosidases. The nano-biocatalysts were successfully used for the hydrolysis of oleuropein, which leads to the formation of its bioactive derivative, hydroxytyrosol (up to 2.4 g L-1), which is a phenolic compound with numerous health benefits. The bio-nanoconjugates exhibited high thermal and operational stability (up to 240 hours of repeated use), which indicated that they are efficient tools for various bio-transformations.

Stabilization of Laccase Through Immobilization on Functionalized GO-Derivatives.

This chapter deals with the use of functionalized derivatives of graphene oxide as nanoscaffolds for the immobilization and stabilization of laccase from Trametes versicolor. Covalent and noncovalent immobilization approaches are described, while a novel method for the development of laccase-based multilayer nanoassemblies is also presented. Various biochemical, spectroscopic, and microscopic techniques were applied to characterize the nanobiocatalytic systems in respect to their microstructure and catalytic performance. Laccase-GO nanosystems were characterized with FTIR spectroscopy in order to confirm the functionalization of the nanomaterials, as well as to interpret the nanomaterial-enzyme interactions, while the multilayer structure of laccase-based multilayer nanoassemblies was confirmed by atomic force microscopy. The nanobiocatalytic systems presented here demonstrated exceptional stability and reusability compared with the free enzyme form, leading to robust biocatalytic systems appropriate for various applications of industrial interest.

Enriching the biological space of natural products and charting drug metabolites, through real time biotransformation monitoring: The NMR tube bioreactor.

BACKGROUND: Natural products offer a wide range of biological activities, but they are not easily integrated in the drug discovery pipeline, because of their inherent scaffold intricacy and the associated complexity in their synthetic chemistry. Enzymes may be used to perform regioselective and stereoselective incorporation of functional groups in the natural product core, avoiding harsh reaction conditions, several protection/deprotection and purification steps. METHODS: Herein, we developed a three step protocol carried out inside an NMR-tube. 1st-step: STD-NMR was used to predict the: i) capacity of natural products as enzyme substrates and ii) possible regioselectivity of the biotransformations. 2nd-step: The real-time formation of multiple-biotransformation products in the NMR-tube bioreactor was monitored in-situ. 3rd-step: STD-NMR was applied in the mixture of the biotransformed products to screen ligands for protein targets. RESULTS: Herein, we developed a simple and time-effective process, the "NMR-tube bioreactor", that is able to: (i) predict which component of a mixture of natural products can be enzymatically transformed, (ii) monitor in situ the transformation efficacy and regioselectivity in crude extracts and multiple substrate biotransformations without fractionation and (iii) simultaneously screen for interactions of the biotransformation products with pharmaceutical protein targets. CONCLUSIONS: We have developed a green, time-, and cost-effective process that provide a simple route from natural products to lead compounds for drug discovery. GENERAL SIGNIFICANSE: This process can speed up the most crucial steps in the early drug discovery process, and reduce the chemical manipulations usually involved in the pipeline, improving the environmental compatibility.

Mapping the interactions and bioactivity of quercetin-(2-hydroxypropyl)-ß-cyclodextrin complex.

Natural products have served as a rich source for drug discovery and development. In the last decade their fruitful integration in the drug discovery pipeline declined due to their reduced bioavailability, mainly attributed to their poor aqueous solubility. We synthesized a quercetin (QUE)-(2-hydroxypropyl)-ß-cyclodextrin (HP-ß-CD) complex that enabled amplification of its solubility and in the same time retained its bioactivity in T24 human bladder cancer cell line. The stability of the complex and the molecular basis of the interactions developed in this host-guest complex were assayed by incorporating an array of analytical techniques and Molecular Dynamics (MD) experiments. 2D DOSY NMR experiment revealed that the diffusion coefficient of free HP-ß-CD was 3.55×10(-10)m(2)s(-1) while that of QUE-HP-ß-CD inclusion complex 3.09×10(-10)m(2)s(-1), indicating the formation of a complex. Solid and liquid high resolution NMR spectroscopy data showed that the most pronounced differences in chemical shifts at carbons and protons correspondingly during complexation occur in the aromatic ring Α (bearing the two phenolic hydroxyl groups meta to each other). The chemical shift differences in the aromatic ring Β (bearing the two phenolic hydroxyl groups ortho to each other) were less pronounced. The MD results confirmed the experimental data.

Regioselective chemical and rapid enzymatic synthesis of a novel redox ­ Antiproliferative molecular hybrid.

Recent science evidenced the interlinkage of oxidative stress and cancer. Due to the inherent complexity of cancer and its accompanying effect of oxidative stress, novel molecules, containing combinatorial functionalities should be targeted. Herein, we synthesized gemcitabine-5'-O-lipoate derived from a regioselective coupling of the chemotherapeutic drug gemcitabine (GEM), a first-line agent for cancer therapy and α-lipoic acid (LA), a potent antioxidant. gemcitabine-5'-O-lipoate was obtained in 4 chemical steps. To avoid the tedious and laborious chemical steps we also utilized biocatalysis using immobilized Candida antarctica lipase B (CALB), and the optimum conditions for the regioselective and one-pot synthesis of gemcitabine-5'-O-lipoate were established by exploiting different solvents (organic solvents and ionic liquids) and enzyme immobilization (acrylic resin and carbon nanotubes). Cytotoxic activity of co-administrating GEM and LA was proven to be synergistic against non-small cell lung cancer cells whereas antagonistic for bladder cancer cells. In contrast, the gemcitabine-5'-O-lipoate hybrid was found to be superior to the parent compounds against both non-small cell lung cancer and bladder cancer cells as also was found to preserve the redox activity of the parent compound LA. The regioselective biotransformation mediated synthesis of the anticancer-antioxidant hybrid illustrates the capacity of biocatalysis to act as an asset in molecular hybridization techniques.

Investigation of the interactions of silibinin with 2-hydroxypropyl-ß-cyclodextrin through biophysical techniques and computational methods.

Cyclodextrins (CDs) are a well-known class of supermolecules that have been widely used to protect drugs against conjugation and metabolic inactivation as well as to enhance the aqueous solubility and hence to ameliorate the oral bioavailability of sparingly soluble drug molecules. The hepatoprotectant drug silibinin can be incorporated into CDs, and here we elucidate the interaction between the drug and the host at the molecular level. The complexation product of silibinin with 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) is characterized by Differential Scanning Calorimetry, mass spectrometry, solid and liquid high-resolution NMR spectroscopy. The chemical shift changes using (13)C CP/MAS on the complexing of the guest with the host provided significant information on the molecular interactions, and they were in agreement with the 2D NOESY results. These results point out that in both solid and liquid forms, the drug is engulfed and interacts with HP-ß-CD in identical manner. Molecular dynamics calculations have been performed to examine the thermodynamic characteristics associated with the silibinin-HP-ß-CD interactions and to study the stability of the complex. To approximate the physiological conditions, the aqueous solubility and dissolution characteristics of the complex at pH states simulating those of the upper gastrointestinal tract have been applied. To evaluate the antiproliferative activity of silibinin-HP-ß-CD complex comparatively to silibinin in MCF-7 human cancer cells, MTT assays have been performed.