OXYGENASE ACTIVITIES FOR THE BIOSYNTHESIS OF AROMATIC ANTIOXIDANTS

Reactions in which organic compounds are oxygenated or hydroxylated are of great value for organic synthesis. The “oxyfunctionalization” of aromatic compounds is a modification of primary interest for the pharmaceutical and food industries allowing to obtain high-value-added compounds, characterized by a wide array of biological activities, starting from cheap and commercially available molecules. Selective oxyfunctionalization of organic substrates, however, can be a significant problem in organic synthesis, as these reactions are often carried out with strong oxidizing agents and occur with little chemo-, regio-, and enantio- selectivity. Thus, growing attention has been dedicated in the last years to the development of biotransformations, also known as bioconversions, which make use of the metabolic versatility of either purified enzymes or whole microorganisms to perform the oxyfunctionalization of organic substrates of industrial interest. These methodologies, compared with already established chemical processes, are appealing alternatives to obtain active aromatic compounds under mild experimental conditions and without employing toxic reagents. In this thesis project several aspects of this kind of biotransformation were analyzed; more in detail the attention was focused on: - The use of the bacterial multicomponent monooxygenase ToMO from Pseudomonas sp. OX1 for the production of novel hydroxylated antioxidants starting from commercially available aromatic precursors such as 2-phenoxyethanol, 2,3-dihydrobenzofuran, 2-indanol and phtalan. The antioxidant potential of the hydroxylated compounds obtained in ToMO-catalyzed bioconversion was assessed both in vitro, by using the DPPH assay, and ex vivo on the embryonic rat cardiomyoblast cell line H9c2 subjected to oxidative stress induced by sodium arsenite. Not all compounds showed antioxidant potential in the DPPH assay; however, when cells where incubated with any of them, a differential protective effect towards the oxidative stress induced by sodium arsenite was observed. - The recombinant expression of ToMO in the GRAS host microorganism Bacillus subtilis to analyze the potential use of this bacterium for the industrial scale-up of ToMO-catalyzed hydroxylation of aromatic substrates of interest. To this purpose, ToMO gene cluster was cloned in two different shuttle vectors, a non-integrative plasmid indicated as pHT01, and Pr19, a vector that allows instead the direct integration of the recombinant gene in B.subtilis chromosomal DNA through single crossing-over. In this latter case, no integration was observed. When using a non-integrative shuttle vector, the ToMO system was efficiently expressed in E.coli, but RT-PCR experiments showed that almost no mRNA corresponding to the tou gene cluster appeared to be transcribed when the plasmid was inserted in B.subtilis. - The expression and purification of the Baeyer Villiger 3,6-diketocamphane 1,6 monooxygenase (3,6 DKCMO) and its flavin reductase component from Pseudomonas putida NCIMB 10007. The optimization of the expression and the purification of these proteins will pave the way to the future biochemical characterization of this flavoenzyme and to its biotechnological use for the oxyfunctionalization of aromatic compounds of industrial interest

Alternative use of Bacillus subtilis spores: Protection against environmental oxidative stress in human normal keratinocytes

Inorganic trivalent arsenic is a major environmental pollutant and exposure to human results in many pathologies, including keratosis and carcinoma. Here, we analyzed the effects of B. subtilis spores on human normal keratinocytes in the presence of sodium arsenite oxidative stress. Pre-treatment of cells with spores before inducing oxidative stress was able to keep normal levels of intracellular ROS, GSH and lipid peroxidation, as well as to inhibit the activation of the MAPK cascade. Moreover, spores showed a positive effect on cell proliferation, probably due to their binding on the cell surface and the activation of intracellular catalases. We found that spores exert their protective effect by the nuclear translocation of Nrf-2, involved in the activation of stress response genes. This, in turn, resulted in a protective effect against sodium arsenite stress injury, as oxidative stress markers were reported to physiological levels when cells were stressed before incubating them with spores. Therefore, B. subtilis spores can be considered as a new agent to counteract oxidative stress on normal human keratinocytes

Display of the peroxiredoxin Bcp1 of Sulfolobus solfataricus on probiotic spores of Bacillus megaterium.

Bacterial spores displaying heterologous proteins have been proposed as a safe and efficient method for delivery of antigens and enzymes to animal mucosal surfaces. Initial studies have been performed using Bacillus subtilis spores, but other spore forming organisms have also been considered. B. megaterium spores have been shown capable of displaying large amounts of a model heterologous protein (Discosoma red fluorescent protein mRFP) that in part crossed the exosporium to localize in the space between the outer coat layer and the exosporium. Here, B. megaterium spores have been used to adsorb Bcp1 (bacterioferritin comigratory protein 1), a peroxiredoxin of the archaeon Sulfolobus solfataricus, known to have an antioxidant activity. The spores were highly efficient in adsorbing the heterologous enzyme which, once adsorbed, retained its activity. The adsorbed Bcp1 localized beneath the exosporium, filling the space between the outer coat and the exosporium. This unusual localization contributed to the stability of the enzyme-spore interaction and to the protection of the adsorbed enzyme in simulated intestinal or gastric conditions

CotG Mediates Spore Surface Permeability in Bacillus subtilis

: Proteins and glycoproteins that form the surface layers of the Bacillus spore assemble into semipermeable arrays that surround and protect the spore cytoplasm. Such layers, acting like molecular sieves, exclude large molecules but allow small nutrients (germinants) to penetrate. We report that CotG, a modular and abundant component of the Bacillus subtilis spore coat, controls spore permeability through its central region, formed by positively charged tandem repeats. These repeats act as spacers between the N and C termini of the protein, which are responsible for the interaction of CotG with at least one other coat protein. The deletion but not the replacement of the central repeats with differently charged repeats affects the spore resistance to lysozyme and the efficiency of germination-probably by reducing the coat permeability to external molecules. The presence of central repeats is a common feature of the CotG-like proteins present in most Bacillus species, and such a wide distribution of this protein family is suggestive of a relevant role for the structure and function of the Bacillus spore. IMPORTANCE Bacterial spores are quiescent cells extremely resistant to a variety of unphysiological conditions, including the presence of lytic enzymes. Such resistance is also due to the limited permeability of the spore surface, which does not allow lytic enzymes to reach the spore interior. This article proposes that the spore permeability in B. subtilis is mediated by CotG, a modular protein formed by a central region of repeats of positively charged amino acid acting as a "spacer" between the N and C termini. These, in turn, interact with other coat proteins, generating a protein layer whose permeability to external molecules is controlled by the distance between the N and C termini of CotG. This working model is most likely expandable to most sporeformers of the Bacillus genus, since they all have CotG-like proteins, not homologous to CotG of B. subtilis but similarly characterized by central repeats

Denatured lysozyme-coated carbon nanotubes: a versatile biohybrid material

Carbon nanotubes (CNTs) are among the most versatile nanomaterials, but their exploitation is hindered by limited dispersibility, especially in aqueous solvents. Here, we show that AP-LYS, a highly cationic soluble derivative of denatured hen egg lysozyme, is a very effective tool for the unbundling and solubilisation of CNTs. AP-LYS proved to mediate the complete and stable dispersion of CNTs at protein: CNT ratios ≥1: 3 (w:w) in very mild conditions (10-20 minutes sonication in ammonium acetate buffer, pH 5.0). Electrophoretic mobility and ζ-potential measurements confirmed that dispersed CNTs were coated by the protein, whereas molecular docking was used to study the interactions between AP-LYS and CNTs. AP-LYS-coated CNTs proved to be a very effective microbial cell-flocculating agent with an efficiency similar to that of chitosan, one of the best available flocculating agents, thus suggesting that this hybrid could find industrial applications in the treatment of wastewaters contaminated by microbial cells, or to remove microbial cells after fermentation processes. Moreover, we exploited the low stability of AP-LYS-coated CNT dispersions in eukaryotic cell culture media to prepare scaffolds with an extracellular matrix-like rough surface for the cultivation of eukaryotic cells

Exploring the Anticancer Potential of Premna resinosa (Hochst.) Leaf Surface Extract: Discovering New Diterpenes as Heat Shock Protein 70 (Hsp70) Binding Agents

Premna, a genus consisting of approximately 200 species, predominantly thrives in tropical and subtropical areas. Many of these species have been utilized in ethnopharmacology for diverse medicinal applications. In Saudi Arabia, Premna resinosa (Hochst.) Schauer (Lamiaceae) grows wildly, and its slightly viscid leaves are attributed to the production of leaf accession. In this study, we aimed to extract the surface accession from fresh leaves using dichloromethane to evaluate the anticancer potential. The plant exudate yielded two previously unknown labdane diterpenes, Premnaresone A and B, in addition to three already described congeners and four known flavonoids. The isolation process was accomplished using a combination of silica gel column chromatography and semi preparative HPLC, the structures of which were identified by NMR and HRESIMS analyses and a comparison with the literature data of associated compounds. Furthermore, we employed a density functional theory (DFT)/NMR approach to suggest the relative configuration of different compounds. Consequently, we investigated the possibility of developing new chaperone inhibitors by subjecting diterpenes 1–5 to a Surface Plasmon Resonance-screening, based on the knowledge that oridonin, a diterpene, interacts with Heat Shock Protein 70 (Hsp70) 1A in cancer cells. Additionally, we studied the anti-proliferative activity of compounds 1–5 on human Jurkat (human T-cell lymphoma) and HeLa (epithelial carcinoma) cell lines, where diterpene 3 exhibited activity in Jurkat cell lines after 48 h, with an IC50 of 15.21 ± 1.0 µM. Molecular docking and dynamic simulations revealed a robust interaction between compound 3 and Hsp70 key residues

Outer Membrane Vesicles Derived from Klebsiella pneumoniae Influence the miRNA Expression Profile in Human Bronchial Epithelial BEAS-2B Cells

: Klebsiella pneumoniae is an opportunistic pathogen that causes nosocomial and community-acquired infections. The spread of resistant strains of K. pneumoniae represents a growing threat to human health, due to the exhaustion of effective treatments. K. pneumoniae releases outer membrane vesicles (OMVs). OMVs are a vehicle for the transport of virulence factors to host cells, causing cell injury. Previous studies have shown changes of gene expression in human bronchial epithelial cells after treatment with K. pneumoniae OMVs. These variations in gene expression could be regulated through microRNAs (miRNAs), which participate in several biological mechanisms. Thereafter, miRNA expression profiles in human bronchial epithelial cells were evaluated during infection with standard and clinical K. pneumoniae strains. Microarray analysis and RT-qPCR identified the dysregulation of miR-223, hsa-miR-21, hsa-miR-25 and hsa-let-7g miRNA sequences. Target gene prediction revealed the essential role of these miRNAs in the regulation of host immune responses involving NF-ĸB (miR-223), TLR4 (hsa-miR-21), cytokine (hsa-miR-25) and IL-6 (hsa-let-7g miRNA) signalling pathways. The current study provides the first large scale expression profile of miRNAs from lung cells and predicted gene targets, following exposure to K. pneumoniae OMVs. Our results suggest the importance of OMVs in the inflammatory response

Ciencia de datos aplicada : Estudio de casos en diversas áreas, focalizado en la industria agropecuaria de la provincia de Rio Negro

Las organizaciones están buscando una nueva manera de producir optimizando y potenciando sus recursos, es por ello que están incorporando tecnologías que conectan el mundo físico con el virtual a través de diversos dispositivos que le permitan acceder a grandes cantidades de datos en tiempo real y tomar mejores decisiones en todo el proceso productivo. Por otra parte, la Ciencia de Datos es un conjunto de principios fundamentales que apoyan y guían la extracción de información y conocimiento a partir de los datos; incluye diversas metodologías, técnicas, algoritmos y herramientas que facilitan el procesamiento avanzado y automático de los mismos; permitiendo identificar información relevante y estratégica, que a simple vista no es detectada. Este proyecto tiene por objetivo articular integradamente mecanismos de proceso de interpretación de grandes masas de información en diversas industrias, (con hincapié en la agropecuaria de la provincia de Río Negro), a través diversas técnicas de tratamiento de datos y su visualización brindada por la Ciencia de Datos.Eje: Bases de Datos y Minería de Datos.Red de Universidades con Carreras en Informátic