28 research outputs found

    ENZYME BIOPROSPECTING OF MICROBIAL GLYCOSIDASES AND DIOXYGENASES FOR BIOCATALYSIS.

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    Main aim of this PhD project is the bioprospecting of different microbial enzymatic activities, to evaluate their biotechnological potential in different bioconversion processes. The characterization of four extradiol ring cleavage dioxygenases (ERCDs) and of a α-L-rhamnosidase isolated from Novosphingobium sp. PP1Y (N. sp. PP1Y) is described. In the last part of the project, gut microorganisms have been used for the identification and characterization of novel glycosyl hydrolases able to degrade the arabinogalactan polysaccharide (AG) of M. tuberculosis cell wall. In this work, the marine microorganism N. sp. PP1Y and the gut microorganism Bacteroides finegoldii were used as source for oxygenases and glycosyl hydrolases. More in detail, the optimization of recombinant expression and purification of four novel ERCDs from strain PP1Y allowed to obtain in good yields the corresponding proteins and to carry out their characterization. The activity screening using different catecholic substrates confirmed that these enzymes are able to catalyze the ring cleavage of a variety of mono- and polycyclic aromatic hydrocarbons with different sizes and conformation. In addition, the bioconversion of catechol estrogens, which could be used as precursors in the production of different steroid families and hormones, was evaluated. The results described in this thesis confirmed that these enzymes can be foreseen as a valuable tool for the modification of complex hydroxylated heterocyclic aromatic compounds, which are a starting point in the production pipeline of many pharmacologically active molecules, such as steroid-like molecules. Noteworthy, site-specific cleavage and subsequent modification of aromatic substrates, obtained by enzymatic biocatalysts, is of great advantage for industrial applications when compared to the complex mixture of products that are released instead during chemical modification procedures. In the second part of the PhD project, the biochemical characterization of RHA-P, a bacterial α-L-rhamnosidase isolated from the microorganism Novosphingobium sp. PP1Y, was performed. The active site topology and substrate specificity of RHA-P were investigated by homology modeling. The enzyme, whose recombinant expression and purification was optimized, resulted to be appealing from a biotechnological point of view for the bioconversion and de-rhamnosylation of natural flavonoids. The biotechnological use of either wild-type or mutant rhamnosidases is currently a need for food and beverages industry to improve the organoleptic properties of processed vegetal products. Moreover, the possibility of using efficient whole cells biocatalysts for expressing RHA-P has been described. This is particularly interesting because whole cells biocatalysts have numerous advantages in industrial bioconversion processes, allowing costs and process steps reduction. Finally, the bioprospecting of novel GHs from microorganisms belonging to the human gut microbiome (HGM), able to degrade the AG of M. tuberculosis cell wall, have been carried out. The isolation and characterization of different galactofuranosidases is described, which are able to completely degrade the mycobacteria galactan moiety of AG. Moreover, evidences for the presence of arabinofuranosidases were found for another HGM bacterium, which may be used as biocatalysts for the complete degradation of mycobacteria AG polysaccharide

    Identification of d -arabinan-degrading enzymes in mycobacteria

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    Bacterial cell growth and division require the coordinated action of enzymes that synthesize and degrade cell wall polymers. Here, we identify enzymes that cleave the D-arabinan core of arabinogalactan, an unusual component of the cell wall of Mycobacterium tuberculosis and other mycobacteria. We screened 14 human gut-derived Bacteroidetes for arabinogalactan-degrading activities and identified four families of glycoside hydrolases with activity against the D-arabinan or D-galactan components of arabinogalactan. Using one of these isolates with exo-D-galactofuranosidase activity, we generated enriched D-arabinan and used it to identify a strain of Dysgonomonas gadei as a D-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave D-arabinan, including members of the DUF2961 family (GH172) and a family of glycoside hydrolases (DUF4185/GH183) that display endo-D-arabinofuranase activity and are conserved in mycobacteria and other microbes. Mycobacterial genomes encode two conserved endo-D-arabinanases with different preferences for the D-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall

    Aptamers and Antisense Oligonucleotides for Diagnosis and Treatment of Hematological Diseases

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    Aptamers or chemical antibodies are single-stranded DNA or RNA oligonucleotides that bind proteins and small molecules with high affinity and specificity by recognizing tertiary or quaternary structures as antibodies. Aptamers can be easily produced in vitro through a process known as systemic evolution of ligands by exponential enrichment (SELEX) or a cell-based SELEX procedure. Aptamers and modified aptamers, such as slow, off-rate, modified aptamers (SOMAmers), can bind to target molecules with less polar and more hydrophobic interactions showing slower dissociation rates, higher stability, and resistance to nuclease degradation. Aptamers and SOMAmers are largely employed for multiplex high-throughput proteomics analysis with high reproducibility and reliability, for tumor cell detection by flow cytometry or microscopy for research and clinical purposes. In addition, aptamers are increasingly used for novel drug delivery systems specifically targeting tumor cells, and as new anticancer molecules. In this review, we summarize current preclinical and clinical applications of aptamers in malignant and non-malignant hematological diseases

    High-yield production in Escherichia coli and convenient purification of a candidate vaccine against SARS-CoV-2

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    Objectives: The aim of the present work was to identify a time-saving, effective, and low-cost strategy to produce in Escherichia coli a protein chimera representing a fusion anti-SARS-CoV-2 candidate vaccine, consisting of immunogenic and antigenic moieties. Results: We overexpressed in E. coli BL21(DE3) a synthetic gene coding for CRM197-RBD, and the target protein was detected in inclusion bodies. CRM197-RBD was solubilized with 1 % (w/v) of the anionic detergent N-lauroylsarcosine (sarkosyl), the removal of which from the protein solution was conveniently accomplished with Amberlite XAD-4. The detergent-free CRM197-RBD was then separated from contaminating DNA using polyethylenimine (PEI), and finally purified from PEI by salting out with ammonium sulfate. Structural (CD spectrum) and functional (DNase activity) assays revealed that the CRM197-RBD chimera featured a native and active conformation. Remarkably, we determined a yield of purified CRM197-RBD equal to 23 mg per litre of culture. Conclusions: To produce CRM197-RBD, we devised the use of sarkosyl as an alternative to urea to solubilize the target protein from E. coli inclusion bodies, and the easy removal of sarkosyl by means of Amberlite XAD-4

    Morphology, molecular interactions and H 2 O diffusion in a poly(lactic-acid)/graphene composite: A vibrational spectroscopy study

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    A composite system made of poly(L-lactic acid) (PLLA) and graphene nanoplatelets (GNP) was investigated by Raman and FTIR spectroscopy. Two compositions were prepared and characterized in comparison to the pristine polymer: they contained, respectively, 0.25 and 0.75 wt% of the nanofiller. The study was focused on the morphological properties of the system, and, in particular, on the level of dispersion and the homogeneity obtainable with the adopted preparation protocol. Furthermore, the possible molecular interactions taking place between the nanofiller and the polymer matrix were considered. Both the above issues were investigated by confocal Raman spectroscopy, with the aid of first-principle calculations to strengthen the spectral interpretation. Finally, the effect of the nanofiller on water diffusion was investigated by time-resolved FTIR spectroscopy, which provided accurate equilibrium and kinetic data, as well as molecular level information on the penetrant-to-substrate interactions. It was found that, for a 0.25 wt% composition, the adopted preparation protocol allowed us to achieve a dispersion at the level of single nanoplatelets, while for a 0.75 wt% composition, the GNP's aggregate into a co-continuous phase. PLLA/GNP interactions were detected by Raman spectroscopy, producing a detectable perturbation of the PLLA conformational equilibrium. Both the diffusivities and the equilibrium water uptake were found to decrease significantly by increasing the filler content

    Standard addition method (SAM) in LC-MS/MS to quantify gluten-derived metabolites in urine samples

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    : A tight adherence to a gluten-free diet (GFD), the most effective treatment currently available for celiac disease, is important to reduce symptoms, avoid nutritional deficiencies and improve quality of life in celiac patients. The development of analytical methods allowing detecting gluten exposure due to occasional or involuntary food transgressions could represent a useful tool to monitor patient habits and conditions and prevent long-term complications. The aim of this work was to develop and validate an approach based on the standard addition methodology (SAM) for the detection and quantification of two main metabolites of alkylresorcinols, 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-propanoic acid (DHPPA), whose presence in urine samples is related to the intake of gluten-containing foods. Analytically, the method consisted of a protein precipitation step followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. The chromatographic method involved the use of a hydrophilic interaction liquid chromatography (HILIC) in a direct phase approach; LC-MS/MS analyses were performed in selected reaction monitoring (SRM) mode. Manipulation and instrumental errors were normalised using stable isotopic standards (ISs). The SAM approach here described requires less than 1 mL of urine per sample, thus greatly reducing the sample volume needed. Noteworthy, despite the small cohort of samples analysed, our data allowed to identify a potential "threshold" value, around 200 ng/mL for DHBA and 400 ng/mL for DHPPA, to discriminate between a GFD and a gluten rich diet (GRD)

    Novel Drug Targets for the Treatment of Cardiac Diseases

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    Background: Cardiovascular disease is the leading cause of morbidity and mortality worldwide in developed countries, and its social and economic burden is expected to increase dramatically over the next decades. Despite significative improvement in the pharmacological treatment, and the huge advances in prevention, the quest for new molecular targets and for novel, more efficient and personalized therapies is still a priority for this group of pathologies. Objective: The paramount complexity of the metabolic networks responsible for the onset and progression of cardiovascular disease is highlighted by the wide and diverse array of new molecular targets recently described in literature. In this brief review, we focused our interest on a subset of promising molecular targets for the development of new pharmacological treatments specific for cardiac diseases such as coronary artery disease, heart failure and myocardial infarction. Conclusion: The global quest for new molecular targets for the treatment of cardiac diseases is leading to an impressive amount of records in the more recent literature. Although several promising molecular pathways have been identified so far, great caution should be used in considering all these targets effective in promoting the production of new drugs. The identification of suitable therapeutic targets is in fact an ongoing challenge that often lacks enough pre-clinical and clinical studies, which hinders the effective utilization of several new drugs due to a lack of efficacy or induction of safety liabilities

    Bioconversion of 4-hydroxyestradiol by extradiol ring-cleavage dioxygenases from Novosphingobium sp. PP1Y

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    Abstract Livestock breeding activities and pharmaceutical wastes lead to considerable accumulation of steroid hormones and estrogens in wastewaters. Here estrogens act as pro-cancerogenic agents and endocrine disruptors interfering with the sexual development of aquatic animals and having toxic effects in humans. Environmental bacteria play a vital role in estrogens degradation. Their wide reservoir of enzymes, such as ring cleavage dioxygenases (RCDs), can degrade the steroid nucleus, catalyzing the meta-cleavage of A, B or D steroid rings. In this work, 4 extra-diol ring cleavage dioxygenases (ERCDs), PP28735, PP26077, PP00124 and PP00193, were isolated from the marine sphingomonad Novosphingobium sp. PP1Y and characterized. Enzymes kinetic parameters were determined on different synthetic catecholic substrates. Then, the bioconversion of catechol estrogens was evaluated. PP00124 showed to be an efficient catalyst for the degradation of 4-hydroxyestradiol (4-OHE2), a carcinogenic hydroxylated derivate of E2. 4-OHE2 complete cleavage was obtained using PP00124 both in soluble form and in whole recombinant E. coli cells. LC–MS/MS analyses confirmed the generation of a semialdehyde product, through A-ring meta cleavage. To the best of our knowledge, PP00124 is the first characterized enzyme able to directly degrade 4-OHE2 via meta cleavage. Moreover, the complete 4-OHE2 biodegradation using recombinant whole cells highlighted advantages for bioremediation purposes

    Bioconversion of 4-hydroxyestradiol by extradiol ring-cleavage dioxygenases from Novosphingobium sp. PP1Y

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    Livestock breeding activities and pharmaceutical wastes lead to considerable accumulation of steroid hormones and estrogens in wastewaters. Here estrogens act as pro-cancerogenic agents and endocrine disruptors interfering with the sexual development of aquatic animals and having toxic efects in humans. Environmental bacteria play a vital role in estrogens degradation. Their wide reservoir of enzymes, such as ring cleavage dioxygenases (RCDs), can degrade the steroid nucleus, catalyzing the meta-cleavage of A, B or D steroid rings. In this work, 4 extra-diol ring cleavage dioxygenases (ERCDs), PP28735, PP26077, PP00124 and PP00193, were isolated from the marine sphingomonad Novosphingobium sp. PP1Y and characterized. Enzymes kinetic parameters were determined on different synthetic catecholic substrates. Then, the bioconversion of catechol estrogens was evaluated. PP00124 showed to be an efcient catalyst for the degradation of 4-hydroxyestradiol (4-OHE2), a carcinogenic hydroxylated derivate of E2. 4-OHE2 complete cleavage was obtained using PP00124 both in soluble form and in whole recombinant E. coli cells. LC–MS/MS analyses confrmed the generation of a semialdehyde product, through A-ring meta cleavage. To the best of our knowledge, PP00124 is the first characterized enzyme able to directly degrade 4-OHE2 via meta cleavage. Moreover, the complete 4-OHE2 biodegradation using recombinant whole cells highlighted advantages for bioremediation purposes
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