A multi-enzymatic cascade reaction for the synthesis of vidarabine 5'-monophosphate

We here described a three-step multi-enzymatic reaction for the one-pot synthesis of vidarabine 5'-monophosphate (araA-MP), an antiviral drug, using arabinosyluracil (araU), adenine (Ade), and adenosine triphosphate (ATP) as precursors. To this aim, three enzymes involved in the biosynthesis of nucleosides and nucleotides were used in a cascade mode after immobilization: uridine phosphorylase from Clostridium perfringens (CpUP), a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), and deoxyadenosine kinase from Dictyostelium discoideum (DddAK). Specifically, CpUP catalyzes the phosphorolysis of araU thus generating uracil and α-d-arabinose-1-phosphate. AhPNP catalyzes the coupling between this latter compound and Ade to form araA (vidarabine). This nucleoside becomes the substrate of DddAK, which produces the 5'-mononucleotide counterpart (araA-MP) using ATP as the phosphate donor. Reaction conditions (i.e., medium, temperature, immobilization carriers) and biocatalyst stability have been balanced to achieve the highest conversion of vidarabine 5'-monophosphate (≥95.5%). The combination of the nucleoside phosphorylases twosome with deoxyadenosine kinase in a one-pot cascade allowed (i) a complete shift in the equilibrium-controlled synthesis of the nucleoside towards the product formation; and (ii) to overcome the solubility constraints of araA in aqueous medium, thus providing a new route to the highly productive synthesis of araA-MP

On the Threat of Systematic Jamming of GNSS

This paper presents a study of the threat of malicious interference to GNSS and examines the special case where the jamming device is incrementally more sophisticated than a typical always-on interference source. The concept of a systematic jamming attack is considered, where the interference signal is intentionally synchronized with the GNSS signals, with the intention of causing maximum disruption with the minimum power expenditure. Various attack methodologies are examined for the case of a civilian L1 receiver. It is shown that, depending on the attack strategy, the target signal and the target receiver, data-recovery, navigation and timing can be denied to a user with some tens of decibels less average power than a traditional jamming attack. It is further shown that some attacks may be capable to effectively deny some receiver functionality in a subtle manner such that presence of the malicious interference goes undetected. Key signal and receiver features that expose a vulnerability are identified and some means of improving receiver robustness are provided

Getting better all the time - The Continued Evolution of the GNSS Software-Defined Radio

Software Defined Radio (SDR) has an infinite number of interpretations depending on the context in which it is designed and used. By way of a starting definition the authors choose to use that of ‘a reconfigurable radio system whose characteristics are partially or fully defined via software or firmware’. In various forms, SDR has permeated a wide range of user groups, from military, business, academia and to the amateur radio enthusiast

Immobilization of old yellow enzymes via covalent or coordination bonds

Ene-reductases (ERs) belonging to the old yellow enzyme (OYE) family have been thoroughly investigated for the stereospecific reduction of activated prochiral C=C double bonds. In this work, OYE3 was immobilized both by covalent binding on glyoxyl-agarose (OYE3-GA), and by affinity-based adsorption on EziG™ particles (OYE3-EziG). The immobilized OYE3-GA was demonstrated to be active (activity recovery = 52%) and to retain almost 100% of its activity under the enzymatic assay conditions (50 mM phosphate buffer pH 7, 28 °C) for six days, whereas the activity of the non-immobilized enzyme dropped to 50% after two days. In the case of EziG™, the highest activity recovery (54%) was achieved by using the most hydrophilic carrier (EziG™ Opal) that was selected for the full characterization of this type of enzyme preparation (stability, recycling, re-use, enzyme leakage). OYE3-EziG was slightly less stable than OYE3-GA under the same experimental conditions. OYE3-GA could be recycled and re-used for up to 12 reaction cycles in the bioreduction of α-methyl-trans-cinnamaldehyde; after 12 runs, the highest conversion achieved was 40%. In the case of the co-immobilized OYE3/GDH-EziG, the conversion dropped to 56% after two reaction cycles. No enzyme leakage was detected over 48 h for both OYE3-GA and OYE3/GDH-EziG (50 mM phosphate buffer pH 7, 28 °C). These seed results pave the way for a true optimization of the immobilization of OYE3, as well as for the use of immobilized OYE3 for preparative applications both in batch and continuous flow conditions

Synthesis of Ribavirin, Tecadenoson, and Cladribine by enzymatic transglycosylation

Despite the impressive progress in nucleoside chemistry to date, the synthesis of nucleoside analogues is still a challenge. Chemoenzymatic synthesis has been proven to overcome most of the constraints of conventional nucleoside chemistry. A purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) has been used herein to catalyze the synthesis of Ribavirin, Tecadenoson, and Cladribine, by a “one-pot, one-enzyme” transglycosylation, which is the transfer of the carbohydrate moiety from a nucleoside donor to a heterocyclic base. As the sugar donor, 7-methylguanosine iodide and its 2′-deoxy counterpart were synthesized and incubated either with the “purine-like” base or the modified purine of the three selected APIs. Good conversions (49-67%) were achieved in all cases under screening conditions. Following this synthetic scheme, 7-methylguanine arabinoside iodide was also prepared with the purpose to synthesize the antiviral Vidarabine by a novel approach. However, in this case, neither the phosphorolysis of the sugar donor, nor the transglycosylation reaction were observed. This study was enlarged to two other ribonucleosides structurally related to Ribavirin and Tecadenoson, namely, Acadesine, or AICAR, and 2-chloro-N6-cyclopentyladenosine, or CCPA. Only the formation of CCPA was observed (52%). This study paves the way for the development of a new synthesis of the target APIs at a preparative scale. Furthermore, the screening herein reported contributes to the collection of new data about the specific substrate requirements of AhPNP

Seroconversion and indolent course of COVID-19 in patients with multiple sclerosis treated with fingolimod and teriflunomide

Non applicale in quanto si tratta di di una Letter to the Edito

Immobilization of γ-Glutamyl Transpeptidase from Equine Kidney for the Synthesis of kokumi Compounds

\u3b3-Glutamyl transpeptidase from equine kidney (ekGGT, E.C. 2.3.2.2) is an intrinsic membrane enzyme which transfers the \u3b3-glutamyl moiety of glutathione to amino acids and peptides, thus producing \u3b3-glutamyl derivatives. An immobilization study of ekGGT was carried out with the aim to develop a robust biocatalyst for the synthesis of \u3b3-glutamyl amino acids which are known as kokumi compounds. Heterofunctional octyl-glyoxyl-agarose resulted in a high immobilization yield and activity recovery (93 % and 88 %, respectively). Immobilized ekGGT retained more than 95 % activity under reaction conditions (Tris-HCl, pH 9, 0.05 M) after 6 days, whereas the residual activity after 6 reaction cycles (18 days) was 85 %. The synthesis of \u3b3-glutamylmethionine catalyzed by octyl-glyoxyl-agarose-ekGGT afforded the product in 42 % yield (101 mg). The immobilized ekGGT was characterized by Raman spectroscopy. The immobilization protocol developed for ekGGT could be of general applicability to membrane proteins

Immobilization of γ-Glutamyl Transpeptidase from Equine Kidney for the Synthesis of Kokumi Compounds

γ-Glutamyl transpeptidase from equine kidney (ekGGT, E.C. 2.3.2.2) is an intrinsic membrane enzyme which transfers the γ-glutamyl moiety of glutathione to amino acids and peptides, thus producing γ-glutamyl derivatives. An immobilization study of ekGGT was carried out with the aim to develop a robust biocatalyst for the synthesis of γ-glutamyl amino acids which are known as kokumi compounds. Heterofunctional octyl-glyoxyl-agarose resulted in a high immobilization yield and activity recovery (93 % and 88 %, respectively). Immobilized ekGGT retained more than 95 % activity under reaction conditions (Tris-HCl, pH 9, 0.05 M) after 6 days, whereas the residual activity after 6 reaction cycles (18 days) was 85 %. The synthesis of γ-glutamylmethionine catalyzed by octyl-glyoxyl-agarose-ekGGT afforded the product in 42 % yield (101 mg). The immobilized ekGGT was characterized by Raman spectroscopy. The immobilization protocol developed for ekGGT could be of general applicability to membrane proteins

Biocatalysis for biomass valorization: peptides and fatty acids from rice bran

Waste upgrading practises have attracted a significant attention in recent years with the aim of managing agrofood by-products in a gainful and sustainable way. We describe here how biocatalysis can assist rice bran valorization, according to the biorefinery concept. [1] Rice is the staple food for over half the world's population. Rice milling generates a massive amount of waste, namely rice bran (70 kg/ton of rice) and rice husk (200 kg/ton of rice). Rice bran (RB), containing fibers (7-11%), proteins (10-16%), lipids (15-22%), carbohydrates (34-52%), micronutrients, represents a second-generation biomass. [2] Rice bran proteins (RBP) have a high nutritional value and optimal digestibility and are gluten-free, hypoallergenic and rich in essential amino acids. However, the first hurdle to be overcome for RBP production and large scale application is their extraction. Structural complexity, poor solubility, and strong aggregation make RBP hardly available. The sequential treatment of RB with carbohydrases and proteases was used to prepare mixtures of water-soluble peptides (RBPHs, RBP Hydrolysates) to be tested as antibacterial, antioxidant and anticholesterol agents, as well as flavour enhancers. [3] Interestingly, sensory analysis revealed that the obtained RBPHs exert only sweet and umami taste. Rice bran oil (RBO) is one of the most underutilized agricultural commodities. We investigated the use of RBO as a feedstock for the production of FFA-derived chemicals (e.g. sugar fatty acid esters). [4] To this aim, RBO was submitted to a preparative lipase-catalyzed hydrolysis to obtain pure FFA. [5] The high acidity of RBO, so far considered as a bottleneck in the exploitation of RBO (i.e. biodiesel production) was here turned into an advantage, making available FFA mixtures as synthetic precursors for high added value products

Design of epidermal growth factor immobilization on 3D biocompatible scaffolds to promote tissue repair and regeneration

Exogenous application of human epidermal growth factor (hEGF) stimulates epidermal wound healing. The aim of this study was to develop bioconjugates based on hEGF mimicking the protein in its native state and thus suitable for tissue engineering applications, in particular for treating skin-related disorders as burns. Ribonuclease A (RNase A) was used to investigate a number of different activated-agarose carriers: cyanogen bromide (CNBr)-activated-agarose and glyoxyl-agarose showed to preserve the appropriate orientation of the protein for receptor binding. EGF was immobilized on these carriers and immobilization yield was evaluated (100% and 12%, respectively). A peptide mapping of unbound protein regions was carried out by LC–MS to take evidence of the residues involved in the immobilization and, consequently, the flexibility and surface accessibility of immobilized EGF. To assess cell proliferative activities, 10, 25, 50, and 100 ng/mL of each immobilized EGF sample were seeded on fibroblast cells and incubated for 24, 48 and 72 h. The immobilized growth factor showed significantly high cell proliferative activity at 50 and 100 ng/mL compared to control and soluble EGF. Although both of the immobilized samples show dose-dependency when seeded with high number of fibroblast cells, CNBr-agarose-EGF showed a significantly high activity at 100 ng/mL and 72 h incubation, compared to glyoxyl-agarose-EGF