22 research outputs found
Nucleósido 2'-desoxirribolsiltransferasa de "Bacillus psychrosaccharolyticus" CECT 4070
No full textTesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, Departamento de Bioquímica y Biología Molecular I, leída el 28-11-2014Sección Deptal. de Bioquímica y Biología Molecular (Biológicas)Fac. de Ciencias BiológicasTRUEunpu
A green synthesis of vidarabine 5’-monophosphate via a one-pot multienzymatic reaction catalyzed by immobilized biocatalysts
No full textIn nature, enzyme cascades can be found in many metabolic pathways. The idea of using multienzymatic
systems to mimic these processes is gaining interest for production of chemical
compounds. A type of multi-enzymatic application is the use of multiple enzymes for shifting
reaction equilibria. This strategy relies on removing intermediates, inhibitory products or byproducts,
via a second enzymatic reaction. In the context of a multi-enzymatic system, a one-pot
process uses more than one enzyme in a single reactor.1
We here describe a three-step sequential 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 immobilized biocatalysts involved in the biosynthesis
of nucleosides and nucleotides were used: uridine phosphorylase from Clostridium perfringens (CpUP),2
a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP),2 and deoxyadenosine kinase
from Dictyostelium discoideum (DddAK).3 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 (Scheme 1).
Reaction conditions (i.e. medium, temperature, immobilization carriers) and biocatalyst stability have
been balanced and optimized to achieve the highest productivity. Vidarabine 5’-monophosphate was
obtained in 95.5% conversion. Optimization of the purification step is in progress
A lateral flow assay for the rapid diagnosis of Mycobacterium bovis infection in wild boar
No full textThe native Eurasian wild boar (Sus scrofa ) is a reservoir of Mycobacterium bovis , the causative agent of animal tuberculosis (TB), a chronic disease in livestock, companion animals and wild mammals. Cases of M. bovis infection in wild boar or feral pig have been reported worldwide, making early detection a priority in the eradication of the disease. Point‐of‐care diagnostic tests, such as low cost lateral flow assays, provide high specificity and sensitivity and can be performed on site , an essential requirement for a rapid screening of wildlife. A lateral flow assay, LFA, (INgezim TB CROM Ab) for the detection of M. bovis ‐specific antibodies in wild boar serum and blood has been developed based on MPB83, one of the major immunogenic antigens of the bacterium. A total of 140 samples of wild boar serum, well‐characterized by Mycobacterium tuberculosis complex culture and TB compatible post‐mortem lesions, have been analysed with LFA, and results were compared with one in‐house and two commercial Enzyme‐linked Immunosorbent Assays (ELISA), INgezim TB Porcine and INgezim Tuberculosis DR. In experimental samples, the achieved values of sensitivity of the different techniques ranged from 84.3% to 92.1% and the specificity was 100% in all of them. In field animals, specificity ranged from 96% to 100%, whereas sensitivity ranged from 48% to 64% in juvenile wild boar, increasing to 93.3%–100% in adult wild boar. In particular, the total sensitivity and specificity values obtained with the new LFA were 83% and 97%, respectively, indicating that INgezim TB CROM Ab could be used as a first approach for the surveillance of TB in wild boar, with a special applicability for animal‐side testing.Part of this research was funded by the EU, Seventh Research Framework Program FP7‐KBBE‐2013‐7 under grant number nº 613799 (WildTBVac). This is a contribution to the WildDriver grant CGL2017‐89866 from MINECO and EU‐FEDER. We are very grateful to Dr. Mercedes Dominguez and her laboratory (Instituto de Salud Carlos III, Unidad de Inmunología Microbiana, Majadahonda, Spain) for their generous supply of P22 protein complex.Peer reviewe
PL3 amidase, a tailor-made lysin constructed by domain shuffling with potent killing activity against Pneumococci and related species
No full text13 p.-7 fig.-1 tab.The emergence and spread of antibiotic-resistant bacteria is pushing the need of
alternative treatments. In this context, phage therapy is already a reality to successfully
fight certain multiresistant bacteria. Among different phage gene products, murein
hydrolases responsible of phage progeny liberation (also called lysins or endolysins)
are weapons that target specific peptidoglycan bonds, leading to lysis and death of
susceptible bacteria when added from the outside. In the pneumococcal system, all
but one phage murein hydrolases reported to date share a choline-binding domain
that recognizes cell walls containing choline residues in the (lipo)teichoic acids. Some
purified pneumococcal or phage murein hydrolases, as well as several chimeric
proteins combining natural catalytic and cell wall-binding domains (CBDs) have been
used as effective antimicrobials. In this work we have constructed a novel chimeric
N-acetylmuramoyl-L-alanine amidase (PL3) by fusing the catalytic domain of the Pal
amidase (a phage-coded endolysin) to the CBD of the LytA amidase, the major
pneumococcal autolysin. The physicochemical properties of PL3 and the bacteriolytic
effect against several pneumococci (including 48 multiresistant representative strain)
and related species, like Streptococcus pseudopneumoniae, Streptococcus mitis, and
Streptococcus oralis, have been studied. Results have shown that low doses of PL3, in
the range of 0.5–5 mg/ml, are enough to practically sterilize all choline-containing strains
tested. Moreover, a single 20-mg dose of PL3 fully protected zebrafish embryos from
infection by S. pneumoniae D39 strain. Importantly, PL3 keeps 95% enzymatic activity
after 4 weeks at 37 C and can be lyophilized without losing activity, demonstrating
a remarkable robustness. Such stability, together with a prominent efficacy against a
narrow spectrum of human pathogens, confers to PL3 the characteristic to be an
effective therapeutic. In addition, our results demonstrate that the structure/functionbased domain shuffling approach is a successful method to construct tailor-made endolysins with higher bactericidal activities than their parental enzymes.Research wasfunded by grants from the Ministerio de Economía y Competitividad (MINECO) to PG (SAF2012-39444-
C02-01) and MM (BFU2012-36825 and BFU2015-70052-R), the Consejería de Educación de la Comunidad de Madrid
(S2010/BMD/2457) to MM. Additional funding was provided by the CIBER de Enfermedades Respiratorias (CIBERES),an initiative of the Instituto de Salud Carlos III (ISCIII).Peer reviewe
One-step enzymatic synthesis of nucleosides from low water-soluble purine bases in non-conventional media
No full textNew insights on nucleoside 2'-deoxyribosyltransferases: a versatile biocatalyst for one-pot one-step synthesis of nucleoside analogs
No full textIn recent years, glycosiltransferases have arisen as standard biocatalysts for the enzymatic synthesis of a wide variety of natural and non-natural nucleosides. Such enzymatic synthesis of nucleoside analogs catalyzed by nucleoside phosphorylases and 2'-deoxyribosyltransferases (NDTs) has demonstrated to be an efficient alternative to the traditional multistep chemical methods, since chemical glycosylation reactions include several protection-deprotection steps. This minireview exhaustively covers literature reports on this topic with the final aim of presenting NDTs as an efficient option to nucleoside phosphorylases for the synthesis of natural and non-natural nucleosides. Detailed comments about structure and catalytic mechanism of described NDTs, as well as their possible biological role, substrate specificity, and advances in detection of new enzyme specificities towards different non-natural nucleoside synthesis are included. In addition, optimization of enzymatic transglycosylation reactions and their application in the synthesis of natural and non-natural nucleosides have been described. Finally, immobilization of NDTs is shown as a practical procedure which leads to the preparation of very interesting biocatalysts applicable to industrial nucleoside synthesis.3.811 JCR (2013) Q1, 32/165 Biotechnology & applied microbiolog
Enzymatic synthesis of nucleoside analogues using immobilized 2′-deoxyribosyltransferase from Lactobacillus reuteri
No full textTwo serological approaches for detection of antibodies to SARS-CoV-2 in different scenarios: a screening tool and a point-of-care test
No full textSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 8 million people worldwide, becoming a pandemic. Detecting antibodies against SARS-CoV-2 is of utmost importance and a good indicator of exposure and circulation of the virus within the general population. Two serological tools based on a double recognition assay [enzyme-linked immunosorbent assay (DR-ELISA) and lateral flow assay (DR-LFA)] to detect total antibodies to SARS-CoV-2 have been developed based on the recombinant nucleocapsid protein. A total of 1065 serum samples, including positive for COVID-19 and negative samples from healthy donors or infected with other respiratory pathogens, were analyzed. The results showed values of sensitivity between 91.2% and 100%, and specificity of 100% and 98.2% for DR-LFA and DR-ELISA, respectively. No cross-reactivity against seasonal coronavirus (HCoV-NL63, HCoV-229E, HCoV-HKU1, HCoV-OC43) was found. These results demonstrate the importance of serology as a complementary tool to polymerase chain reaction for follow-up of recovered patients and identification of asymptomatic individuals
2′-deoxyribosyltransferase from bacillus psychrosaccharolyticus: A mesophilic-like biocatalyst for the synthesis of modified nucleosides from a psychrotolerant bacterium
No full textStructure-function relationships of a novel 2 -deoxyribosyltransferase from the psychrotolerant bacterium Bacillus psychrosaccharolyticus (BpNDT) have been exhaustively studied by biochemical and high resolution crystallographic analyses. Despite BpNDT exhibiting some structural features characteristic of cold-adapted enzymes such as localized flexibility in critical loops, its biochemical properties are typical of mesophilic enzymes. BpNDT is a highly symmetrical homohexamer with tightly associated subunits that possesses flexible and short loops bordering the active sites. The catalytic center is essentially identical to that of other mesophilic homologues. Moreover, BpNDT shows that it is a mesophilic-like enzyme since it is not heat-labile and exhibits an apparent unfolding temperature (T) of 49C, being active during 96 h at 40 and 50C. Finally, BpNDT synthesizes natural and modified nucleosides, with preference for purines as acceptors and pyrimidine nucleosides as donors. Remarkably, the synthesis of several therapeutic nucleosides has been efficiently carried out. In this sense, 5-hydroxymethyl-2 -deoxyuridine (5-HMdUrd), 7-deaza-6-hydroxypurine-2-deoxyriboside (7-DHPdRib) and theophylline-2 -deoxyriboside were synthesized for the first time by an NDT enzyme, showing the biotechnological interest of BpNDT.This work was supported by grant CTQ2009-11543 from the Spanish Ministry of Science and Innovation and grant S2009/PPQ-1752 (CAPOTE) from Comunidad de Madrid. José Miguel Mancheño acknowledges the ESRF for provision of synchrotron radiation facilities.Peer Reviewe
Nucleoside 2'-Deoxyribosyltransferase from Psychrophilic Bacterium Bacillus psychrosaccharolyticus — Preparation of an Immobilized Biocatalyst for the Enzymatic Synthesis of Therapeutic Nucleosides
No full textNucleoside 2'-deoxyribosyltransferase (NDT) from the psychrophilic bacterium Bacillus psychrosaccharolyticus CECT 4074 has been cloned and produced for the first time. A preliminary characterization of the recombinant protein indicates that the enzyme is an NDT type II since it catalyzes the transfer of 2'-deoxyribose between purines and pyrimidines. The enzyme (BpNDT) displays a high activity and stability in a broad range of pH and temperature. In addition, different approaches for the immobilization of BpNDT onto several supports have been studied in order to prepare a suitable biocatalyst for the one-step industrial enzymatic synthesis of different therapeutic nucleosides. Best results were obtained by adsorbing the enzyme on PEI-functionalized agarose and subsequent cross-linking with aldehyde-dextran (20 kDa and 70% oxidation degree). The immobilized enzyme could be recycled for at least 30 consecutive cycles in the synthesis of 2'-deoxyadenosine from 2'-deoxyuridine and adenine at 37 °C and pH 8.0, with a 25% loss of activity. High conversion yield of trifluridine (64.4%) was achieved in 2 h when 20 mM of 2'-deoxyuridine and 10 mM 5-trifluorothymine were employed in the transglycosylation reaction catalyzed by immobilized BpNDT at 37 °C and pH 7.5
