Nucleósido 2'-desoxirribolsiltransferasa de "Bacillus psychrosaccharolyticus" CECT 4070

Tesis 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

Nucleoside 2\u27-Deoxyribosyltransferase from Psychrophilic Bacterium Bacillus psychrosaccharolyticus — Preparation of an Immobilized Biocatalyst for the Enzymatic Synthesis of Therapeutic Nucleosides

Nucleoside 2\u27-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\u27-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\u27-deoxyadenosine from 2\u27-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\u27-deoxyuridine and 10 mM 5-trifluorothymine were employed in the transglycosylation reaction catalyzed by immobilized BpNDT at 37 °C and pH 7.5

PL3 amidase, a tailor-made lysin constructed by domain shuffling with potent killing activity against Pneumococci and related species

13 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

New insights on nucleoside 2'-deoxyribosyltransferases: a versatile biocatalyst for one-pot one-step synthesis of nucleoside analogs

In 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

Synthesis of therapeutic nucleoside analogues using nucleoside 2’-deoxyribosyltransferase from Bacillus psychrosaccharolyticus

No data (2014)UE

Development of an immobilized biocatalyst based on Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine

The immobilization of Bacillus psychrosaccharolyticus nucleoside 2′-deoxyribosyltransferase was deeply investigated and finally optimized. The best immobilization procedure resulted to be ionic adsorption on PEI 600 Da agarose followed by crosslinking with 70% oxidized dextran (20 kDa). The percentage of recovered activity was further improved (from 21% to 33%) by the addition of 20% glycerol to the immobilization mixture. The resulting biocatalyst showed a stability profile similar to that of the soluble enzyme and it was used for the preparative synthesis of trifluridine and decytabine obtaining conversions ranging from 50% to 76%.Comunidad Autónoma de Madrid (Spain) (Grant S2009/PPQ-1752) and Spanish Ministry of Science and Innovation (CTQ2009-11543)4.636 JCR (2016) Q1, 6/72 Chemistry, Applied, 30/146 Chemistry, Physical, 12/135 Engineering, ChemicalUE

A lateral flow assay for the rapid diagnosis of Mycobacterium bovis infection in wild boar

The 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

2’-deoxyribosyltransferases: An efficient biocatalyst for the synthesis of nucleoside analogues

In 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.No data (2013)UE

Nucleoside 2'-Deoxyribosyltransferase from psychrophilic bacterium bacillus psychrosaccharolyticus - Preparation of an immobilized biocatalyst for the enzymatic synthesis of therapeutic nucleosides

Nucleoside 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 7.5.2.416 JCR (2014) Q2, 21/57 Chemistry, organicUE