title: Nucleoside 2'-deoxyribosyltransferase from psychrophilic bacterium Bacillus psychrosaccharolyticus--preparation of an immobilized biocatalyst for the enzymatic synthesis of therapeutic nucleosides.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense University of Madrid, C/José Antonio Novais 2, 28040 Madrid, Spain. albaf@bbm1.ucm.es.
authors.forename: Alba
authors.initials: A
authors.lastname: Fresco-Taboada
authors.affiliation: Department of Drug Sciences and Italian Biocatalysis Center, Università degli Studi di Pavia, Via Taramelli 12, 27100 Pavia, Italy. immacolata.serra@unipv.it.
authors.forename: Immacolata
authors.initials: I
authors.lastname: Serra
authors.affiliation: Department of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense University of Madrid, C/José Antonio Novais 2, 28040 Madrid, Spain. jesus.fernandez2@uem.es.
authors.forename: Jesús
authors.initials: J
authors.lastname: Fernández-Lucas
authors.affiliation: Department of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense University of Madrid, C/José Antonio Novais 2, 28040 Madrid, Spain. acebal@bbm1.ucm.es.
authors.forename: Carmen
authors.initials: C
authors.lastname: Acebal
authors.affiliation: Department of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense University of Madrid, C/José Antonio Novais 2, 28040 Madrid, Spain. arroyo@bbm1.ucm.es.
authors.forename: Miguel
authors.initials: M
authors.lastname: Arroyo
authors.affiliation: Department of Drug Sciences and Italian Biocatalysis Center, Università degli Studi di Pavia, Via Taramelli 12, 27100 Pavia, Italy. marco.terreni@unipv.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Terreni
authors.affiliation: Department of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense University of Madrid, C/José Antonio Novais 2, 28040 Madrid, Spain. idlmata@ucm.es.
authors.forename: Isabel
authors.initials: I
authors.lastname: de la Mata
mesh_terms: D001407:Bacillus / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009705:Nucleosides / Q000138:chemical synthesis* / Q000494:pharmacology*; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D014271:Trifluridine / Q000138:chemical synthesis
chemical_list: D004800:Enzymes, Immobilized; D009705:Nucleosides; D011994:Recombinant Proteins; D010430:Pentosyltransferases; C420226:nucleoside deoxyribosyltransferase II; D014271:Trifluridine
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