title: Enzymatic synthesis of nucleoside analogues using immobilized 2'-deoxyribosyltransferase from Lactobacillus reuteri.
abstract: Covalent attachment of recombinant Lactobacillus reuteri 2'-deoxyribosyltransferase to Sepabeads EC-EP303 leads to the immobilized biocatalyst SLrNDT4, which displayed an enzymatic activity of 65.4 IU/g of wet biocatalyst in 2'-deoxyadenosine synthesis from 2'-deoxyuridine and adenine at 40°C and pH 6.5. Response surface methodology was employed for the optimization of SLrNDT4 activity. Optimal conditions for SLrNDT4 highest activity were observed at 40°C and pH 6.5. Immobilized biocatalyst retained 50% of its maximal activity after 17.9 h at 60°C, whereas 96% activity was observed after storage at 40°C for 110 h. This novel immobilized biocatalyst has been successfully employed in the enzymatic synthesis of different natural and therapeutic nucleosides effective against cancer and viral diseases. Among these last products, enzymatic synthesis of therapeutic nucleosides such as 5-ethyl-2'-deoxyuridine and 5-trifluorothymidine has been carried out for the first time. Importantly for its potential application, SLrNDT4 could be recycled for 26 consecutive batch reactions in the synthesis of 2,6-diaminopurine-2'-deoxyriboside with negligible loss of catalytic activity.
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Biológicas, Universidad Complutense de Madrid, c/José Antonio Novais 2, 28040 Madrid, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Fernández-Lucas
authors.forename: Alba
authors.initials: A
authors.lastname: Fresco-Taboada
authors.forename: Carmen
authors.initials: C
authors.lastname: Acebal
authors.forename: Isabel
authors.initials: I
authors.lastname: de la Mata
authors.forename: Miguel
authors.initials: M
authors.lastname: Arroyo
mesh_terms: D055162:Biocatalysis; D001709:Biotechnology / Q000379:methods; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000096:biosynthesis* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D052200:Limosilactobacillus reuteri / Q000201:enzymology*; D009705:Nucleosides / Q000737:chemistry / Q000378:metabolism*; D010430:Pentosyltransferases / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004800:Enzymes, Immobilized; D009705:Nucleosides; D011994:Recombinant Proteins; D010430:Pentosyltransferases; C420226:nucleoside deoxyribosyltransferase II