Immobilization and stabilization of recombinant multimeric uridine and purine nucleoside phosphorylases from Bacillus subtilis.
We selected the PnpI/PupG (PNP) with specificity for ribo- and deoxyriboguanosine and ribo- and deoxyriboinosine and the Up/Pdp (UP) with specificity for uridine, thymidine, and deoxyuridine from the purine and pyrimidine salvage pathway of the Gram-positive bacterium Bacillus subtilis. Then, an extensive study of the UP (uridine phosphorylase) and PNP (purine nucleoside phosphorylase) immobilization and stabilization was carried out: optimal UP preparation was achieved by immobilization onto Sepabeads coated with poly(ethyleneimine) and finally cross-linked with aldehyde dextran (UP-Sep-PEI-Dx); optimal immobilized PNP was prepared onto glyoxyl-agarose. Both derivatives were highly stable and active even under drastic experimental conditions (pH 10, 45 degrees C) unlike the free enzymes which were promptly inactivated. The derivatives prepared were successfully used in the synthesis of 2'-deoxyguanosine by enzymatic transglycosylation in aqueous solution between 2'-deoxyuridine and guanine.
Rocchietti
Silvia
S
Innovate Biotechnology srl, Parco Scientifico Tecnologico, Strada Savonesa 9, I-15050 Rivalta Scrivia (AL), Italy.
Ubiali
Daniela
D
Terreni
Marco
M
Albertini
Alessandra M
AM
Fernández-Lafuente
Roberto
R
Guisán
José M
JM
Pregnolato
Massimo
M
D001412:Bacillus subtilis / Q000201:enzymology*; D055592:Biophysical Phenomena; D001703:Biophysics; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003849:Deoxyguanosine / Q000737:chemistry; D003857:Deoxyuridine / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry; D006031:Glycosylation; D006147:Guanine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry*; D012685:Sepharose / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014529:Uridine / Q000737:chemistry*; D014543:Uridine Phosphorylase / Q000737:chemistry*
D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006147:Guanine; D012685:Sepharose; D011683:Purine-Nucleoside Phosphorylase; D014543:Uridine Phosphorylase; D003849:Deoxyguanosine; D003857:Deoxyuridine; D014529:Uridine