title: Stabilization of thymidine phosphorylase from Escherichia coli by immobilization and post immobilization techniques.
abstract: Homodimeric thymidine phosphorylase from Escherichia coli (TP, E.C. 2.4.2.4) was immobilized on solid support with the aim to have a stable and recyclable biocatalyst for nucleoside synthesis. Immobilization by ionic adsorption on amine-functionalized agarose and Sepabeads(®) resulted in a very high activity recovery (>85%). To prevent undesirable leakage of immobilized enzyme away from the support, the ionic preparations were cross-linked with aldehyde dextran (MW 20 kDa) and the influence of the dextran oxidation degree on the resulting biocatalyst activity was evaluated. Although in all cases the percentage of expressed activity after immobilization drastically decreased (≤ 25%), this procedure allowed to obtain an active catalyst which resulted up to 6-fold and 3-fold more stable than the soluble (non immobilized) enzyme and the just adsorbed (non cross-linked) counterpart, respectively, at pH 10 and 37°C. No release of the enzyme from the support could be observed. Covalent immobilization on aldehyde or epoxy supports was generally detrimental for enzyme activity. Optimal TP preparation, achieved by immobilization onto Sepabeads(®) coated with polyethyleneimine and cross-linked, was successfully used for the one-pot synthesis of 5-fluoro-2'-deoxyuridine starting from 2'-deoxyuridine or thymidine (20mM) and 5-fluorouracil (10mM). In both cases, the reaction proceeded at the same rate (3 μmol min(-1)) affording 62% conversion in 1h.
authors: Dipartimento di Scienze del Farmaco and Italian Biocatalysis Center, Via Taramelli 12, Università degli Studi di Pavia, 27100 Pavia, Italy.
authors: Immacolata
authors: I
authors: Serra
authors: Carla Daniela
authors: CD
authors: Serra
authors: Silvia
authors: S
authors: Rocchietti
authors: Daniela
authors: D
authors: Ubiali
authors: Marco
authors: M
authors: Terreni
mesh_terms: D000327:Adsorption; D002384:Catalysis; D003432:Cross-Linking Reagents; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005467:Floxuridine / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods; D008863:Microspheres; D012685:Sepharose; D013939:Thymidine Phosphorylase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D005467:Floxuridine; D012685:Sepharose; D013939:Thymidine Phosphorylase