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Serra I.,University of Pavia | Serra C.D.,Innovate Biotechnology srl | Serra C.D.,University of Milan | Rocchietti S.,Innovate Biotechnology srl | And 2 more authors.
Enzyme and Microbial Technology | Year: 2011

Homodimeric thymidine phosphorylase from Escherichia coli (TP, E.C. 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 20kDa) 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 (3μmolmin -1) affording 62% conversion in 1h. © 2011 Elsevier Inc. Source

Bavaro T.,University of Pavia | Bavaro T.,Italian Biocatalysis Center | Ubiali D.,University of Pavia | Ubiali D.,Italian Biocatalysis Center | And 12 more authors.
Biocatalysis and Biotransformation | Year: 2010

Commercial lipases from the yeast Candida rugosa have been compared with two recombinant C. rugosa lipases, rCRL1 and rCRL1lid3, with respect to their immobilization and exploitation in biotransformations aimed at the synthesis of pyrimidine nucleosides. Immobilization on octyl-agarose and decaoctyl-Sepabeads but not on Eupergit® C gave comparable results to commercial lipases for rCRL1, while only a low percentage (12%) of rCRL1lid3 was efficiently immobilized. When immobilized on decaoctyl-Sepabeads, rCRL1 showed a markedly higher stability to chemical inactivation, since it could maintain 100% activity after 180 h incubation in 30% (v/v) acetonitrile. Hydrolysis of peracylated uridine and cytidine and their fluorinated counterparts proceeded with high regioselectivity and good yield, and even improved when rCRL1 was immobilized on decaoctyl-Sepabeads. © 2010 Informa UK Ltd. Source

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