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Stabilization of thymidine phosphorylase from Escherichia coli by immobilization and post immobilization techniques

Paper ID Volume ID Publish Year Pages File Format Full-Text
17556 42677 2011 7 PDF Available
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 (20 mM) and 5-fluorouracil (10 mM). In both cases, the reaction proceeded at the same rate (3 μmol min−1) affording 62% conversion in 1 h.

Keywords
E. coli thymidine phosphorylase; Enzyme immobilization; Transglycosylation; 5-fluoro-2′-deoxyuridine
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Stabilization of thymidine phosphorylase from Escherichia coli by immobilization and post immobilization techniques
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 49, Issue 1, 10 June 2011, Pages 52–58
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us