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Cloning and characterization of purine nucleoside phosphorylase in Escherichia coli and subsequent ribavirin biosynthesis using immobilized recombinant cells

Paper ID Volume ID Publish Year Pages File Format Full-Text
17453 42670 2011 7 PDF Available
Title
Cloning and characterization of purine nucleoside phosphorylase in Escherichia coli and subsequent ribavirin biosynthesis using immobilized recombinant cells
Abstract

With improved enzymatic activity and easy accessibility, the recombinant purine nucleoside phosphorylase (PNPase) could be a very promising alternative for nucleoside biosynthesis. In our work, the deoD gene encoding PNPase was successfully cloned from Escherichia coli MG1665 and overexpressed in E. coli BL 21(DE3). After optimization of expression conditions including temperature, induction timing and isopropyl-thio-β-d-galactoside (IPTG) concentration, over 70% of expressed total protein was His-tagged PNPase, in the soluble and functional form. Followed assays indicated that the recombinant enzyme exhibited similar substrate specificity and pH preference as the wild type PNPase. Furthermore, the immobilization technology was applied to develop the possible application of recombinant enzyme. Agar from four different polymer carriers was selected as a suitable matrix for whole recombinant cell entrapment. Subsequent enzyme assays, kinetic analysis and stability evaluation of free and immobilized recombinant cells were compared. The results indicated that although the immobilization process reduced the substrate affinity and catalytic efficiency of recombinant cells, it could significantly enhance the stability and reusability of these cells. Finally, the immobilized whole cell biocatalyst was applied to produce ribavirin, as a model nucleoside synthesis reaction. The obtained relative high productivity of rabavirin and quick reaction time suggested the great potential and feasibility of immobilized PNPase in efficient and valuable industrial utilizations.

Keywords
PNPase, purine nucleoside phosphorylase; IPTG, isopropyl-thio-β-d-galactoside; UPase, uridine phosphorylase; TCA, triazole carboxyl amide; LB, Luria–Bertani; OD, optical density; DCW, dry cell weight; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel el
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Cloning and characterization of purine nucleoside phosphorylase in Escherichia coli and subsequent ribavirin biosynthesis using immobilized recombinant cells
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 48, Issues 6–7, 6 May 2011, Pages 438–444
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