کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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70681 | 48841 | 2011 | 7 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Stabilization of Escherichia coli uridine phosphorylase by evolution and immobilization Stabilization of Escherichia coli uridine phosphorylase by evolution and immobilization](/preview/png/70681.png)
Mutation and immobilization techniques were applied to uridine phosphorylase (UP) from Escherichia coli in order to enhance its thermal stability and hence productivity in a biocatalytic reaction. UP was evolved by iterative saturation mutagenesis. Compared to the wild type enzyme, which had a temperature optimum of 40 °C and a half-life of 9.89 h at 60 °C, the selected mutant had a temperature optimum of 60 °C and a half-life of 17.3 h at 60 °C. Self-immobilization of the native UP as a Spherezyme showed a 3.3 fold increase in thermostability while immobilized mutant enzyme showed a 4.4 fold increase in thermostability when compared to native UP. Combining UP with the purine nucleoside phosphorylase from Bacillus halodurans allows for synthesis of 5-methyluridine (a pharmaceutical intermediate) from guanosine and thymine in a one-pot transglycosylation reaction. Replacing the wild type UP with the mutant allowed for an increase in reaction temperature to 65 °C and increased the reaction productivity from 10 to 31 g l−1 h−1.
Figure optionsDownload as PowerPoint slideResearch highlights▶ Uridine phosphorylase from Escherichia coli was evolved using iterative saturation mutagenesis. ▶ The most beneficial mutant uridine phosphorylase showed a 20 °C increase in optimum thermal activity and a 3.7 fold increase in thermal stability at 60 °C when compared to the wild-type enzyme. ▶ Uridine phosphorylase and purine nucleoside phosphorylase, both multiple-subunit enzymes, were successfully immobilised by Spherezyme formation at high specific activity. ▶ Co-immobilized enzymes showed retention of productivity and yield characteristics for the production of 5-methyluridine by transglycosylation. ▶ Use of the thermally enhanced uridine phosphorylase gave a 3-fold improvement in reaction productivity (up to 31 g l−1 h−1).
Journal: Journal of Molecular Catalysis B: Enzymatic - Volume 68, Issues 3–4, March 2011, Pages 279–285