Stabilization of Escherichia coli uridine phosphorylase by evolution and immobilization

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.

Graphical abstractFigure optionsDownload full-size imageDownload 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).