Stabilization of Escherichia coli uridine phosphorylase by evolution and immobilization |
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| Institution: | 1. Faculty of Information Technology, Ho Chi Minh City University of Technology, Ho Chi Minh City, Vietnam;2. College of Electronics and Information Engineering, Sejong University, Seoul, Republic of Korea;3. Division of Data Science, Ton Duc Thang University, Ho Chi Minh City, Vietnam;4. Faculty of Information Technology, Ton Duc Thang University, Ho Chi Minh City, Vietnam;5. Department of Electrical and Computer Engineering, University of Alberta, Edmonton, T6R 2V4 AB, Canada;6. Department of Electrical and Computer Engineering, Faculty of Engineering, King Abdulaziz University, Jeddah, 21589, Saudi Arabia;7. Systems Research Institute, Polish Academy of Sciences, Warsaw, Poland |
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| Abstract: | 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. |
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