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- W3209284179 abstract "• A bioprocess for efficient production of 2’-deoxy-2’-fluoroadenosine was developed. • Selecting mutation sites were based on molecular docking and dynamics simulations. • An efficient screening method was established. • The major impact of random coil on the active site of PNP was discovered. Nucleoside analogs are commonly used drugs for the treatment of cancer and viral infections. Purine nucleoside phosphorylase (PNP) is one of key enzymes required for the biosynthesis of 2’-deoxy-2’-fluoroadenosine. To improve PNP activity, two methods for selecting mutation sites were used based on molecular docking and dynamic simulations of PNP and various substrates. An efficient PNP mutant screening method was established. The variant E166 F/M167D 2 M had the highest activity and was selected for further characterization. The enzymatic activity and reaction rate of transglycosylation catalyzed by this variant were increased by 47.6 % and 38.8 % compared with the wild-type PNP, respectively. Two representative variants were used for analysis of conformational differences. We discovered that the flexibility of the random coil where Phe159 was located had a significant impact on the active center. In addition, the synthesis of 2’-deoxy-2’-fluoroadenosine was scaled up using 500 mL of phosphate buffer (100 mM, pH 7.0) containing 62.5 mM adenine, 25 mM 2’-deoxy-2’-fluorouridine, 3 mg thymidine phosphorylase (TP), and 3 mg E166 F/M167D 2 M. The concentration of the product reached 14.35 mM and the conversion rate reached 57.4 %. Thus, this process represents a promising approach for industrial production of 2’-deoxy-2’-fluoroadenosine." @default.
- W3209284179 created "2021-11-08" @default.
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- W3209284179 date "2021-12-01" @default.
- W3209284179 modified "2023-09-23" @default.
- W3209284179 title "Site-directed mutation of purine nucleoside phosphorylase for synthesis of 2'-deoxy-2'-fluoroadenosine" @default.
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- W3209284179 doi "https://doi.org/10.1016/j.procbio.2021.10.028" @default.
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