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.

核苷类似物是治疗癌症和病毒感染的常用药物。嘌呤核苷磷酸化酶 (PNP) 是 2'-脱氧-2'-氟腺苷生物合成所需的关键酶之一。为了提高 PNP 活性，基于 PNP 和各种底物的分子对接和动态模拟，使用了两种选择突变位点的方法。建立了一种高效的PNP突变体筛选方法。变体 E166 F/M167D 2 M 具有最高活性，并被选中用于进一步表征。与野生型 PNP 相比，该变体催化的转糖基化的酶活性和反应速率分别提高了 47.6% 和 38.8%。两种代表性变体用于分析构象差异。我们发现 Phe159 所在的无规线圈的柔韧性对活性中心有显着影响。此外，使用含有 62.5 mM 腺嘌呤、25 mM 2'-脱氧-2'-氟尿苷、3 mg 的 500 mL 磷酸盐缓冲液（100 mM，pH 7.0）放大 2'-脱氧-2'-氟腺苷的合成胸苷磷酸化酶(TP)和3mg E166 F/M167D 2 M。产物浓度达到14.35 mM，转化率达到57.4 %。因此，该过程代表了一种用于工业生产 2'-脱氧-2'-氟腺苷的有前途的方法。3 mg E166 F/M167D 2 M。产物浓度达到14.35 mM，转化率达到57.4%。因此，该过程代表了一种用于工业生产 2'-脱氧-2'-氟腺苷的有前途的方法。和3 mg E166 F/M167D 2 M。产物浓度达到14.35 mM，转化率达到57.4%。因此，该过程代表了一种用于工业生产 2'-脱氧-2'-氟腺苷的有前途的方法。