N-脱氧核糖转移酶II催化合成克拉屈滨及其定向进化

目的:使用表达N-脱氧核糖转移酶Ⅱ (Nucleoside deoxyribose transferase, NDT)的大肠杆菌重组工程菌E.coli BL21/pET-NDT,作为催化剂,合成克拉屈滨(Cladribine)。同时构建高通量的酶活筛选体系,利用其改造NDT,以期提高克拉屈滨的合成效率。方法:首先用野生型NDT催化克拉屈滨的合成。接着以黄嘌呤氧化酶和辣根过氧化物酶联合作用来检测NDT酶活。最后构建NDT随机突变体库,并筛选出突变体。结果:在10%DMSO的体系中,野生型NDT催化2-氯腺嘌呤合成克拉屈滨的转化率达到93%。同时使用构建的筛选体系在突变体库中筛选到了酶活发生改变的突变体。结论:本研究使用NDT作为催化剂,成功地一步合成了克拉屈滨。同时本研究构建的高通量筛选方法成功应用于改造NDT的酶学性质,为拓展NDT合成核苷类似物的能力提供了一种新的方法。

Synthesis of Cladribine by N-deoxyribosyltransferase II and Directed Evolution of the Enzyme

ABSTRACT Objective: N-deoxyribosyltransferase II (NDT) was produced by recombinant strain E.coli BL21/pET-NDT. This recombinant strain was used as catalysis to synthesize cladribine. High-throughput screening system was established to engineer NDT, in order to increase the yield of cladribine. Methods: First, synthesize cladribine with wild NDT. Then combine Xanthine Oxidase with Horseradish Peroxidase to assay the activity of NDT in a high-throughput manner. Finally, construct a random mutant library of NDT, and screen the mutants of interest. Results: In the reaction mixture of 10 % DMSO, wild NDT catalysed the synthesis of cladribine, and the yield was 93%. The constructed random library was screened, and a mutant with altered activity was found. Conclusion: In this study, NDT was employed as a catalyst, and cladribine was synthesized successfully in one step. And the high-throughput screening system constructed in this study was successfully applied to the alternation of the NDT activity, which offers a new way to expand the ability of NDT in preparation of unnatural nucleoside analogs.