合成利巴韦林的关键酶嘌呤核苷磷酸化酶的原核表达及活性分析

Prokaryotic Expression and Functional Analysis of Purine Nucleoside Phosphorylase of the Key Enzyme for Ribavirin Produced

目的:在枯草芽孢杆菌中表达嘌呤核苷磷酸化酶(PNPase)并分析其活性。方法:将PNPase的编码基因deoD克隆入pDG148表达载体,构建原核穿梭型表达载体pDG148-deoD,采用电转化方法将表达载体转入枯草芽孢杆菌WB600后诱导表达重组PNPase;研究重组PNPase的活性。结果与结论:获得的重组PNPase活性较对照提高了193.9%,其最适催化条件为65℃、pH7.5、500μmol/L底物浓度和1%1,2,4-三氮唑-3-羧甲酰胺;对重组菌的发酵条件进行了初步优化,IPTG诱导6h后在发酵液中添加0.5%的Tween-80能大幅度提高重组PNPase的酶活力。

Objective： To express purine nucleoside phosphorylase（PNPase） in Bacillus subtilis and to analyze its activity.Methods： The gene deoD encoding PNPase was identified and cloned into vector pDG148, and the prokaryotic shuttle vector pDG148-deoD was constructed and transformed to B.subtilis WB600, and the recombinant PNPase expression was induced. The activity of the recombinant PNPase was analyzed by temperature and pH perturbation difference spectra. Resuits and Conclusion： The results showed that the enzyme activity of the recombinant PNPase was increased by 193.9%, and the optimum temperature was 65℃, the optimum pH was 7.5, the optimum substrate concentration was 500 μmol/L and the optimum formamide was 1%. The enzyme activity of recombinant PNPase was increased with Tween-80 treatment after recombinant bacteria was induced by the addition of IPTG.