枯草芽孢杆菌嘌呤核苷磷酸化酶酶学性质研究及在利巴韦林酶法合成中的应用

利用PCR技术从枯草芽孢杆菌基因组DNA中扩增出其编码嘌呤核苷磷酸化酶的两种基因deoD和punA,构建工程菌并采用金属螯合层析纯化PNP₇₀₂和PNP₈₁₆,酶学性质研究表明:二者具有一致的最适反应温度(60℃)和最适反应pH值(7~8),PNP₈₁₆磷酸解肌苷的催化效率(k_cat/K_m)比PNP₇₀₂高出11.12倍。底物特异性试验表明:PNP₇₀₂为高分子量的六聚体,而PNP₈₁₆为低分子量的三聚体。分别以纯化酶和工程菌菌体为酶源,以肌苷或鸟苷为核糖基供体,TCA(1,2,4-三氮唑-3-甲酰胺)为底物,酶法合成核苷类抗病毒药物利巴韦林,PNP₈₁₆和工程菌XL-Blue(pPNP₈₁₆)较PNP₇₀₂和工程菌XL-Blue(pPNP₇₀₂)具有更高的催化速度和底物转化率,表明来源于微生物的低分子量的三聚体PNP在核苷类药物和中间体微生物酶法合成中具有更高的应用价值。

Characterization of Purine Nucleoside Phosphorylase from Bacillus subtilis and Application in Enzymic Production of Ribavirin

The purine nucleoside phosphorylas (PNP,EC.2.4.2.1) genes deoD and punA which amplified from the Bacillus subtilis168 genome by polymerase chain reaction were identified, cloned and expressed in E. coli XL-Blue, respectively. Recombinant purine nucleoside phosphorylases PNP₇₀₂ and PNP₈₁₆ were purified by Ni⁺-NTA column, and several characteristics were determined. The results revealed that PNP₇₀₂ and PNP₈₁₆ both have the same optimal temperature (60℃) and pH (7~8). Enzymic kinetics experiment showed that the catalytic efficiency (K_cat/K_m) of PNP₈₁₆ is 11.12 times higher than that of PNP₇₀₂ toward inosine. Compared with PNP₈₁₆, PNP₇₀₂ has broader substrate specificity. The engineering strain XL-Blue (pPNP₈₁₆) has much higher catalytic activity than the XL-Blue (pPNP₇₀₂) in enzymatic synthesizing of the nucleoside antiviral drugs ribavirin, indicating that the low molecular weight homologous trimer PNP derived from microorganisms has the same or higher value in the microbial enzymatic synthesis of nucleoside drugs and intermediates.