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  

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

作　　者：夏俊刚[1] 何逵夫[1] 谢希贤[1] 徐庆阳[1] 陈宁[1] 

机构地区：[1]天津科技大学生物工程学院教育部工业微生物重点实验室,天津300457

出　　处：《中国生物工程杂志》2010年第12期53-59,共7页China Biotechnology

基　　金：天津市高等学校科技发展基金计划项目(20070903)资助项目

摘　　要：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（kcat/Km）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-B lue（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。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（Kcat/Km）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.

关 键 词：枯草芽孢杆菌 嘌呤核苷磷酸化酶 酶学性质 利巴韦林 酶法 

分 类 号：Q936[生物学—微生物学]