Ge Chongtao, OuYang Liming, Ding Qingbao, Ou Ling
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.
Appl Biochem Biotechnol. 2009 Oct;159(1):168-77. doi: 10.1007/s12010-008-8429-3. Epub 2008 Dec 20.
The genes encoding purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase), and thymidine phosphorylase (TPase) from Escherichia coli K12 were cloned respectively into expression vector pET-11a or pET-28a. The recombinant plasmids were transformed into the host strain E. coli BL21(DE3) to construct four co-expression recombinant strains. Two of them had double recombinant plasmids (DUD and DAD) and the other two had tandem recombinant plasmid (TDU and TDA) in them. Under the repression of antibiotic, recombinant plasmids stably existed in host strains. Enzymes were abundantly expressed after induction with IPTG and large amount of target proteins were expressed in soluble form analyzed with SDS-PAGE. Compared with the host strain, enzyme activity of the recombinant strains had been notably improved. In the transglycosylation reaction, yield of 2,6-diaminopurine-2'-deoxyriboside (DAPdR) from 2,6-diaminopurine (DAP) and thymidine reached 40.2% and 51.8% catalyzed by DAD and TDA respectively; yield of 2,6-diaminopurine riboside (DAPR) from DAP and uridine reached 88.2% and 58.0% catalyzed by TDU and DUD respectively.
将来自大肠杆菌K12的编码嘌呤核苷磷酸化酶（PNPase）、尿苷磷酸化酶（UPase）和胸苷磷酸化酶（TPase）的基因分别克隆到表达载体pET-11a或pET-28a中。将重组质粒转化到宿主菌株大肠杆菌BL21(DE3)中，构建了4种共表达重组菌株。其中两种含有双重组质粒（DUD和DAD），另外两种含有串联重组质粒（TDU和TDA）。在抗生素的抑制作用下，重组质粒稳定存在于宿主菌株中。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导后，酶大量表达，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析，大量目标蛋白以可溶形式表达。与宿主菌株相比，重组菌株的酶活性显著提高。在转糖基化反应中，由DAD和TDA催化时，2,6-二氨基嘌呤（DAP）和胸苷生成2,6-二氨基嘌呤-2'-脱氧核糖苷（DAPdR）的产率分别达到40.2%和51.8%；由TDU和DUD催化时，DAP和尿苷生成2,6-二氨基嘌呤核糖苷（DAPR）的产率分别达到88.2%和58.0%。
