嘌呤核苷磷酸化酶与嘧啶核苷磷酸化酶的融合表达及酶法合成嘌呤核苷类产物

Fusion expression of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase for the synthesis of purine nucleoside products

将嘌呤核苷磷酸化酶与嘧啶核苷磷酸化酶进行融合,提高生物酶法催化合成克拉屈滨等嘌呤核苷类产物的产率。设计不同的刚性、柔性连接短肽(Linker),将嘧啶核苷磷酸化酶EcUP与嘌呤核苷磷酸化酶AmPNP连接融合,考察酶融合蛋白的可溶性表达与活性情况。使用EEEEEEKKK短肽连接的融合蛋白EcUP-L4-AmPNP可溶性表达水平较高,并对嘌呤核苷与嘧啶核苷均具有反应活性,对嘌呤核苷的水解率远小于嘧啶核苷。研究发现:融合酶EcUP-L4-AmPNP催化合成克拉屈滨等嘌呤核苷类产物的产率70.4%~98.3%,合成产率比分别加入酶EcUP和酶AmPNP的游离双酶体系提高了约1.5~2.0倍。核苷磷酸化酶的融合蛋白有利于实现高效催化合成嘌呤核苷产物,生物催化合成克拉屈滨等几种嘌呤核苷类产物的产率显著提高。

Fusion of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase could improve the yield of purine nucleoside drugs such as cladribine.Different typical linkers were designed to combine purine nucleoside phosphorylase(AmPNP) with pyrimidine nucleoside phosphorylase(EcUP).The soluble expression of the fusion enzyme and the activity of the fusion enzyme were also inresrestigated. The soluble expression level of the fusion enzyme EcUP-L4-AmPNP combined by rigidity linker EEEEEEKKK was improved,and it could show relatively high activity towards both purine nucleosides and pyrimidine nucleosides.The hydrolysis activity of purine nucleoside was much lower than pyrimidine nucleoside by fusion enzyme EcUP-L4-AmPNP,which was favor to efficient biosynthesis of purine nucleoside products.The yield of purine nucleosides including cladribine synthesized by fusion enzyme EcUP-L4-AmPNP,increased from 70.4% to 98.3%,which was about 1.5-2.0 times higher than that of free dual enzyme.The yield of purine nucleosides such as cladribine synthesized by using fusion enzyme EcUP-L4-AmPNP as biocatalyst was significantly improved.