Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China.
Biotechnol Lett. 2011 Jun;33(6):1107-12. doi: 10.1007/s10529-011-0535-6. Epub 2011 Jan 29.
Purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of various purine nucleosides is widely distributed in prokaryotes and eukaryotes. Four pnp genes from Bacillus subtilis 168, Escherichia coli K-12 and Pseudoalteromonas sp. XM2107 were cloned by PCR and expressed in E. coli XL1-Blue. Recombinant PNPs (rPNPs) were purified by Ni(2+)-NTA chromatography. Compared with other rPNPs, PNP(816) was a low-molecular-mass homotrimer, which exhibited 11-, 4- and 1.5-fold higher values in k (cat)/K (m) using inosine as the substrate at 37°C. The PNP(816) or engineered strain XBlue (pQE-816) had a higher catalytic activity than other rPNPs or engineered strains during the enzymatic synthesis of ribavirin, which suggested that the low-molecular-mass homotrimer derived from microorganisms has higher catalytic activity for synthesis of nucleoside antiviral drugs.
嘌呤核苷磷酸化酶（PNP）能催化各种嘌呤核苷的可逆磷酸解，广泛分布于原核生物和真核生物中。通过 PCR 从枯草芽孢杆菌 168、大肠杆菌 K-12 和假交替单胞菌 XM2107 中克隆了 4 个 pnp 基因，并在大肠杆菌 XL1-Blue 中表达。通过 Ni(2+)-NTA 层析法纯化了重组 PNPs（rPNPs）。与其他 rPNPs 相比，PNP(816)是一种低分子量的三聚体，在 37°C 时以肌苷为底物，其 k (cat)/K (m) 值分别高出 11 倍、4 倍和 1.5 倍。在利巴韦林的酶促合成过程中，PNP(816)或工程菌株 XBlue（pQE-816）的催化活性高于其他 rPNPs 或工程菌株，这表明来源于微生物的低分子量三聚体具有更高的合成核苷抗病毒药物的催化活性。
