Wang Guanglu, Xia Jungang, Xie Xixian, Xu Qingyang, Chen Ning
College of Biotechnology, Tianjin University of Science and Technology, Key Laboratory of Industrial Microbiology of Education Ministry, Tianjin 300457, China.
Wei Sheng Wu Xue Bao. 2010 Feb;50(2):222-7.
Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) is an important enzyme which is applied in nucleoside medication and intermediate biosynthesis. In this paper, we aimed to obtain the PNP gene from cold-adapted marine bacterium Pseudoalteromonas sp. XM2107 and study the characteristics of enzyme for applying in nucleoside medication and intermediate biosynthesis.
Purine nucleoside phosphorylas gene which amplified from the cold-adapted marine bacterium Pseudoalteromonas sp. XM2107 genome by homology-based PCR cloning was cloned, sequenced and expressed at E. coli BL21 (DE3) by using expression vector pET-His. The recombinant purine nucleoside phosphorylas enzyme (XmPNP) was purified by metal chelate chromatography and its several characteristics were determined completely.
Analysis of entire sequences of XmPNP revealed that the whole sequence is 702 bp and coded a peptide of 233 amino acids with a calculated molecular mass of 25 kDa. Compared with mesophilic counterparts, XmPNP showed a lower temperature optimum (50 degrees C). The optimal pH for inosine phosphorolysis catalyzed by XmPNP was around 7.6 at sodium phosphate buffer. XmPNP showed the highest activity toward inosine (K(m) value, 0.382 mmol/L, at 37 degrees C) and the activity decreased in the order of guanosine and adenosine. Furthermore, XmPNP still expressed high catalytic activity and excellent thermalstability at ordinary temperature.
Both high catalytic activity and good thermalstability at ordinary temperature indicated that it will provide attractive candidate for prodrug activation and nucleoside medication biotransformation.
嘌呤核苷磷酸化酶（PNP，EC 2.4.2.1）是一种重要的酶，应用于核苷药物和中间体生物合成。本文旨在从适冷海洋细菌假交替单胞菌属XM2107中获取PNP基因，并研究该酶的特性，以应用于核苷药物和中间体生物合成。
通过基于同源性的PCR克隆从适冷海洋细菌假交替单胞菌属XM2107基因组中扩增出嘌呤核苷磷酸化酶基因，进行克隆、测序，并利用表达载体pET-His在大肠杆菌BL21（DE3）中表达。重组嘌呤核苷磷酸化酶（XmPNP）通过金属螯合层析进行纯化，并全面测定其多种特性。
对XmPNP的全序列分析表明，其全长为702 bp，编码一个233个氨基酸的肽段，计算分子量为25 kDa。与嗜温同类酶相比，XmPNP的最适温度较低（50℃）。在磷酸钠缓冲液中，XmPNP催化肌苷磷酸解的最适pH约为7.6。XmPNP对肌苷的活性最高（37℃时K(m)值为0.382 mmol/L），对鸟苷和腺苷的活性依次降低。此外，XmPNP在常温下仍表现出较高的催化活性和优异的热稳定性。
高催化活性和常温下良好的热稳定性表明，它将为前药激活和核苷药物生物转化提供有吸引力的候选物。
