假交替单胞菌XM2107嘌呤核苷磷酸化酶基因克隆表达、重组蛋白纯化及酶学性质  

Molecular cloning,gene expression and characterization of purine nucleoside phosphorylase from Pseudoalteromonas sp.XM2107

作　　者：王光路[1] 夏俊刚[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：《微生物学报》2010年第2期222-227,共6页Acta Microbiologica Sinica

基　　金：天津市高等学校科技发展基金计划项目(20070903)~~

摘　　要：【目的】嘌呤核苷磷酸化酶(PNP,EC.2.4.2.1)在酶法合成核苷类药物及中间体中具有广泛应用。本文研究的目标是,获得极地嗜冷菌假交替单胞菌Pseudoa lteromonas sp.XM2107嘌呤核苷磷酸化酶编码基因,并对该酶酶学性质进行研究,以考察该酶在核苷类中间体及药物合成中的潜在应用价值。【方法】利用同源序列PCR技术从Pseudoa lteromonas sp.XM2107基因组DNA中扩增出其编码嘌呤核苷磷酸化酶基因,测序获得编码序列。将该基因在大肠杆菌BL21(DE3)中进行重组表达以及金属螯合层析纯化,对其酶学性质进行初步研究。【结果】经过测序获得了该酶编码基因序列,全长702 bp,共编码233个氨基酸,大小为25 kDa,Genbank登录号为GQ475485。酶学性质研究发现,该重组酶最适反应温度为50℃,最适酶促反应pH为7.6(25 mmol/L磷酸盐缓冲液),最适酶促反应底物为肌苷(Km值0.389 mmol/L,37℃),且对底物腺苷和鸟苷也有磷酸解活性,在普通温度下具有较高催化活性和较好热稳定性。【结论】来源于Pseudoa lteromonas sp.XM2107的嘌呤核苷磷酸化酶在普通温度条件下具有较高的催化活性及良好热稳定性性质,在核苷类中间体和药物合成中具有较广泛的应用价值。[ Objective] 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 co/d-adapted marine bacterium Pseudoalteromonas sp. XM2107 and study the characteristics of enzyme for applying in nucleoside medication and intermediate biosynthesis. [ Methods] 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. [ Results] 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℃）. 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, value,0. 382 mmol/L, at 37℃ ） and the activity decreased in the order of guanosine and adenosine. Furthermore, XmPNP still expressed high catalytic activity and excellent thermalstability at ordinary temperature. [ Conclusion] Both high catalytic activity and good thermalstability at ordinary temperature indicated that it will provide attractive candidate for prodrug activation and nucleoside medication biotransformation.

关 键 词：假交替单胞菌 嘌呤核苷磷酸化酶 酶学性质 

分 类 号：Q93[生物学—微生物学] Q78