title: [Molecular cloning, gene expression and characterization of purine nucleoside phosphorylase from Pseudoalteromonas sp. XM2107].
abstract: 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 cold-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 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.
  
  
  CONCLUSIONS
  Both high catalytic activity and good thermalstability at ordinary temperature indicated that it will provide attractive candidate for prodrug activation and nucleoside medication biotransformation.
authors.affiliation: College of Biotechnology, Tianjin University of Science and Technology, Key Laboratory of Industrial Microbiology of Education Ministry, Tianjin 300457, China. wangguanglu@163.com
authors.forename: Guanglu
authors.initials: G
authors.lastname: Wang
authors.forename: Jungang
authors.initials: J
authors.lastname: Xia
authors.forename: Xixian
authors.initials: X
authors.lastname: Xie
authors.forename: Qingyang
authors.initials: Q
authors.lastname: Xu
authors.forename: Ning
authors.initials: N
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D031161:Pseudoalteromonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011683:Purine-Nucleoside Phosphorylase