[Molecular cloning, gene expression and characterization of purine nucleoside phosphorylase from Pseudoalteromonas sp. XM2107].
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.
Wang
Guanglu
G
College of Biotechnology, Tianjin University of Science and Technology, Key Laboratory of Industrial Microbiology of Education Ministry, Tianjin 300457, China. wangguanglu@163.com
Xia
Jungang
J
Xie
Xixian
X
Xu
Qingyang
Q
Chen
Ning
N
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
D001426:Bacterial Proteins; D011683:Purine-Nucleoside Phosphorylase