Extremophiles

, Volume 12, Issue 3, pp 325–333

Purine nucleoside phosphorylase from Pseudoalteromonas sp. Bsi590: molecular cloning, gene expression and characterization of the recombinant protein

Original Paper

DOI: 10.1007/s00792-007-0131-9

Cite this article as:
Li, X., Jiang, X., Li, H. et al. Extremophiles (2008) 12: 325. doi:10.1007/s00792-007-0131-9

Abstract

The gene encoding purine nucleoside phosphorylase (PNP) from the cold-adapted marine bacterium Pseudoalteromonas sp. Bsi590 was identified, cloned and expressed in Escherichia coli. The gene encodes a polypeptide of 233 amino acids with a calculated molecular weight of 25,018 Da. Pseudoalteromonas sp. Bsi590 PNP (PiPNP) shares 60% amino sequence identity and conservation of amino acid residues involved in catalysis with mesophilic Escherichia coli deoD-encoded purine nucleoside phosphorylase (EcPNP). N-terminal his-tagged PiPNP and EcPNP were purified to apparent homogeneity using Ni2+-chelating column. Compared with EcPNP, PiPNP possessed a lower temperature optimum and thermal stability. As for PNP enzymes in general, PiPNP and EcPNP displayed complicated kinetic properties; PiPNP possessed higher Km and catalytic efficiency (kcat/Km) compared to EcPNP at 37°C. Substrate specificity results showed PiPNP catalyzed the phosphorolytic cleavage of 6-oxopurine and 6-aminopurine nucleosides (or 2-deoxynucleosides), and to a lesser extent purine arabinosides. PiPNP showed a better activity with inosine while no activity toward pyrimidine nucleosides. The protein conformation was analyzed by temperature perturbation difference spectrum. Results showed that PiPNP had lower conformation transition point temperature than EcPNP; phosphate buffer and KCl had significant influence on PiPNP protein conformation stability and thermostability.

Keywords

Pseudoalteromonas Escherichia coli Purine nucleoside phosphorylase Characterization 

Abbreviations

PiPNP

Pseudoalteromonas sp. Bsi590 purine nucleoside phosphorylase

EcPNP

Escherichia coli purine nucleoside phosphorylase

Copyright information

© Springer 2008

Authors and Affiliations

  • Xiaohui Li
    • 1
  • Xinyin Jiang
    • 1
  • Huirong Li
    • 2
  • Daming Ren
    • 1
  1. 1.The State Key Laboratory of Genetic Engineering, Institute of GeneticsFudan UniversityShanghaiChina
  2. 2.SOA Key Laboratory for Polar SciencePolar Research Institute of ChinaShanghaiChina

Personalised recommendations