2. Academic Validation
  3. A simple assay for determining activities of phosphopentomutase from a hyperthermophilic bacterium Thermotoga maritima

A simple assay for determining activities of phosphopentomutase from a hyperthermophilic bacterium Thermotoga maritima

  • Anal Biochem. 2016 May 15:501:75-81. doi: 10.1016/j.ab.2016.02.013.
Hanan M A Moustafa 1,  Taha I Zaghloul 2,  Y-H Percival Zhang 3
Affiliations

Affiliations

  • 1 Department of Biological Systems Engineering, Virginia Tech, Blacksburg, VA 24061, USA; Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, El-Chatby, Alexandria 21526, Egypt.
  • 2 Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, El-Chatby, Alexandria 21526, Egypt.
  • 3 Department of Biological Systems Engineering, Virginia Tech, Blacksburg, VA 24061, USA; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin Airport Economic Area, Tianjin 300308, China. Electronic address: ypzhang@vt.edu.
PMID: 26924489 DOI: 10.1016/j.ab.2016.02.013
Abstract

Phosphopentomutase (PPM) catalyzes the interconversion of α-D-(deoxy)-ribose 1-phosphate and α-D-(deoxy)-ribose 5-phosphate. We developed a coupled or uncoupled enzymatic assay with an enzyme nucleoside Phosphorylase for determining PPM activities on D-ribose 5-phosphate at a broad temperature range from 30 to 90 °C. This assay not only is simple and highly sensitive but also does not require any costly special instrument. Via this technology, an open reading frame TM0167 from a thermophilic bacterium Thermotoga maritima putatively encoding PPM was cloned. The recombinant PPM was overexpressed in Escherichia coli Rosetta. This enzyme has the highest activity at 90 °C. MnCl2 (0.1 mM) and 50 μM α-D-glucose 1,6-bisphosphate are cofactors. The kinetic parameters of Km and kcat are 1.2 mM and 185 s(-1) at 90 °C, respectively. The enzyme has a half-life time of up to 156 min at 90 °C. This enzyme is the most active and thermostable PPM reported to date.

Keywords

Biocatalysis; Enzymatic assay; Phosphopentomutase; Thermophilic enzyme; Thermotoga maritima.

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