Moustafa Hanan M A, Zaghloul Taha I, Zhang Y-H Percival
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.
Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, El-Chatby, Alexandria 21526, Egypt.
Anal Biochem. 2016 May 15;501:75-81. doi: 10.1016/j.ab.2016.02.013. Epub 2016 Feb 26.
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.
磷酸戊糖变位酶（PPM）催化α-D-（脱氧）-核糖1-磷酸和α-D-（脱氧）-核糖5-磷酸的相互转化。我们开发了一种偶联或非偶联酶法，利用核苷磷酸化酶在30至90°C的宽温度范围内测定PPM对5-磷酸-D-核糖的活性。该方法不仅简单且高度灵敏，而且不需要任何昂贵的特殊仪器。通过这项技术，克隆了来自嗜热细菌海栖热袍菌的一个推测编码PPM的开放阅读框TM0167。重组PPM在大肠杆菌Rosetta中过表达。该酶在90°C时具有最高活性。MnCl2（0.1 mM）和50 μMα-D-葡萄糖1,6-二磷酸是辅因子。在90°C时，Km和kcat的动力学参数分别为1.2 mM和185 s(-1)。该酶在90°C时的半衰期长达156分钟。该酶是迄今为止报道的活性最高且最耐热的PPM。
