The open reading frame TM1080 from Therm otoga maritima encoding ribose-5-phosphate isomerase type B (RpiB) was cloned and over-expressed in Escherichia cob BL21 (DE3). After optimization of cell culture conditions, more than 30% of intracellular proteins were soluble recombinant RpiB. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 70 degrees C and pH 6.5-8.0. Under its suboptimal conditions (60 degrees C and pH 7.0), k(cat), and K-m values were 540 s(-1) and 7.6 mM, respectively; it had a half lifetime of 71 h, resulting in its turn-over number of more than 2 x 10(8) mol of product per mol of enzyme. This study suggests that it is highly feasible to discover thermostable enzymes from exploding genomic DNA database of extremophiles with the desired stability suitable for in vitro synthetic biology projects and produce high-purity thermoenzymes at very low costs. (C) 2012 Elsevier Inc. All rights reserved.